InteractiveFly: GeneBrief

sans fille : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Fruitless alternative pre-mRNA splicing (AS) isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, this study shows that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ~1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior (Wang, 2016).

How gene regulation modulates neuronal activities leading to cognition and behavior is an important question in biology. Although many behavior-associated genes and neuronal cell types have been identified, a detailed understanding that links the molecular events of gene regulation to specific behaviors is still lacking. Alternative pre-mRNA splicing (AS) is a crucial gene regulatory mechanism that enables a single gene to generate functionally distinct messenger RNA transcripts (mRNAs) and protein products. The nervous system makes extensive use of AS to generate diverse and complex neural mRNA expression patterns that determine numerous neuronal cell types and functions. AS is regulated by the small nuclear ribonucleoprotein complexes (snRNPs) that compose the spliceosome for intron recognition and removal, as well as a large repertoire of non-snRNP RNA-binding proteins that affect decisions on splice site use. This dynamic and complex AS regulatory network modulates diverse neuronal functions, like synaptic transmission and signal processing, hence further impacting higher brain functions, such as cognition and behavioral control (Wang, 2016).

The Drosophila KH-domain RNA binding splicing factor P-element somatic inhibitor (PSI) is best known for regulating tissue-specific AS of the Drosophila P-element transposon transcripts to restrict transposition activity to germ-line tissues. PSI directly interacts with the U1 snRNP through a 70-aa tandem direct repeat domain at the C terminus of the PSI protein (termed the 'AB' domain). Deletion of the AB domain in transgenic flies resulted in male sterility and male courtship defects. U1 snRNP, as an essential component of the spliceosome that binds to 5' splice sites (5'SS), defines exon-intron boundaries, and initiates spliceosome assembly for intron removal. U1 snRNP further affects AS decisions and suppresses pre-mRNA premature cleavage and polyadenylation through binding to pseudo-5'SS (5'SS-like motifs that are not used for splicing) that are abundantly distributed throughout the transcriptome. It remains a mystery how U1 snRNP differentiates the vast number of functional 5'SS and pseudo-5'SS in the transcriptome that leads to functionally distinct AS patterns. In the case of Drosophila P-element transposon AS regulation, the PSI-U1 snRNP interaction enables PSI to modulate the competitive binding of U1 snRNP between the accurate 5'SS in the third intron and an upstream pseudo-5'SS in the transposon pre-mRNA, and thus influence the final AS decision. It is possible that PSI may play a more general role in specifically localizing U1 snRNP to the transcriptome for AS regulation beyond the P-element transposon, and thus exert a more broad influence over fruit fly physiology (Wang, 2016).

AS patterns are often controlled by the interaction of RNA binding proteins (RBPs) with nascent pre-mRNA transcripts. These RNA-protein interactions can determine where the spliceosomal U1 and U2 snRNPs bind to the transcriptome, and thus dictate AS decisions and constitute an important mechanism for gene regulation. RBPs, such as PSI or TIA-1, which directly interact with U1 snRNP, are good candidates for proteins controlling AS patterns in this manner, and changes in these RBP-snRNP associations can have profound phenotypic effects. For example, this study shows that a subtle mutation that abolishes the PSI-U1 snRNP interaction dramatically changed the AS patterns of hundreds neuronal pre-mRNAs and resulted in highly abnormal male courtship behaviors. Given the diverse number of cell types, gene-expression patterns, and the extensive AS that occurs in animal nervous systems, it is anticipated that AS regulation will play critical roles in both the normal physiological or disease states of neurons (Wang, 2016).

The PSI-U1 snRNP interaction may further play crucial roles in other pre-mRNA processing pathways. For example, U1 snRNP was recently ascribed a new function in regulating global mRNA 3' end termination and suppression of premature pre-mRNA cleavage and polyadenylation near the 5' ends of transcripts in humans, mice, and Drosophila through selective binding to 5'SS-like motifs, a process called telescripting. It has remained a mystery how U1 snRNP discriminates the numerous potential 5'SS sites across the transcriptome. PSI may be one example of RBP regulators that alter the binding of U1 snRNP to pre-mRNA sites through direct protein-protein interactions, and thus changing pre-mRNA splicing, polyadenylation, or other pre-mRNA processing patterns (Wang, 2016).

These findings further reveal that even broadly expressed RBPs, such as PSI, can affect gene regulation in restricted subsets of neurons in the Drosophila brain that modulate specific behaviors, such as courtship and mating. The work presented here also provides the first identification of the PSI protein as a transacting RNA splicing factor controlling male-specific fruitless splicing (Wang, 2016).

Taken together, these results link the molecular interaction between PSI and U1 snRNP to specific phenotypic effects on Drosophila courtship behavior through the coordination of an AS program in the brain. These results provide important insights into the mechanisms controlling gene activity in the nervous system, leading to the precise control of complex animal behaviors (Wang, 2016).

Earlier treatment of Sans fille in The Interactive Fly

From the standpoint of developmental biology, the alternative splicing of exons is intimately connected with the spice of life: variety. This overview will discuss the complex, variety-producing process known as alternative splicing, and aspects of the process involving the gene snf.

In the most general terms, splicing is the removal of an intron, a specified portion of the nucleotide chain, from between two flanking exons (portions of the pre-messenger RNA that code for proteins). The process is regulated by certain proteins called splice factors, and depends upon the successful construction of splice machinery, in particular a structure known as a spliceosome, a multiprotein complex that is assembled step by step during the splicing process. Alternative splicing generates alternative messenger RNA species, depending on the pattern of intron removal. For example, by alternative splicing in which exons are skipped, unwanted exons can be excluded altogether from mRNA species coding for a particular polypeptide chain, thus generating a functionally alternative protein.

By way of an oversimplified analogy, imagine a nucleotide chain of people (introns and exons) holding hands. As some people leave the line, those remaining will join hands (or be spliced) with new neighbors on one side or the other or both. Depending on the specific intron(s) removed, the exons will find themselves adjacent to different exon neighbors. This brings about a re-arrangement of the nucleic acid pre-splice sequence; from this new sequence, depending on how and where the exons have been shuffled, an altered code is now in place, providing different proteins from a unified pre-splice, pre-messenger RNA. These changes in coding sequences and hence, changes in the proteins produced, can mean the difference between an embryo developing into a male or a female; cell fate rarely gets more basic than this.

The splice machinery of the cell is found in the nucleoplasm of the cell. The idea that the nucleoplasm, as distinct from the DNA (the bearer of the genetic code of the cell), is a determiner of cell fate is an idea whose time has come. By generating alternative versions of proteins, for examples, alternative versions of splice factors or transcription factors, the nucleoplasm-based splice machinery regulates cell fate. For a more detailed version of how the nucleoplasm constructs the spliceosome, the protein machinery that carries out splicing, see Transformer 2.

sans fille is now known as a splice factor. It has revealed itself to interested investigators only very slowly. Its initial identity, established from mutation studies, was as a sex determination gene (Oliver, 1988). Only when snf was cloned, and its sequence identified, was its even more basic nature revealed. SNF is similar in sequence and functionally identical to U1A snRNP (small nuclear ribonucleoprotein), a protein of major importance in splicing. SNF's role in alternative splicing places it directly in the sex determination hierarchy, as a factor involved in Sex lethal's autoregulation (Flickinger, 1994).

How does snf function in sex determination? snf or U1A snRNP as it is usually called when refering to it as a splice factor, is one of the few major proteins responsive to factors involved in alternative splicing. snf's role in germline sex determination was discovered directly from the genetic analysis of a single allele, snf1621. Females homozygous for snf1621 are sterile. In the mutant neither the oocyte nor the nurse cells differentiate. Instead the germinal cells continue to divide, resulting in the formation of ovarian tumors. A similar phenotype is seen in fused, ovo, and otu mutants. The similarity of snf and Sxl mutant phenotypes in the germline, and the fact that they interact genetically, suggests that they disrupt the same process, germline sex determination (Salz, 1992 and references).

In contrast to snf's role in germline sex determination, its role in somatic sex differentiation can only be inferred by an unusual genetic interaction between mutations at the snf and Sxl loci. Although doubly heterozygous females have reduced viability, many surviving females show signs of sex transformations. snf is required for the activation of Sex-lethal in both the germline and the soma (Salz, 1992).

Genetic evidence alone was insufficient to pinpoint the role of snf in sex determination. A clue was provided in a study of SXL ovarian expression in snf1621 mutants. The splicing pattern of SXL transcripts in ovaries mutant for snf1621 shows substantial levels of male-spliced SXL mRNA. In wild type females, three major classes of SXL mRNAs are observed. In snf1621 mutants, one of these three classes is not detectable and a second is reduced (Salz, 1992). The germ cells populating snf1621 mutants have little or no SXL protein. This result is consistent with the idea that regulation of SXL splicing is impaired in germ cells mutant for snf1621 (Salz, 1992 and Bopp, 1993).

Further analysis revealed aberrant Sex-lethal regulation in snf mutants in late embryogenesis. The role of snf somatic sex determination is only evident when the probability of Sxl activation is reduced. In this case, embryonic female-specific lethality is caused by a lack of maternal snf function. By examining both the RNA and protein expression pattern of Sxl in embryos mutant for snf, it is found that Sxl is not stably activated. Although both the early female-specific pattern of Sxl mRNA and the early SXL protein expression appear normal, there is little or no accumulation of the female-specific spliced form of the late transcripts, and consequently little or no SXL protein is found late in embryogenesis (Albrecht, 1993).

Cloning of snf reveals a protein with two RNA recognition motifs (RRMs); both are similar to two closely related small nuclear ribonuclear proteins (snRNPs): U1A and U2B". These two RNA-binding proteins bind different RNAs. U1a binds U1 snRNA, and U2B" binds U2 snRNA. Both proteins and their bound RNAs are intimately involved is splicing. In many respects, SNF resembles U2B" more closely than U1A. Not only does SNF have a similar overall structure and molecular weight to U2B" but it also contains a 5-amino-acid motif within the amino-terminal RRM that is identical to that found in U2B". In the human protein, this region has been shown to be necessary for the U1A protein to discriminate between U1 and U2 RNA. Functional studies demonstrate the true identity of SNF as a U1 snRNP. In both Drosophila and human cell extracts, an epitope-tagged SNF protein immunoprecipitates from U1 small nuclear RNA (snRNA) but not U2 snRNA (Harper, 1992).

How does SNF relate to alternative splicing? The splicing of SXL pre-mRNA is autoregulated by Sex lethal protein. SNF cooperates with SXL to block utilization of the male-specific exon of the SXL pre-mRNA, suggesting a model in which the SXL protein blocks spliceosome assembly by forming a non-productive snRNA/SXL complex. By blocking the male-specific exon, SXL assures that only the female mRNA, capable of coding for a complete SXL protein, is made in females.

Initially, SXL protein binds to intron sequences far distant from the male exon (exon 3), and by so doing, it blocks the utilization of male exon splice sites. SXL proteins then bind to the poly U tracts in the introns upstream and downstream of the SXL male exon. SXL proteins in the intron then make contact with the U1 snRNPs associated with the male exon splice junctions, presumably via protein-protein interactions with SNF. These contacts would prevent the snRNPs at the male exon splice junctions from participating in subsequent splicing steps (Saltz, 1996 and Deshpande, 1996).

The role of U1 snRNP in alternative splicing is not confined to SXL pre-mRNA. Members of the SR family of proteins can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. Individual SR proteins have distinct abilities to promote interaction of U1 snRNP with alternative 5' splice junctions. (See snf Evolutionary homologs for additional information). In Drosophila, the proteins Transformer and Transformer 2 have conserved SR domains and function in an identical fashion to other SR proteins in influencing alternative splicing of the Doublesex pre-mRNA. SR proteins act as a bridge between U1, associated with the 5' splice site and U2AF65/U2AF35 (see U2 small nuclear riboprotein auxiliary factor 50) assembled at the 3' splice site. The highly conserved SR protein RBP1 targets sequences within the DSX repeat region that are required for the efficient splicing of DSX pre-mRNA (Heinrichs, 1995). Essentially SR proteins are involved in the assembly of the spliceosome. It is thought that SR proteins can regulate alternative splicing in many pre-mRNAs.

U1 snRNP is really a multiprotein complex, of which the A protein (Sans fille) is but one component. The A protein makes direct physical contact with the U1 snRNA, the RNA component of the snRNP. This RNA component is involved in direct recognition of the conserved sequence of the 5' splice sites of pre-mRNA introns. The A component is also known to interact with proteins involved in mRNA polyadenylation. The U1-70K subunit is known to directly interact with SR proteins in vertebrates. U1-C lacks an RNA binding domain and does not appear to bind directly to U1 snRNA. However, at the amino terminal end protein C contains a zinc finger-like structure of the CC-HH type. Several lines of evidence indicate that the zinc finger-like structure is essential for the binding of protein C to U1 snRNP particles. U1 snRNP is known to make physical contact with the cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts. CBC is required for the efficient recognition of the 5' splice site by U1 snRNP during formation of a complex on the first intron of pre-mRNA. The number and complexity of known U1 snRNP interactions will continue to rise as more information is acquired on spliceosome biology.

REGULATION

Transcritional Regulation

Targets of activity

Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific Sex lethal RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is Sans-fille (Snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether Snf acts as part of an intact snRNP or a free protein. Evidence is provided that Sex lethal interacts with Sans-Fille in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with Sex lethal. Moreover, Sex lethal associates with other integral U1 snRNP components, and genetic evidence is provided to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link Sex lethal with the heterodimeric splicing factor, U2AF (see U2 small nuclear riboprotein auxiliary factor 50). These studies point specifically to a mechanism by which Sex lethal represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, these studies are difficult to reconcile with the current model that proposes that the Sex lethal blocks splicing at the second catalytic step, and instead argue that the Sex lethal protein acts after splice site recognition, but before catalysis begins (Nagengast, 2003).

The Sxl male exon is unusual in that it contains two 3' AG dinucleotides separated by a short polypyrimidine tract. Interestingly, although the upstream 3' splice site is used almost exclusively for exon ligation in tissue-culture cells, both 3' splice sites are required for Sxl-mediated male-exon skipping. Moreover, crosslinking studies in HeLa cell extracts have shown that the U2AF heterodimer binds to the downstream 3' splice site and the intervening polypyrimidine tract, suggesting that U2AF may play an active role in Sxl regulation. These biochemical data have been validated by demonstrating that the Sxl protein can associate with the Drosophila U2AF orthologs. More importantly, genetic data provide compelling support for the biological relevance of these interactions by demonstrating that in females, the small subunit is important for both Sxl male-exon skipping and female viability. In addition to demonstrating a role for U2AF in Sxl autoregulation, this genetic result is notable because previous studies have failed to find RNA splicing defects associated with small subunit mutations. Whether this success reflects substrate-specificity or sensitivity of the assay remains to be determined (Nagengast, 2003).

In addition to controlling the use of the male exon 3' splice site, these studies suggest that Sxl controls the use of the male-exon 5' splice site by interacting with the U1 snRNP. This connection was established in three ways: (1) it was found that mutation of a single residue in the N-terminal RRM of SNF compromises both complex formation with Sxl and assembly into the U1 snRNP, thus suggesting that the two events are linked; (2) it has been demonstrated that, in addition to SNF, Sxl can associate with other integral U1 snRNP components, including the U1-70K protein and the U1 snRNA in whole cell extracts; (3) genetic interaction data provide evidence that U1-70K, like SNF, is important for the successful establishment of the Sxl autoregulatory splicing loop in females (Nagengast, 2003).

Although the discovery that SNF is an snRNP protein was the first clue that Sxl might act by associating with components of the general splicing machinery, the role of SNF has remained enigmatic. The role of SNF has been clarified by demonstrating that its contribution to the function of the U1 snRNP is not absolutely essential for viability of either sex, and that Sxl can associate with the U1 snRNP through a SNF-independent mechanism. Nevertheless, in vivo analysis continues to support a role for snf in Sxl splicing autoregulation by demonstrating that Sxl splicing defects are detectable under specific conditions. Interestingly, the phenotypic consequences of these Sxl splicing defects are more severe in the germline than in the soma. One possible explanation for this difference is that the requirements for Sxl splicing autoregulation are fundamentally different in the two tissue types. It is thought more likely that the mechanism is the same, but that the additional interaction with the U1 snRNP provided by SNF becomes critical when Sxl protein levels are low. This hypothesis is based on the fact that, in the germline, the majority of Sxl protein is cytoplasmic, and thus low levels of nuclear Sxl protein are the norm. By contrast, in other tissues, the Sxl protein accumulates in the nucleus, enabling the Sxl-U1 snRNP complex to form even when SNF is not stably associated with the U1 snRNP. The finding that these snf mutant females rarely survive if they are also heterozygous for Sxl, provides additional support for the idea that SNF function is only critical when Sxl protein levels are low (Nagengast, 2003).

Together, these studies argue that interactions between Sxl, the U1 snRNP and U2AF underlie the mechanism by which Sxl promotes skipping of the male exon. Based on these studies, a model is proposed in which Sxl acts not by preventing assembly of the U1 snRNP or U2AF onto the pre-mRNA, but instead interacts with the U1 snRNP bound to the male-exon 5' splice site, and U2AF at the male-exon 3' splice site, to form complexes that block these general splicing factors from assembling into a functional spliceosome. These 5' and 3' Sxl blocking complexes might function independently or they might interact across the exon to form a larger inhibitory complex. Furthermore, because it has not been possible to demonstrate that Sxl interacts directly with either U1-70K or U2AF, it is speculated that one or more bridging proteins are required to link Sxl to the general splicing machinery (Nagengast, 2003).

Although the in vivo approach cannot directly address when in the pathway of spliceosome assembly Sxl acts, biochemical studies have shown that during the course of spliceosome assembly, U2AF and the U1 snRNP are only transiently associated with splicing substrates, and are released before the formation of a functional spliceosome. Therefore, based on these studies, it seems reasonable to propose that Sxl acts by blocking splicing after splice site recognition but before catalysis begins. The data are therefore difficult to reconcile with the recent model, which proposes that Sxl blocks splicing after spliceosome assembly, at the second catalytic step of the reaction. Using RNA interference in Drosophila tissue culture cells it has been demonstrated that efficient male exon skipping depends on the presence of SPF45, a protein that is known to be required for the second step of splicing. Together with studies that show that SPF45 can bind to the upstream 3' splice site of the Sxl male exon and physically interact with Sxl, these data point to a role for SPF45 in Sxl splicing regulation. However, the primary evidence that Sxl blocks the splicing reaction during the second step rests on the results of in vitro splicing assays in which Sxl was shown to inhibit splicing of a chimeric splicing substrate that contains only a small region of the intronic region required for successful autoregulation in vivo. It is suspected that by looking at this 48 bp region, which contains a dispensable Sxl-binding site in addition to the two potential 3' splice sites, out of context, a failsafe mechanism was uncovered that comes into play when Sxl-mediated splicing regulation is otherwise compromised. Additional studies investigating the function of SPF45 in vivo will be required to determine the importance of this second step blocking mechanism and should provide insight into whether multiple mechanisms are needed to drive efficient regulated exon skipping (Nagengast, 2003).

Somatic inhibition restricts splicing of the Drosophila P-element third intron (IVS3) to the germ line. This simple system has been exploited to provide a model for a mechanism of alternative pre-mRNA splicing. Biochemical complementation experiments reveal that Drosophila somatic extracts inhibit U1 snRNP binding to the 5' splice site. U1 snRNP binds to a pseudo-5' splice site in the 5' exon and multiprotein complexes bind to an adjacent site. Binding of these factors appears to mediate the inhibitory effect, because mutations in the pseudo-5' splice sites block binding and activate splicing in vitro. Likewise, wild-type, but not mutant, 5' exon RNA titrates inhibitory factors away from the pre-mRNA and activates splicing. Thus, the pseudo-5' splice sites have been defined as crucial components of the regulatory element; there is a correlation between inhibitory activity and specific RNA binding factors from Drosophila somatic cells. The pseudo-5' splice sites also provide a mechanistic description of somatic inhibition. Because the inhibitory activity involves general splicing functions such as protein recognition of 5' splice site sequences and changes in the distribution of bound U1 snRNP, these data may also provide insights into how splice sites are selected (Siebel, 1992).

Alternative splicing of pre-mRNAs is a versatile regulatory mechanism that can achieve quantitative control of gene expression and functional diversification of gene products. Much progress has been made toward understanding the basic splicing reaction and recognizing exon/intron boundaries, but the mechanisms that regulate alternative splicing are only beginning to be elucidated. Recognition of the 5' splice site by U1 snRNP and of the branchpoint near the 3' splice site by U2 snRNP auxiliary factor (U2AF) are two critical early steps that are regulated in cell- or stage-specific alternative splicing. The picture emerging from biochemical and genetic studies is that splice site selection results from the combined action of conserved consensus sequences that base-pair with the U snRNAs together with protein-protein and protein-RNA interactions that stabilize snRNP binding and mediate bridging interactions between snRNPs at the 5' and 3' splice sites. These interactions involve a growing list of non-snRNP factors, some of which may be responsible for developmental regulation of splice site selection (Burnette, 1999 and references).

Members of the SR family of RNA-binding proteins are required for multiple steps of the splicing reaction in vitro and their concentration can influence splice site competition both in vitro and in overexpression assays using cultured cells. SR proteins are required for the activity of at least some splicing enhancers that stimulate the use of weak 5' or 3' splice sites, and there is evidence for distinct specificities in these interactions. Members of the hnRNP A/B family of RNA-binding proteins also influence splice site selection in a concentration-dependent manner in vitro and when overexpressed in cultured cells. In these assays the hnRNP RNA binding proteins can antagonize the action of SR proteins. These observations have suggested that SR proteins and hnRNP A/B proteins function in vivo as concentration-dependent regulators of alternative splicing. Another possibility is that members of these families serve as cofactors or targets for the actual regulators. Particular SR proteins have been proposed to interact with developmentally specific factors to promote regulation of splicing (Burnette, 1999 and references).

Although a framework of hypotheses is evolving, little is known about regulators of alternative splicing and how they function in vivo. Notable exceptions are Sxl and Tra, proteins that control alternative splicing decisions during sex determination in Drosophila. Because few developmentally specific regulators of alternative splicing have been identified, it is possible that many -- if not most -- alternative splicing decisions are regulated by relatively subtle variations in the levels of general, widely distributed factors, perhaps acting cooperatively or antagonistically as proposed for SR and hnRNP A/B proteins. This is consistent with much correlative evidence and many in vitro observations, but conclusive proof that either type of protein normally regulates an alternative splicing decision in vivo has yet to be obtained. Although null alleles of the Drosophila SR protein gene B52 (homolog of human SRp55) show it to be essential for viability, examination of multiple constitutively and alternatively spliced RNAs has failed to reveal any alterations of splicing even in the absence of detectable protein (Burnette, 1999 and references).

The homeotic gene Ultrabithorax (Ubx) of Drosophila melanogaster was used as a model for regulation of alternative splicing in large and complex transcription units. The six alternative Ubx mRNAs share large protein-coding 5' and 3' exons but differ in the pattern of incorporation of three elements: B is located between two alternative donor sites at the end of the first common exon, whereas mI and mII are internal cassette exons. Within the central nervous system (CNS), different neurons express distinct ratios of Ubx isoforms. The complex and quantitative nature of this regulation is unlike that of other well-studied model systems in Drosophila (e.g., sex-specific splicing in the sex determination hierarchy or germ line-specific splicing of P-element transcripts) but resembles that of many other genes in vertebrates and invertebrates. It seems most likely that this type of alternative splicing is controlled not by highly tissue- and gene-specific splicing regulators but by developmental variations in the concentration or activity of broadly distributed multifunctional factors that may act combinatorially. Hence, Ubx should be a valuable model where genetic approaches can be used to dissect this type of regulation (Burnette, 1999 and references).

Strong reductions of function for the postulated type of regulatory factors would probably cause lethal phenotypes that would be uninterpretable in terms of their effects on Ubx splicing. However, the Ubx splicing pattern should be sensitive to partial reductions in the concentration or activity of these regulatory factors. This may also be true for factors that play important accessory roles in regulation as targets or as constitutively expressed components of regulatory complexes. Two approaches were used to identify such factors. (1) First, a test was carried out to see if the Ubx alternative splicing pattern is altered in heterozygotes for strong loss-of-function mutations. Such mutations are found in a set of genes implicated in the control of alternative splicing in Sxl and P-element RNAs. (2) To identify the location of additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested for dominant enhancement of the haploinsufficient Ubx haltere phenotype; it was then asked whether the Ubx splicing pattern is altered in heterozygotes for the interacting deficiencies, and the phenotypic interaction and effect on splicing was traced to specific genes when mutations existed in reasonable candidates. Inclusion of the cassette exons in Ubx mRNAs is reduced strongly in heterozygotes for hypomorphic alleles of hrp48, which encodes a member of the hnRNP A/B family and is implicated in control of P-element splicing. Significant reductions of mI and mII inclusion were also observed in heterozygotes for loss-of-function alleles of virilizer, fl(2)d, and crooked neck. The products of virilizer and fl(2)d are also required for Sxl autoregulation at the level of splicing; crooked neck encodes a protein with structural similarities to yeast-splicing factors Prp39p and Prp42p. Deletion of at least five other loci caused significant reductions in the inclusion of mI and/or mII (Burnette, 1999).

Coupled RT-PCR assays were used to analyze the pattern of Ubx alternative splicing in heterozygous third instar larvae and in adults. The isoform ratios in third instar larvae were in close agreement with those determined previously using nuclease protection assays. Types Ia and IIa are the predominant Ubx mRNAs and those lacking both mI and mII (isoforms IVa and b) make up only a small fraction of the total. Adults contain a significantly higher proportion of class IV mRNAs than larvae; this differs from previous reports and probably reflects the very early and narrow age distribution of the adults used in this study. It is important to note that the Ubx isoform ratios did not vary significantly between different wild-type strains nor between these and several control strains that carried different balancer chromosomes and irrelevant mutations. These results demonstrate that the mechanism that controls Ubx alternative splicing is robust, a conclusion that is consistent with the faithful conservation of Ubx isoform structure and expression among Drosophila species spanning 60 million years of evolution. The fact that the quantitative isoform pattern revealed by this assay is insensitive to considerable variation in genetic background highlights the significance of the effects described below for specific mutations and deficiencies. Although amplified Ubx cDNA fragments that contain mI but not mII (i.e., hypothetical isoforms IIIa and IIIb) should have the same length as isoforms IIa and IIb, such amplifiers would be expected to exhibit distinctly slower mobility due to the difference in nucleotide sequence (Burnette, 1999).

The products of Sxl, tra, and tra-2 are known regulators of alternative splicing decisions in Drosophila but they are not essential for processes other than sex determination (and dosage compensation, in the case of Sxl) because males that are null for these genes are viable and appear phenotypically normal. However, additional genes [fl(2)d, virilizer, and l(2)49Db] are required for correct control of alternative splicing decisions by Sxl are also essential for viability in both sexes; hence, their products may also have roles in other alternative splicing events. To determine whether these include the control of Ubx alternative splicing, it was asked whether the Ubx isoform ratios are altered in heterozygotes for mutations in these genes. In contrast to the stability described in the preceding section, the Ubx splicing pattern is altered significantly when the expression or function of virilizer or fl(2)d is reduced. The strongest effect is observed with virilizer, using a loss-of-function allele (vir³) that is recessive lethal in both sexes. In heterozygous larvae the proportion of Ubx class I mRNAs declines while that of classes II and IV increases. The proportion of class I that contains the B element is not altered. The increase in classes II and IV indicates that inclusion of both mI and mII is reduced but that the effect on mI exceeds that on mII. Inclusion of mI is also reduced in adults, although the effect was weaker than in larvae. More modest but statistically significant reductions of mI and mII inclusion are also observed in larvae heterozygous for the fl(2)d² mutation, which is also a loss-of-function allele that is recessive lethal in both sexes (Burnette, 1999).

hrp48 plays a critical role in the inclusion of mI and mII: hrp48 is a member of the hnRNP-A/B family of RNA-binding proteins and forms part of a protein complex that regulates splicing of intron 3 (IVS3) in P-element transcripts. Although repression of IVS3 splicing in somatic tissues is dictated by PSI, which is a soma-specific component of the regulatory complex, the hrp48 protein binds specifically to sequences within the cis-acting regulatory element in the RNA. hrp48 was originally identified as a general component of heterogeneous nuclear ribonucleoprotein particles and the hrp48 gene is essential for viability, so it must perform additional functions unrelated to P-element expression; these functions might include regulation of other splicing decisions. The five known mutant alleles of hrp48 are all P-element insertions in the upstream regulatory region and are not null. Nevertheless, inclusion of mI and mII in Ubx mRNAs is reduced markedly in larvae and adults heterozygous for the strong recessive lethal allele hrp48¹; weaker alleles, some of which are viable as homozygotes, have similar but more modest effects. The effect of hrp48 mutations resembles that of vir and fl(2) mutations: inclusion of mII is affected more weakly than mI, and the proportion of isoform I that contains the B element is not altered. Heterozygosity for hrp48¹ reduces inclusion of mI by 27%; this is the strongest effect observed for any mutation or deficiency in this study, indicating that normal levels of hrp48 are critical for inclusion of the internal exons, especially mI, in Ubx mRNAs (Burnette, 1999).

One enhancer, Df(1)64c18g, deletes the genes crooked neck (crn) and kurz (kz), which are located at 2F1 and are both candidate RNA-processing factors. The crn gene encodes a protein with 16 tetratrichopeptide repeats, a motif implicated in protein-protein interactions. Although CRN protein has been proposed to function as a transcription factor involved in cell cycle control, recent data show that it is closely related to the yeast splicing factors Prp39p and Prp42p, which associate with yeast U1 snRNP and are required for splicing. The kz gene encodes a protein with extensive homology to yeast ATP-dependent splicing factors Prp2p, Prp16p, and Prp22p. These proteins define a distinct subfamily of ATP-dependent putative RNA-helicases. Because mutant alleles of these genes are available, a direct test was carried out to see if deletion of one or both might be responsible for enhancement of the Ubx haltere phenotype and whether they affect the Ubx splicing pattern. Like the deficiency, two hypomorphic, recessive lethal alleles of crn (EA130 and RC63) act as dominant zygotic enhancers of Ubx¹⁹⁵/+ and Ubx^9.22/+. RT-PCR analysis shows that inclusion of mI, but not mII, is reduced significantly in larvae heterozygous for crn^EA130. The second allele, crn^RC63, has similar effects on the Ubx phenotype and splicing pattern. A recessive lethal allele of kz (DF942) behaves as a weak dominant enhancer of Ubx¹⁹⁵/+ and Ubx^9.22/+, but RT-PCR analyses does not reveal a significant dominant effect on the Ubx splicing pattern (Burnette, 1999).

The inclusion of mI and mII in Ubx mRNAs is regulated by competition between 5' splice sites that flank each of these exons after they are joined to E5'. As the RNA is transcribed, mI and subsequently mII are spliced constitutively to the upstream exon but can then be removed, together with the downstream intron, using an upstream 5' splice site within E5' or at the junction with this exon. For the majority of nascent RNAs (those initially spliced using 5' splice site a in E5'), a strong 5' splice site is regenerated at the junction between E5' and mI or mII that competes with the mI or mII 5' splice site located 51 nt downstream. For a minority of nascent RNAs (those initially spliced using 5' splice site b in E5') the a site is still present in E5' and can compete with the mI or mII 5' splice site located 78 nt downstream; use of the a site then removes the B element along with mI or mII. Developmental regulation of mI and mII inclusion is achieved by modulating the competition between the upstream and downstream 5' splice sites that flank these exons (Burnette, 1999).

Reduction of function in all of the factors identified in this work leads to reduced inclusion of mI (and in most cases also mII). This suggests roles in suppression of the upstream sites (which strongly match the 5' splice site consensus) or stimulation of the downstream sites (which match the consensus more weakly). It is interesting that three of the factors identified in this study that are required for inclusion of mI and mII in Ubx mRNAs may also be required for suppression of 5' splice site utilization in other RNAs: the functions of virilizer and fl(2)d are required for Sxl to repress splicing of the male-specific exon in its own RNA, and hrp48 is implicated as part of a complex that mediates repression of a 5' splice site in P-element RNA. In addition, heterozygosity for a null allele of sans-fille (snf^J210) does not alter the Ubx splicing pattern, but the antimorphic allele snf^e8H, which interferes with autoregulation of Sxl splicing, enhances the Ubx haltere phenotype and increases exclusion of mI and mII (Burnette, 1999 and references).

The products of virilizer, fl(2)d, and snf might function as parts of a complex that mediates active repression of 5' splice site utilization through interactions with U1 snRNP. Formation or stabilization of this repression complex could be directed to different target splice sites through the action of distinct factors that, like Sxl, bind to cis-acting regulatory signals and interact with components of the complex. An intriguing possibility is that hrp48 interacts (directly or indirectly) with a U1 snRnp/Snf/Vir/Fl(2)D complex to target suppression of splicing at the upstream sites that are used to remove mI. The strong reduction of mI inclusion (27%) observed in hrp48¹ heterozygotes suggests a critical role for hrp48 in modulating competition between the regenerated and downstream 5' splice sites that flank this exon. Although hrp48 is an hnRNP protein that probably binds nonspecifically to many RNAs, it is also known to form part of a specific complex that blocks use of the 5' splice site for the third intron of P-element RNA in somatic cells. This regulatory complex prevents U1 snRNP from binding at the 5' splice site and recruits it instead, nonproductively, to the more upstream of two overlapping pseudo-5' splice sites within the exon; hrp48 itself makes contact with the downstream pseudo-5' splice site, F2. Splicing of P-element IVS3 in a reporter transgene is partially derepressed in adult escapers homozygous for a semilethal hrp48 allele, indicating that hrp48 is necessary for efficient suppression of the 5' splice site. Hence, it may be significant that a sequence within mI that overlaps the regenerated 5' splice site matches F2 and flanking nucleotides at 8 of 10 positions; this sequence is conserved among four Drosophila species that diverged up to 60 million years but maintain identical regulation of mI inclusion. hrp48 might bind to this sequence and help to recruit U1 snRNP nonproductively to the regenerated 5' splice site at the E5'/mI junction; in intermediates where mI has been spliced to the b site of E5', this complex could also block access to the a site located 27 nt upstream. This would explain why the hrp48, vir, and fl(2)d mutations reduce mI inclusion but do not alter the proportion of class I mRNAs that contain the B element: failure to assemble the repression complex at the E5'/mI junction would allow inappropriate use of both the regenerated site (used to remove mI from E5'a/mI and E5'b/mI intermediates) and the a site (used to remove mI and the B element from E5'b/mI intermediates) (Burnette, 1999 and references).

The effect of hrp48, vir, and fl(2)d mutations on inclusion of exon mII, which does not contain an F2-like element, may not be the result of resplicing at the E5'/mII junction. The reduction of mII inclusion (detected as an increase in class IV mRNAs rather than a decrease in class II) could be explained if the repression complex must remain assembled at the E5'/mI junction to prevent subsequent removal of mI and mII together during splicing of intron 3. Intermediates from which mI is removed during splicing of intron 2 would retain mII. The net result would be an increase in both class II and class IV mRNAs, as observed. In addition, it is noted that the effect of hrp48 mutations on mI and mII inclusion is the opposite of what one would expect from the simple idea that hnRNP A/B proteins generally promote exon skipping (and use of upstream 5' splice sites), antagonizing a general effect of SR proteins that promote exon inclusion (or use of downstream 5' splice sites). The observations presented here are more consistent with a specific role for hrp48 acting through cis-regulatory elements to prevent resplicing of mI. It is more difficult to speculate on the roles of crn or the still-unidentified factors deleted by deficiencies that alter the Ubx splicing pattern. In principle, these could participate in repression of the regenerated 5' splice sites or stimulation of the competing downstream site. They could also be involved in interactions between mI and mII that seem to be required for effective use of the downstream 5' splice site located at the mI/intron 2 boundary. Although a weak homology to the homeodomain led to the proposal that the crooked neck protein functions as a transcription factor, its 16 tetratrichopeptide repeats form a distinct subfamily with those of Prp39p and Prp42p, two splicing factors from yeast that interact with U1 snRNP but appear not to bind RNA directly. A third yeast member of this group has been identified that has more extensive homology to crn ; it will be interesting to learn whether this also functions as a splicing factor (Burnette, 1999 and references).

P-element somatic inhibitor (PSI) is a KH domain-containing splicing factor highly expressed in Drosophila somatic tissues. A direct association of PSI with the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) particle has been detected in somatic nuclear extracts. This interaction is mediated by highly conserved residues within the PSI C-terminal AB motif and the U1 snRNP-specific 70K protein. Through the AB motif, PSI modulates U1 snRNP binding on the P-element third intron (IVS3) 5' splice site and its upstream exonic regulatory element. Ectopic expression experiments in the Drosophila female germline demonstrate that the AB motif also contributes to IVS3 splicing inhibition in vivo. These data show that the processing of specific target transcripts, such as the P-element mRNA, is regulated by a functional PSI-U1 snRNP interaction in Drosophila (Labourier, 2001).

U1 snRNP determines mRNA length and regulates isoform expression

U1 snRNP (U1), in addition to its splicing role, protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation (PCPA) at cryptic polyadenylation signals (PASs) in introns. In this study, a high-throughput sequencing strategy of differentially expressed transcripts (HIDE-seq) mapped PCPA sites genome wide in divergent organisms, including Drosophila and mammalian cultured cells. Surprisingly, whereas U1 depletion terminates most nascent gene transcripts within ~1 kb, moderate functional U1 level decreases, insufficient to inhibit splicing, dose-dependently shifts PCPA downstream and elicits mRNA 3' UTR shortening and proximal 3' exon switching characteristic of activated immune and neuronal cells, stem cells, and cancer. Activated neurons' signature mRNA shortening could be recapitulated by U1 decrease and antagonized by U1 overexpression. Importantly, it was shown that rapid and transient transcriptional upregulation inherent to neuronal activation physiology creates U1 shortage relative to pre-mRNAs. Additional experiments suggest cotranscriptional PCPA counteracted by U1 association with nascent transcripts, a process termed telescripting, ensuring transcriptome integrity and regulating mRNA length (Berg, 2012).

Messenger RNAs (mRNAs) in eukaryotic cells are produced from precursor transcripts (pre-mRNAs) by posttranscriptional processing. In metazoans, two processing reactions -- splicing of introns and alternate cleavage and polyadenylation -- are particularly extensive and contribute most significantly to mRNA transcriptome diversity. Splicing is performed by a spliceosome that assembles on each intron and predominantly comprises small nuclear RNPs (snRNPs), U1, U2, U4, and U5, and U6 snRNPs in equal stoichiometry. U1 snRNP (U1) plays an essential role in defining the 5' splice site (5'ss) by RNA:RNA base pairing via U1 snRNA's 5' nine nucleotide (nt) sequence. Using antisense morpholino oligonucleotide complementary to U1 snRNA's 5' end (U1 AMO) that interferes with U1 snRNP's function in human cells, accumulation of introns was observed in many transcripts, as expected for splicing inhibition. However, in addition, the majority of pre-mRNAs terminated prematurely from cryptic polyadenylation signals (PASs) in introns, typically within a short distance from the transcription start site (TSS). These findings indicated that nascent transcripts are vulnerable to premature cleavage and polyadenylation (PCPA) and that U1 has a critical function in protecting pre-mRNA from this potentially destructive process. It was further shown that PCPA suppression is a separate, splicing-independent and U1-specific function, as it does not occur when splicing is inhibited with U2 snRNA AMO or the splicing inhibitor, spliceostatin A (SSA) (Berg, 2012).

These observations were made by transcriptome profiling using partial genome tiling arrays, which provided limited information. In this study, to define the parameters involved in PCPA and its suppression, a strategy (HIDE-seq) was devised to select and sequence only differentially expressed transcripts, identifying changes that occur upon U1 decrease to various levels and in different organisms. The sequence information obtained from HIDE-seq provided genome-wide PCPA maps, and these, together with direct experiments, revealed that U1's PCPA suppression is not only essential for protecting nascent transcripts but is also a global gene expression regulation mechanism. Unexpectedly, PCPA position varied widely with the degree of U1 decrease, trending to usage of more proximal PASs with greater reduction. This yielded mRNAs with shorter 3' untranslated regions (3' UTRs) and alternatively spliced isoforms resulting from usage of more proximal alternative polyadenylation (APA) sites, which is characteristic of activated immune, neuronal, and cancer cells. It was demonstrated that U1 decrease can recapitulate such specific mRNA changes that occur during neuronal activation. Indeed, it was shown that the rapid transcriptional upregulation during neuronal activation is a physiological condition that creates U1 shortage relative to nascent transcripts. Furthermore, U1 overexpression inhibits activated neurons' mRNA signature shortenings. It is suggested that by determining the degree of PCPA suppression, U1 levels play a key role in PAS usage and hence mRNA length. A model is proposed whereby U1 binds to nascent pre-mRNAs cotranscriptionally to explain how U1 shortage results in a corresponding loss of distal PASs suppression from the cleavage and polyadenylation machinery that is associated with the RNA polymerase II (polII) transcription elongation complex (Berg, 2012).

Protein Interactions

A complete set of seven U1-related sequences have been cloned and characterized from Drosophila melanogaster. These sequences, coding for U1 snRNA's, the RNA component of U1A, are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously studied: one U1 gene at 21D that encodes the prototype U1 sequence (U1a), one U1 gene at 82E that encodes a U1 variant with a single nucleotide substitution (U1b), and a pseudogene at 82E. The four previously uncharacterized genes comprise another U1b gene at 82E, two additional U1a genes at 95C, and a U1 gene at 95C that encodes a new variant (U1c) with a distinct single nucleotide change relative to U1a. Three blocks of 5' flanking sequence similarity are common to all six full length genes. The U1b RNA is expressed in Drosophila Kc cells and is associated with snRNP proteins, suggesting that the U1b-containing snRNP particles are able to participate in the process of pre-mRNA splicing. The expression throughout Drosophila development of the two U1 variants has been observed relative to the prototype sequence. The U1c variant is undetectable, while the U1b variant exhibits a primarily embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse (Lo, 1990).

Most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II, but U6 and a few others are synthesized by RNA polymerase III. Transcription of snRNA genes by either polymerase is dependent on a proximal sequence element (PSE) located upstream of position -40, relative to the transcription start site. In contrast to findings in vertebrates, sea urchins, and plants, the RNA polymerase specificity of Drosophila snRNA genes is intrinsically encoded in the PSE sequence itself. The differential interaction of the Drosophila melanogaster PSE-binding protein (DmPBP) with U1 and U6 gene PSEs has been investigated. By using a site specific protein-DNA photo-cross-linking assay, three polypeptide subunits of DmPBP with apparent molecular masses of 95, 49, and 45 kDa have been identified that are in close proximity to the sequence element. Two additional putative polypeptides of 230 and 52 kDa have been identified that may be integral to the complex. The 95-kDa subunit cross-links at positions spanning the entire length of the PSE, but the 49- and 45-kDa subunits cross-links only to the 3' half of the PSE. The same polypeptides cross-link to both the U1 and U6 PSE sequences. However, there are significant differences in the cross-linking patterns of these subunits at a subset of the phosphate positions, depending on whether binding is to a U1 or U6 gene PSE. These data suggest that RNA polymerase specificity is associated with distinct modes of interaction of DmPBP with the DNA at U1 and U6 promoters (Wang, 1998).

Sans fille (SNF), a Drosophila homolog of mammalian U1A and U2B" snRNP proteins, is an integral component of the machinery required for splice site recognition in all pre-mRNAs. Mutations in snf disrupt the establishment of Sex lethal mRNA female-specific splicing pattern in both the germ line and soma. Because snf is required to establish the female-specific splice site choice, snf is likely to function in conjunction with SXL to bias the general splice machinery against the recognition of the male 5' splice site. SNF cooperates with SXL to block utilization of the male-specific exon of the SXL pre-mRNA, suggesting a model in which the SXL protein blocks spliceosome assembly by forming a non-productive snRNA/SXL complex. This suggests that snRNPs, like transcription factors, can have antagonistic roles in controlling gene expression (Salz, 1994 and Salz, 1996).

SXL and SNF proteins can interact directly in vitro, and these proteins are part of an RNase-sensitive complex in vivo that can be immunoprecipitated with anti-SXL antibody. The SNF protein associated with SXL protein is in a large, rapidly sedimenting complex. These complexes contain additional small nuclear ribonucleoprotein particle protein and the U1 and U2 small nuclear RNAs. Sxl transcripts can also be immunoprecipitated by anti-SXL antibodies. A model is presented for SXL mRNA splicing regulation. SXL protein binds to intron sequences far from the male exon (exon 3) and blocks the utilization of male exon splice sites. SXL proteins would bind to the poly U tracts in the introns upstream and downstream of the SXL male exon. SXL proteins in the intron would then make contact with the U1 and U2 snRNPs associated with the male exon splice junctions, presumably via protein-protein interactions with SNF. These contacts would prevent the snRNPs at the male exon splice junctions from participating in subsequent splicing steps (Deshpande, 1996).

Specific recognition and pairing of the 5' and 3' splice sites are critical steps in pre-mRNA splicing. The splicing factors SC35 and SF2/ASF, both functioning as SR proteins, specifically interact with both the integral U1 small nuclear ribonucleoprotein (snRNP U1-70K) and with the 35 kd subunit of the splicing factor U2AF (U2AF35). Previous studies indicated that the U1 snRNP binds specifically to the 5' splice site, while U2AF35-U2AF65 heterodimer binds to the 3' splice site. Together, these observations suggest that SC35 and other members of the SR family of splicing factors may function in splice site selection by acting as a bridge between components bound to the 5' and 3' splice sites. Interestingly, SC35, SF2/ASF, and U2AF35 also interact with the Drosophila splicing regulators Transformer (Tra) and Transformer-2 (Tra2), suggesting that protein-protein interactions mediated by SR proteins may also play an important role in regulating alternative splicing (Wu, 1993).

Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. A splicing 'coactivator', SRm160/300, has been identified that contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independent of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound 'activators' and the snRNP machinery of the spliceosome (Eldridge, 1999).

Exonic splicing enhancer (ESE) sequences are important for the recognition of adjacent splice sites in pre-mRNA and for the regulation of splice site selection. It has been proposed that ESEs function by associating with one or more serine/arginine-repeat (SR) proteins that stabilize the binding of the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor (U2AF) to the polypyrimidine tract upstream of the 3' splice site. This model was tested by analyzing the composition of splicing complexes assembled on an ESE-dependent pre-mRNA derived from the Drosophila doublesex gene. Several SR proteins and hTra2beta, a human homolog of the Drosophila alternative splicing regulator Transformer-2, associate with this pre-mRNA in the presence, but not in the absence, of a purine-rich ESE. By contrast, the 65-kDa subunit of U2AF (U2AF-65 kDa) binds equally to the pre-mRNA in either the presence or absence of the ESE. Time course experiments reveal differences in the levels and kinetics of association of individual SR proteins with the ESE-containing pre-mRNA: U2AF-65 kDa binds prior to most SR proteins and hTra2b and its level of binding does not change significantly during the course of the splicing reaction. Binding of U2AF-65 kDa to the ESE-dependent pre-mRNA is, however, dependent on U1 snRNP. The results indicate that an ESE promotes spliceosome formation through interactions that are distinct from those required for the binding of U2AF-65 kDa to the polypyrimidine tract (Li, 1999).

The factor requirements for binding of U2AF-65 kDa to the dsx substrate were investigated. U1 snRNP is required for the binding of U2 snRNP to the dsx(GAA)₆ pre-mRNA used in the present study. U1 snRNP can promote the cross-linking of U2AF-65 kDa to an upstream polypyrimidine tract across an exon and also to the polypyrimidine tract of a constitutively spliced pre-mRNA containing a single intron. Would U1 snRNP also be required for the binding of U2AF-65 kDa, within the context of cross-intron interactions during ESE-dependent splicing on the biotinylated dsx(GAA)₆ pre-mRNA? Splicing complexes assembled on this substrate were affinity selected from splicing reactions depleted of individual snRNPs and then immunoblotted with the anti-U2AF-65 kDa antibody. Depletion of U1 snRNP results in a significant reduction in the level of U2AF-65 kDa binding to the dsx(GAA)₆ pre-mRNA compared with its level of binding in a 'mock'-depleted extract. This reduction is not due to a nonspecific loss since depletion of U2 snRNP does not reduce the level of U2AF-65 kDa binding, and mixing equal amounts of the U1 and U2 snRNP-depleted extracts restores binding to the level observed in the mock-depleted reaction. These results indicate that U1 snRNP functions in stabilizing the binding of U2AF-65 kDa to the dsx(GAA)₆ pre-mRNA (Li, 1999).

It is proposed that U1 snRNP promotes two distinct sets of interactions during ESE-dependent splicing. One set involves ESE-independent interactions that are required for the binding of U2AF-65 kDa to the polypyrimidine tract, which then promotes partial binding of U2 snRNP to the branch site. This set of interactions likely involves cross-intron interactions mediated by the branch site-binding factor SF1/mBBP, which interacts with U2AF-65 kDa and is also required for the stable binding of U2 snRNP to the branch site. The other set of interactions promoted by U1 snRNP simultaneously requires the ESE and functions to further stabilize the binding of U2 snRNP to the branch site; this set of interactions also promotes the association of SRm160/300 with the pre-mRNA. This set of interactions does not influence the binding of U2AF to the pre-mRNA. Although depletion of SRm160/300 or U2 snRNP weakens but does not prevent the association of the other component with the dsx(GAA)6 pre-mRNA, these two components interact specifically. Thus, instead of promoting splicing complex formation through interactions mediated by the U2AF heterodimer, one or more ESE-associated components, including the SRm160/300 splicing coactivator, may promote splicing by interacting directly with the snRNP machinery of the spliceosome (Li, 1999).

Transcriptional regulation of U1 small nuclear RNA gene, encoding the RNA component of U1 snRNP

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, and located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduces transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increases the efficiency of the U1 promoter and simultaneously relieves the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter has no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter (Zamrod, 1993)

Most of the major spliceosomal small nuclear RNAs (snRNAs) (i.e. U1, U2, U4 and U5) are synthesized by RNA polymerase II (pol II). In Drosophila melanogaster, the 5'-flanking DNA of these genes contains two conserved elements: the proximal sequence element A (PSEA) and the proximal sequence element B (PSEB). The PSEA is essential for transcription and is recognized by DmSNAPc, a multi-subunit protein complex. Previous site-specific protein-DNA photo-cross-linking assays have demonstrated that one of the subunits of DmSNAPc, DmSNAP43, remains in close contact with the DNA for 20 bp beyond the 3' end of the PSEA, a region that contains the PSEB. Mutation of the PSEB does not abolish the cross-linking of DmSNAP43 to the PSEB. Thus the U1 PSEA alone is capable of bringing DmSNAP43 into close contact with this downstream DNA. However, mutation of the PSEB perturbs the cross-linking pattern. In concordance with these findings, PSEB mutations result in a 2- to 4-fold reduction in U1 promoter activity when assayed by transient transfection (Lai, 2005).

Conserved elements analogous to the PSEB have not been identified in the Pol II-transcribed snRNA genes of other metazoans in which functional studies have been carried out. However, snRNA genes of other insects contain conserved nucleotides in this location. It is possible that fruit flies (and other insects) have taken advantage of utilizing the specificity of the PSEB to modulate the strength of snRNA promoters over an evolutionary time scale. For example, three variant U5 genes, which are probably expressed at low levels, have PSEBs that are among the most divergent from the consensus PSEB (Lai, 2005).

Interestingly, substitution mutations in the -33 to -20 region of a human U2 gene have been found to have minor effects on the transcription start site. It is therefore possible that the D. melanogaster PSEB, which is located within this region, may play a role in helping to establish the correct start site. Transcription of the D. melanogaster U1 gene has been shown to require the TBP. Due to the location of the PSEB (-25 to -32) and its 8 bp length, it seems possible that the PSEB may be a site of DNA interaction with TBP. The PSEB may represent a 'compromise' sequence that allows it to be co-occupied simultaneously both by DmSNAP43 and TBP (Lai, 2005).

To see if this might be possible, TBP bound to DNA was modeled as if the PSEB were a TATA-box. Then, taking into consideration that the PSEA is separated from the PSEB by exactly 8 bp, the sites were identified where DmSNAP43 would cross-link with the DNA. The modeling illustrates that the phosphates that cross-link to DmSNAP43 are not occluded by TBP, and further suggests that DmSNAP43 could interact with the DNA both 'behind' and 'beneath' TBP. Further experiments will be required to examine the validity of this working model (Lai, 2005).

Functioning of the Drosophila Wilms'-tumor-1-associated protein homolog, Fl(2)d, in Sex-lethal-dependent alternative splicing

fl(2)d, the Drosophila homolog of Wilms'-tumor-1-associated protein (WTAP), regulates the alternative splicing of Sex-lethal (Sxl), transformer (tra), and Ultrabithorax (Ubx). Although WTAP has been found in functional human spliceosomes, exactly how it contributes to the splicing process remains unknown. This study attempts to identify factors that interact genetically and physically with fl(2)d. The Sxl-Fl(2)d protein-protein interaction was examined in detail and evidence is presented suggesting that the female-specific fl(2)d¹ allele is antimorphic with respect to the process of sex determination. fl(2)d was shown to interact genetically with early acting general splicing regulators, and Fl(2)d is present in immunoprecipitable complexes with Snf, U2AF50, U2AF38, and U1-70K. By contrast, no Fl(2)d complexes were detected containing the U5 snRNP protein U5-40K or with a protein that associates with the activated B spliceosomal complex SKIP. Significantly, the genetic and molecular interactions observed for Sxl are quite similar to those detected for fl(2)d. Taken together, these findings suggest that Sxl and fl(2)d function to alter splice-site selection at an early step in spliceosome assembly (Penn, 2008).

Alternative splicing of pre-mRNAs requires the default splicing machinery to choose between different potential 5' and 3' splice-site combinations. Factors like Sxl that force the selection of alternative 5' and 3' splice-site combinations must exert their effects through interactions with components of the general splicing machinery. However, since the splicing of pre-mRNAs is a multi-step process that depends upon the assembly and remodeling of a large and highly dynamic RNA protein complex, these regulatory interactions could potentially occur at many different points in the processing reaction. Previous genetic and molecular studies have implicated several general splicing factors in Sxl-dependent alternative splicing. These include Snf, U1-70k, U2AF, and Spf45. Of these proteins, only Snf is expected to be present in all of the intermediate steps in the splicing reaction. In contrast, studies on spliceosomal intermediates in humans indicate that the three other Sxl interactors are associated with the spliceosome only during the early stages of the splicing reaction (Jurica, 2003; Deckert, 2006). Both Snf and U1-70k are components of the U1 snRNP and will be present when the U1 snRNP first associates with the 5' splice site of the pre-mRNA to form the prespliceosome E complex. After U1 interacts with the 5' splice site, U2AF is thought to bind to the polypyrimidine tract upstream of the 3' splice site and recruit the U2 snRNP to the pre-mRNA to form spliceosome complex . In addition to Snf, U1-70k, and U2AF, this complex in humans also includes the SPF45 protein. In the next step, a complex containing three other snRNPs, the U4/U6,U5 tri-snRNP, associates with the spliceosome to form the B complex. This is followed by extensive structural rearrangements in which the U1 and subsequently U4 snRNPs are displaced. U170k together with the U1-associated Snf should be lost from the complex with disassociation of the U1 snRNP. The subsequent unwinding of the U4/U6 base pairs and dissociation of U4 permits base pairing between U6 and the 3' splice site and the U2 snRNA. This generates the active complex B*, which catalyzes the first transesterification reaction to generate complex C. Both U2AF and SPF45 appear to be missing from complex B*, while the only Sxl cofactor that is expected to remain until the final splicing step should be the Snf protein associated with the U2 snRNP. Thus, with the exception of Snf, the proteins known to be important for Sxl-dependent alternative splicing appear to function prior to the formation of the activated B* complex and the first splicing reaction (Penn, 2008).

Several lines of evidence suggest that this is also likely to be true for Fl(2)d. First, it was found that Fl(2)d is in an immunoprecipitable complex with Snf, U1-70K, and both of the U2AF subunits, U2AF50 and U2AF38. All of these proteins are expected to be present in one or more of the complexes (E, A, or B) that are formed early in the splicing reaction. Second, it has recently been found that U2AF not only is present in the early complexes E and A, but also can be detected in the inactive B complex (Deckert, 2006). Consistent with this expectation, complexes between U2AF50 and the U5 snRNP protein U5-40k were detected. In contrast, complexes between U2AF50 and Fl(2)d could be detected, complexes between could not be detected U5-40k and Fl(2)d. This finding would suggest that Fl(2)d is stably associated with the E and/or A complex, but is not stably associated with the B complex. Third, the SKIP protein associates with the inactive and activated B complexes, but is absent from the A complex (Jurica, 2003; Deckert, 2006). As was the case for U5-40k, interactions could not be detected between SKIP and Fl(2)d. With the caveat that these negative results must be interpreted with caution, these findings, taken together, would argue that Fl(2)d functions at an early step(s) in the splicing reaction prior to the formation of complex B (Penn, 2008).

Since Fl(2)d is expressed in both sexes and has functions in alternative splicing that are not connected to Sxl, it could be argued that the physical interactions detected between Fl(2)d and different components of the splicing apparatus do not reflect its functioning in Sxl-dependent alternative splicing. While this is a potential concern, there are a number of reasons why this is believed to be unlikely. For one, these complexes appear to be physiologically relevant to Sxl-dependent alternative splicing; female-specific genetic interactions were observed between fl(2)d and the genes encoding several of these proteins. In the case of snf, not only a null allele was tested, but also two mutations, snf¹⁴⁸ and snf^5mer, which differentially affect Snf protein interactions with the U1 or the U2 snRNPs, respectively. When mothers heterozygous for the U1-deficient snf¹⁴⁸ are mated to Sxl^- fathers, there is a marked reduction in the viability of female offspring. This female lethality is enhanced by the antimorphic allele fl(2)d¹, but not by the null allele fl(2)d². In contrast to snf¹⁴⁸, there is little, if any, female-specific lethality in the progeny of snf^5mer/+ females and Sxl^- fathers; however, the snf^5mer allele shows a very strong synergistic female lethal interaction with fl(2)d¹. Likewise, a strong synergistic interaction was observed between fl(2)d¹ and U2AF38^δE18 (Penn, 2008).

Another reason to believe that the association of Fl(2)d with early splicing regulators is relevant to how it promotes Sxl-dependent alternative splicing is the fact that Sxl is found in complexes with the same set of splicing factors in nuclear extracts as Fl(2)d. As noted above, these factors include Snf, U170k, and the two U2AF subunits U2AF38 and U2AF50. In the case of Snf, it has been shown that Sxl is in an immunoprecipitable complex with Snf in nuclear extracts. Although this is also true for Fl(2)d, there are some differences in how Fl(2)d and Sxl interact with Snf. For one, Fl(2)d:Snf interactions in nuclear extracts are insensitive to RNase, while Sxl:Snf interactions are RNase sensitive. While it was not possible to test whether Fl(2)d:Snf interactions involve direct protein contacts, both Fl(2)d and Snf can interact directly with Sxl in vitro. Fl(2)d and Sxl also differ in their interactions with the Snf mutant proteins 148 and 5mer. Sxl can associate with the Snf^5mer protein, but does not form a complex with Snf¹⁴⁸. By contrast, Fl(2)d complex formation with the Snf¹⁴⁸ mutant protein appears to be equivalent to that observed for wild-type Snf, while complexes with Snf^5mer appear to be destabilized and are present in reduced yield. In addition, it was also found that Sxl resembles Fl(2)d in that it is not stably associated either with the U5 snRNP protein U5-40k or with SKIP (Penn, 2008).

On the basis of in vitro splicing experiments (using a chimeric pre-mRNA consisting of an adenovirus 5' exon/intron fused to a short sequence spanning the 3' splice site of the Sxl male exon) it has been suggestedthat Sxl autoregulation depends upon Sxl inhibition of the second catalytic step of splicing, i.e., the joining of the 5' splice site of the Sxl second exon to the 3' splice site of the male exon and the release of the second intron lariat intermediate. In this model, Sxl was proposed to block this catalytic step by inhibiting the SPF45 factor bound to one of the two AG sequences in the male exon 3' splice site. It was suggested that this would force the splicing machinery to bypass the male exon 3' splice site and instead join the free 5' splice site of exon 2 to the 3' splice of exon 4 located slightly more than a kilobase downstream of the male exon. In addition to the fact that using a 3' AG for the second catalytic step that is located ~1 kb from the branch point would be highly unusual, it is difficult to reconcile this model for Sxl autoregulation with the results presented in this study, which argue that Sxl must act at a much earlier point during the initial assembly of the splicing apparatus on target pre-mRNAs. Other findings also seem to be inconsistent with this model. For one, the Sxl-binding sites located in the polypyrimidine tract of the male exon 3' splice site that were used in the in vitro splicing experiments are completely dispensable for female splicing of the Sxl pre-mRNAs in vivo. In fact, the critical sites for Sxl binding are located in the upstream and downstream intron sequences flanking the male exon >200 bases from the male 3' splice site. In addition, Sxl regulation in vivo seems to pivot on blocking the use of the male exon 5' splice site, while controlling the use of the male exon 3' splice site plays at most only a subordinate role in regulation. Finally, although SPF45 is present in purified B spliceosome complexes from humans, it is apparently absent from the catalytically active B* and C complexes (Penn, 2008).

Another question of interest is the nature of the relationship between Sxl and Fl(2)d. Like Snf, Fl(2)d can interact directly with Sxl in vitro. For Snf, the first Sxl RRM domain R1 mediates this interaction while for Fl(2)d the interaction appears to depend upon a combination of the Sxl N terminus and the R1 RRM domain. Although the in vitro interactions between recombinant Sxl and Snf proteins are not dependent on (or stimulated by RNA), RNase treatment completely disrupts Sxl:Snf interactions in nuclear extracts. By contrast, RNase treatment appears to significantly enhance Sxl:Fl(2)d interactions in nuclear extracts. Since the two Sxl RRM domains undergo a substantial rearrangement when they bind to RNA, it is possible that Sxl:Fl(2)d interactions occur prior to the binding of the Sxl protein to the pre-mRNA, while Sxl:Snf interactions occur after Sxl has associated with its target sequences. If this is the case, then one plausible idea would be that Fl(2)d helps recruit Sxl into the assembling spliceosome. This mechanism could potentially account for the finding that fl(2)d mutations dominantly suppress the female lethal effects of the antimorphic Nβ-gal trangene: there would be less of Nβ-GAL fusion protein incorporated into the Sxl splicing complex when fl(2)d activity is reduced. However, since it was not possible to demonstrate an association between Fl(2)d and the Nβ-GAL fusion protein in vivo, other mechanisms for suppression cannot be excluded and further studies will be required to fully understand how Fl(2)d functions in Sxl-dependent alternative splicing (Penn, 2008).

Given that Sxl and key cofactors like Fl(2)d associate with early acting general splicing regulators that function to define the 5' and the 3' splice sites, it is possible that Sxl promotes female-specific splicing of its own pre-mRNAs by inhibiting the process of exon definition. Presumably it would do so by specifically targeting the U1 snRNP associated with the male exon 5' splice site and SPF45 and the U2AF heterodimer associated with the male exon 3' splice site. Exon definition is thought to be particularly important when a small exon is surrounded by large introns as is the case for the Sxl male exon. It is only ~190 bp in length and is flanked by large introns. Interestingly, exon definition cannot occur for exons >500 nucleotides and, if a large exon is surrounded by large introns, such an exon is often skipped entirely. Consistent with these studies, several of the gain-of-function mutations in Sxl are transposon insertions that increase the size of the male exon. In these mutants, Sxl is not required for female-specific splicing and the male exon is skipped, even in the absence of Sxl protein. Also supporting the notion that male exon definition might be an especially sensitive step that would make it a good target for Sxl regulation is the fact that both the 3' and 5' male exon splice sites are known to be suboptimal. In fact, when the male exon (plus the associated splice sites) is placed into a heterologous intron, the male exon is not recognized by the splicing machinery unless the splice sites are optimized to more closely resemble the consensus sequence. Even then, the male exon is not efficiently recognized by the default splicing machinery and is skipped most of the time. Further studies will be required to explore this possible mechanism for Sxl regulation (Penn, 2008).

DEVELOPMENTAL BIOLOGY

Embryonic

In contrast a nuclear distribution during most of development, antibody staining of early embryos (0-30 minutes of development) reveals that SNF is non-nuclear. Several mitotic division later, SNF is restricted to the nuclei (Flickinger, 1994).

Evolution of an RNP assembly system: a minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster

In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2-8. The evolution of this machinery was studied using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the Drosophila contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. These data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction (Kroiss, 2008).

Splicing of pre-mRNAs is catalyzed by the spliceosome, a macromolecular machine consisting of a large number of protein factors and the uridine-rich small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/6, and U5. The biogenesis of these particles occurs in a stepwise manner. First, nuclear-transcribed, m⁷G-capped snRNAs U1, U2, U4, and U5 are exported into the cytoplasm, where a conserved sequence motif in these RNAs (Sm site) serves as a binding platform for the seven Sm proteins B/B', D1, D2, D3, E, F, and G. As a consequence, a ring-shaped Sm core domain is formed. This domain is crucial for subsequent steps in the biogenesis of UsnRNPs, such as formation of the hypermethylated m^2,2,7G cap and import of the assembled particle into the nucleus. At a yet to be defined step, additional factors are recruited to form the mature UsnRNP particles that function in splicing (Will, 2001; Kroiss, 2008 and references therein).

Previous studies have shown that Sm proteins bind spontaneously, albeit in a hierarchical manner, onto UsnRNAs in vitro). However, in cellular extracts, this process depends on ATP and the activity of the multisubunit SMN complex. Recently, a systematic interaction study on the human SMN complex has established its basic architecture. A modular composition was deduced where the three factors SMN, Gemin2, and Gemin8 form the backbone of the entire complex. Onto this core, the peripheral building blocks Gemin3/4 and Gemin6/7/UNRIP bind to form the functional unit. In support of this modular architecture, Gemin-containing subcomplexes have been identified composed of SMN/Gemin2, Gemin3-Gemin5, and Gemin6/7/UNRIP (Kroiss, 2008 and references therein)

The SMN complex not only functions in the assembly of the Sm core domain, but also influences additional steps in the biogenesis pathway of UsnRNPs. One such step is the nuclear import of the assembled UsnRNP, which is mediated by the SMN complex (or parts thereof) in conjunction with the import factor importin β. In addition, specific UsnRNP proteins and the cap hypermethylase Tgs1 have been found in association of the SMN complex (Mouaikel, 2003). This observation indicates that the SMN complex coordinates various events during UsnRNP biogenesis by assuming the role of a binding platform for the respective assisting factors (Kroiss, 2008).

The multisubunit composition of the human SMN complex has impeded the mechanistic dissection of the UsnRNP assembly process. Thus, although RNA interference studies indicated essential roles of several Gemins in the assembly reaction, their precise contributions remain unclear. To facilitate mechanistic studies and to gain insight into the evolution of the SMN complex, genomic databases were mined for organisms that lack individual Gemins and hence may contain a simpler assembly machinery. Indeed, this was the case for different organisms including dipterans. Drosophila was chosen for further investigation because of the wealth of genetic resources. An affinity chromatography strategy has permitted the purification of an assembly-active complex composed of SMN and Gemin2 only. Remarkably, this complex not only facilitated assembly of the Sm core domain but also discriminates between cognate and noncognate RNAs. Thus, a combined bioinformatic and biochemical approach revealed that the assembly reaction requires only two core proteins in vitro, even though SMN complexes from most metazoans are of considerable complexity. It is speculated that Gemins 3-8 have been recruited to the SMN complex in the course of evolution to integrate assembly with additional steps in the biogenesis of UsnRNPs (Kroiss, 2008).

To understand how the UsnRNP assembly machinery has evolved, homology searches were performed for all Gemin proteins constituting the human SMN complex in genomic databases of a variety of organisms. Because of its diverse functions and its transient cytoplasmic interaction with the SMN complex, the UNRIP protein has been excluded from this analysis (Kroiss, 2008).

SMN and Gemin2 orthologs (termed Yab8p and Yip1p) but no other Gemins can be found in the fungus Schizosaccharomyces pombe. Importantly, both orthologs interact physically and may hence form a functional unit. In Saccharomyces cerevisiae, however, only the distantly related Gemin2 ortholog Brr1p, but no SMN ortholog, could be identified. Brr1p has been proposed to be an ortholog of human Gemin2. However, because of its limited homology to Yip1p this finding has been questioned. Taking advantage of the dramatically increased genome databases and novel search algorithms (PSI BLAST), Brr1p can be defined as the single significant homolog of human Gemin2 and S. pombe Yip1p. Because reciprocal searches further support this homology, Brr1p is the ortholog of human Gemin2. Thus, S. cerevisiae retained only one Gemin and hence is unlikely to form a functional SMN complex. Interestingly, like S. pombe, the plants Arabidopsis thaliana and Oryza sativa contained orthologs of SMN and Gemin2 only. It is therefore concluded that SMN and Gemin2 represent the most primitive and ancestral version of the SMN complex. Surprisingly, the genome of Dictyostelium discoideum, a facultative multicellular organism, encoded orthologs of Gemin3 and Gemin5. Given that D. discoideum is basal to fungi and metazoans, the bioinformatic data suggest that both Gemins have been lost during evolution in the fungi branch but were retained in metazoans. Interestingly, the presence of a Gemin5 ortholog in Ostreococcus tauri but its absence in land plants indicates an independent gene loss in this phylum. Moreover, it was found that SMN and Gemins 2, 3, 5, 6, 7, and 8 are present in the cnidarian Nematostella vectensis, a basic metazoan. Gemin4 first appeared in the sea urchin Strongylocentrotus purpuratus, whereas it is absent in all ecdysozoans under study. This suggests that Gemin4 has joined the SMN complex only recently in evolution, most likely with the appearance of deuterostomians. Consequently, it was found to be part of the SMN complex in vertebrates such as Danio rerio but also cephalochordates like Branchiostoma floridae and Ciona intestinalis (urochordates). Thus, plants and some fungi possess a core complex composed of SMN and Gemin2 only, whereas an elaborate SMN complex has developed only in animal branches by addition of Gemin proteins (Kroiss, 2008).

The bioinformatic data indicated an evolutionary trend in the animal kingdom toward a multisubunit SMN complex (see Evolution of an RNP assembly system). Interestingly, however, no orthologs of most Gemins were found in the dipterans Drosophila melanogaster and Anopheles gambiae although they were present in closely related Apis mellifera and Nasonia vitripennis. Further analysis was restricted to D. melanogaster in this study. Besides the known SMN ortholog (Miguel-Aliaga, 2000; Rajendra, 2008), a Gemin2 ortholog was found encoded by CG10419 and putative orthologs of Gemin3 (Dhh1) and Gemin5 (Rigor mortis). Dhh1 protein shows high conservation in the N-terminal DEAD box helicase domain but possesses a diverged C terminus. Rigor mortis displays moderate homology to Gemin5 over the entire protein length. A phylogenetic analysis revealed that both evolve significantly faster than their orthologs in other organisms. This released evolutionary pressure might indicate the emergence of a novel function or the loss of a common one for these factors. These data suggest that D. melanogaster possesses a much simpler SMN complex as compared with vertebrates (Kroiss, 2008).

To investigate whether Dhh1 and Rigor mortis have retained their function in the context of the D. melanogaster SMN (dSMN) complex, use was made of a novel epitope tag. This tag consists of the first 30 aa of human SMN protein, which are specifically recognized by the monoclonal antibody 7B10 (Meister, 2000). Importantly, competition with synthetic peptide comprising this epitope allows native elution of tagged proteins from this antibody. Because D. melanogaster SMN protein lacks these 30 aa, a plasmid was constructed allowing the expression and subsequent purification of a protein fused to this epitope (termed TagIt epitope) after stable transfection of Schneider2 cells. In a TagIt-Dhh1 affinity purification, only small amounts of dSMN protein could be detected under physiological conditions but not at salt concentrations exceeding 250 mM. Thus, Dhh1 is only weakly associated with dSMN. Similarly, the role of Rigor mortis was investigated. No binding of Rigor mortis to dSMN has been observed, arguing against a stable association of this protein with the dSMN complex. These data suggest that Rigor mortis either functions in UsnRNP core formation in a manner different from vertebrate Gemin5 or has completely lost its function in the pathway of UsnRNP biogenesis (Kroiss, 2008).

To gain detailed insight into the composition of the D. melanogaster SMN complex, a TagIt-dSMN-expressing Schneider2 cell line was generated. Importantly, TagIt-dSMN was incorporated into a high-molecular-weight complex that also contained Gemin2. This implied that the tagged dSMN protein engages in interactions similar to those of its endogenous counterpart. The SMN complex was affinity-purified from extracts by means of 7B10 affinity chromatography. Affinity-purified proteins were separated by SDS-PAGE under reducing and nonreducing conditions and identified by protein mass spectrometry and Western blotting. Whereas the tagged SMN protein and its interactor dGemin2 could be readily identified, neither Dhh1 nor Rigor mortis was found under the purification conditions applied in this study (Kroiss, 2008).

It is known that the human SMN complex consists of the core machinery (i.e., SMN and Gemins) as well as the transiently interacting substrates that are transferred onto the UsnRNA during assembly. These are the Sm proteins and some UsnRNP-specific proteins. Strikingly, the entire set of Sm proteins, namely SmB, SmD1 (gene snRNP69D), SmD2 (CG1249), SmD3, SmE (CG18591), SmF (DebB), and SmG (CG9742), was prominently present in the elution. Furthermore, the UsnRNP-specific factors U1 70KU2A', the U2B''/U1A ortholog SNF, and the ortholog of the U5 specific protein (CG4849) U5 116kD were found reproducibly in the purified complex. However, the abundance of these specific proteins varied among preparations and was often substoichiometric (Kroiss, 2008).

During UsnRNP assembly, the SMN complex physically contacts the UsnRNAs (Fischer, 1997). In vertebrates, this interaction has been proposed to be mediated, at least in part, by Gemin5 and to occur in the cytoplasm. Interestingly, despite the absence of Rigor mortis in the TagIt-dSMN complex, snRNAs U1, U2, U4, and U5 were specifically coprecipitated with dSMN and dGemin2 antibodies from total Schneider2 cell extract. Identical results were obtained when the SMN complex was purified from the cytosol, where SMN is predominantly localized. Hence, in D. melanogaster, the SMN complex is sufficient to recruit a set of substrate proteins similar to those in vertebrates. In addition, the complex interacts specifically with U snRNAs in the cytoplasm, which reflects a situation previously observed in Xenopus laevis oocytes (Kroiss, 2008).

Previous studies have indicated that Gemins interact within the SMN complex in a modular manner. Interestingly, homology searches for components of the SMN complex in a variety of organisms have recapitulated this finding on an evolutionary scale. The most simple SMN-containing complex is composed of SMN and Gemin2 only and can be found in unicellular organisms such as the fission yeast S. pombe and in plants. The next level of complexity is characterized by the appearance of Gemin3 in D. discoideum, thus predating the emergence of the Fungi/Metazoa clade. The absence of Gemin5 from genomes of fungi and land plants and its presence in the green algae O. tauri and in D. discoideum indicate independent secondary gene loss in fungi and plants. This may be due to a role of Gemin5 outside of the SMN complex, which is not retained in these organisms (Kroiss, 2008).

Only later in evolution at the level when first metazoans developed, the building block composed of Gemins 6, 7, and 8 was added to the set of the Gemin family. From this time on, organisms had the potential to express an SMN complex similar in architecture to the human one. The only component that was not present at that point was Gemin4, which can be found only in the genomes of deuterostomians. Thus, the data suggest that the SMN complex evolved by a blockwise addition of Gemins to an ancient core complex of SMN and Gemin2 in a manner corresponding to their mutual biochemical association (Kroiss, 2008).

In striking contrast to this overall evolutionary trend, a remarkable simplification of this complex in the dipterans was found A. gambiae and D. melanogaster. In these animals, no orthologs of Gemin4 were found, as expected, but also of Gemins 6-8 were absent. However, these latter Gemins were clearly present in hymenopterans. Although orthologs of Gemin3 and Gemin5 were found in dipterans, they show a significantly higher evolutionary rate in dipterans than in other clades. These computational findings have been experimentally challenged by a biochemical approach that has allowed isolation of an assembly-active SMN complex from D. melanogaster. Indeed, the composition of the complex was remarkably simple and consisted of SMN and Gemin2 as the only stoichiometric components. Dhh1 (Gemin3) bound to this core complex only at low salt concentrations, and Rigor mortis (Gemin5) was not present at all. The D. melanogaster SMN complex therefore equals its counterpart in S. pombe and plants although the function of SMN and Gemin2 orthologs in these organisms has not been demonstrated. It is conceivable that Dhh1 and Rigor mortis have adopted novel functions in a different context because they rapidly diverge from their ancestors. Consistent with this notion, a function of Rigor mortis (Gates, 2004) in ecdysone signaling has been described (Kroiss, 2008).

Despite the obvious simplicity of the SMN complex in D. melanogaster, this study has provided evidence that this unit is functionally related to the SMN complex of mammals. First, a set of UsnRNP-related substrates, namely the common Sm proteins, UsnRNP specific factors (U1 70K, U2A', U2B''/U1A, and U5 115K), and UsnRNAs were found to be part of the complex. Most of these factors have previously been shown to bind to SMN complexes of vertebrates (Meister, 2002). Second, affinity-purified dSMN complex mediate the assembly of the Sm core domain in vitro. Similar to the situation in humans, a strong dependence was found of UsnRNP core assembly on temperature but not on ATP (Meister, 2002). However, at present the possiblility cannot be ruled out that assembly of UsnRNPs in D. melanogaster cytosolic extracts requires ATP hydrolysis as observed for the same reaction in vertebrates (Kroiss, 2008).

The obvious simplicity of the assembly system of D. melanogaster allowed the reconstitution of the dSMN complex from recombinant proteins and the investigation of its mode of action. Interestingly, strong cooperativity was observed in Sm protein binding onto the complex. Heterooligomers D1/D2 and B/D3 had only little affinity for the complex, but binding was greatly enhanced in the presence of recombinant Sm heterooligomer E/F/G. Further studies are required to determine the precise binding sites of all Sm proteins on the SMN complex and to test the influence of arginine methylation on Sm protein binding. It was an open question why UsnRNP assembly is strictly dependent on the SMN complex in vivo even though this reaction is spontaneous in vitro. Assembly studies with the D. melanogaster SMN complex show that precise assembly of the Sm core domain on UsnRNA was possible only when Sm proteins were prebound to the SMN complex, whereas misassembly of isolated Sm proteins occurred under the same conditions. In addition, human SMN and Gemin2 are likewise sufficient to specifically transfer Sm proteins onto UsnRNA. Hence, these data and similar studies performed in vertebrates argue for a dual role of the SMN complex as an RNP assembler and chaperone (Kroiss, 2008).

From an evolutionary point of view, these findings raise the question why dipterans can afford a minimized assembly system, whereas apparently other branches in the animal kingdom require a multicomponent SMN complex. The most plausible explanation for this paradox is that Gemins 3-8 are not primarily involved in the assembly reaction per se but rather in other steps during the UsnRNP biogenesis. Thus, it is known that the human SMN complex integrates several steps in biogenesis, such as cap hypermethylation and nuclear import. It is speculated that these steps will occur in dipterans independent of the SMN complex and may hence allow for the omission of individual Gemins. Further studies will be needed to test whether this is indeed the case (Kroiss, 2008).

In conclusion, these studies have shown that the integration of bioinformatics and biochemistry can be used to analyze cellular pathways functionally and evolutionarily. Similar strategies may prove to be powerful tools in the analysis of even more complex systems such as the spliceosome (Kroiss, 2008).

Adult

All nuclei in both the germ-line and the somatic components of the ovary are NSF protein positive. High levels are found in the cytoplasm of nurse cells, suggesting that the SNF protein is deposited into the developing oocyte when the nurse cells dump their contents (Flickinger, 1994)

EFFECTS OF MUTATION

With a focus on Sex-lethal (Sxl), the master regulator of Drosophila somatic sex determination, a comparison has been carried out between the sex determination mechanism that operates in the germline and that which operates in the soma. In both cell types, Sxl is functional in females (2X2A) and nonfunctional in males (1X2A). Somatic cell sex is determined initially by a dose effect of X:A numerator genes on Sxl transcription. Once initiated, the active state of SXL mRNA is maintained by a positive autoregulatory feedback loop in which Sxl protein ensures its continued synthesis by binding to SXL pre-mRNA and thereby imposing the productive (female) splicing mode. Ectopic expression of Sxl protein triggers the female-specific Sxl mRNA feedback loop in male germ cells without disrupting spermatogenesis. There is no adverse effect on male viability or fertility. The presence of Sxl protein may sometimes retard the rate of differentiation of spermatocytes, but does not abort the process (Hager, 1997).

The gene splicing-necessary factor (sans fille or snf), which encodes a component of U1 and U2 snRNPs, participates in SXL RNA splicing control. An increase in the dose of snf+ can trigger the female Sxl RNA splicing mode in male germ cells and can feminize triploid intersex (2X3A) germ cells. These snf+ dose effects are as dramatic as those of X:A numerator genes on Sxl in the soma and qualify snf as a numerator element of the X:A signal for Sxl in the germline. Female-specific regulation of Sxl in the germline involves a positive autoregulatory feedback loop on RNA splicing, as it does in the soma. Neither a phenotypically female gonadal soma nor a female dose of X chromosomes in the germline is essential for the operation of this feedback loop, although a female X-chromosome dose in the germline may facilitate it. Engagement of the Sxl splicing feedback loop in somatic cells invariably imposes female development. In contrast, engagement of the Sxl feedback loop in male germ cells does not invariably disrupt spermatogenesis; nevertheless, it is premature to conclude that Sxl is not a switch gene in germ cells for at least some sex-specific aspects of their differentiation. In fact, increased doses of snf+ and Sxl+ can feminize germ cells when germ cells have an altered X chromosome to autosome ratio. snf+ and Sxl+ feminize 2X3A germ cells. Flies with the higher doses of snf+ display a greater proportion of yolky germline cysts and eggs. Somatic sex is important in this feminization as the sexual phenotype of all internal and external somatic dimorphic characters appears to be fully female in 2X3A animals carrying a heat-shock transformer transgene. What then is the role of Snf in the germ-line? It seems likely that Snf acts to boost the autoregulatory effectiveness of very low levels of female Sxl protein, rather than acting directly on its own to influence SXL transcript splicing. Ironically, the testis may be an excellent organ in which to study the interactions among regulatory genes such as Sxl, snf, ovo and otu, which control female-specific processes in the ovary (Hager, 1997).

Females homozygous for sans fille1621 (= fs(1)1621) have an abnormal germ line. Instead of producing eggs, the germ-line cells proliferate, either forming ovarian tumors or excessive numbers of nurse cells. The Sex-lethal gene product(s) regulate the branch point of the dosage compensation and sex determination pathways in the soma. The role of Sex-lethal in the germ line is not clear but the germ line of females homozygous for female sterile Sex-lethal alleles or germ-line clones of loss-of-function alleles are characterized by ovarian tumors. Females heterozygous for sans fille1621 or Sex-lethal are phenotypically wild type with respect to viability and fertility but females trans-heterozygous for sans fille1621 and Sex-lethal show ovarian tumors, somatic sexual transformations, and greatly reduced viability (Oliver, 1988). snf's role in germline sex determination was determined directly from the genetic analysis of a single allele, snf1621. Females homozygous for snf1621 are sterile. In the mutant neither the oocyte nor the nurse cells differentiate. Instead the germinal cells continue to divide, resulting in the formation of ovarian tumors. A similar phenotype is seen in fused, ovo, and otu mutants. The similarity of snf and Sxl mutant phenotypes in the germline suggests that they disrupt the same process: germline sex determination (Salz, 1992 and references).

In contrast to snf's role in germline sex determination, its role in somatic sex differentiation and X chromosome dosage compensation can only be inferred by an unusual genetic interaction between mutations at the snf and Sxl loci. Although doubly heterozygous females have reduced viability, many surviving females show signs of sex transformations. snf is required for the activation of Sex-lethal in both the germline and the soma (Salz, 1992).

To determine unequivocally the null phenotype of snf, deletions were prepared of the entire snf-coding sequence. Animals homozygous for such a deletion suffer embryonic lethality (Flickinger, 1994).

In Drosophila, females require products of the gene Sxl for sex determination, dosage compensation and fertility. The X-chromosomal gene snf, located in 4F1 to 4F11 and previously called fs(1)1621, provides maternal and zygotic functions necessary for Sxl activity in germ line and soma. In XX animals, the mutation SxlM1 which was reported to express the female-specific functions of Sxl constitutively can rescue all phenotypes resulting from lack of snf product. XY animals carrying SxlM1 and lacking maternal or zygotic snf activity survive as males with some female traits. A stock was constructed in which the females are snf SxlM1/snf SxlM1 and males snf SxlM1/Y. This shows that SxlM1 is not truly expressed constitutively in animals with an X:A ratio of 0.5, but requires activity of snf for initiation or maintenance (Steinmann-Zwicky, 1988).

The two sexes of Drosophila melanogaster are distinguished by a two-fold difference in the dose of a small set of specific X-linked genes -- the so-called numerator elements -- which collectively determine the transcriptional state of the switch gene Sex-lethal (Sxl). During a 45-minute window of time very early in development the numerator elements do this through their actions on the Sxl 'establishment' promoter, Sxl_Pe. The double dose of numerator elements in chromosomal females (XX) triggers transcription at Sxl_Pe whereas the single dose in chromosomal males (XY) leaves this promoter off. However, a very different mechanism then operates to maintain the functional state of Sxl. This maintenance process exhibits Sxl gene dosage effects with levels snf⁺ gene product. Thus, although Sxl interacts with a variety of RNAs to control a diversity of functions, only the autoregulatory aspect of Sxl is affected by increased Snf. Encoded by sans fille, Snf is the Drosophila homolog of mammalian U1A and U2B" and is an integral component of U1 and U2 small nuclear ribonucleoprotein particles (snRNPs). Surprisingly, changes in the level of this housekeeping protein can specifically affect autoregulatory activity of the RNA-binding protein Sex-lethal (Sxl) in an action that must be physically separate from Snf's functioning within snRNPs. This observation adds to evidence that the functional relationship between these two genes is very different from that between Sxl and other genes that affect Sxl pre-mRNA splicing (Cline, 1999)

Exploiting an unusual new set of mutant Sxl alleles in an in vivo assay, Snf has been shown to be rate-limiting for Sxl autoregulation when Sxl levels are low. In such situations, increasing either the maternal or zygotic snf dose enhances the positive autoregulatory activity of Sxl for Sxl somatic pre-mRNA splicing without affecting Sxl activities toward its other RNA targets. In contrast, increasing the dose of genes encoding either the integral U1 snRNP protein U1-70k, or the integral U2 snRNP protein SF3a⁶⁰, has no effect. Increased snf⁺ enhances Sxl autoregulation even when U1-70k and SF3a⁶⁰ are reduced by mutation to levels that, in the case of SF3a⁶⁰, demonstrably interfere with Sxl autoregulation. The observation that increased snf does not suppress other phenotypes associated with mutations that reduce U1-70k or SF3a⁶⁰ is additional evidence that snf dose effects are not caused by increased snRNP levels. Mammalian U1A protein, like Snf, has a snRNP-independent function (Cline, 1999).

From the effects of raising the dose of the wild-type snf gene above normal levels, it is inferred that the integral snRNP protein encoded by snf acts outside of the snRNP in controlling pre-mRNA splicing for Sxl. One would not pick snf as a gene likely to display phenotypic effects of increased dose because snf encodes only one of many proteins that make up U1 and U2 snRNPs. In the genetically sensitized system used here to reveal snf⁺ dose effects, these complex multimeric assemblies are at levels that suffice for all of the needs of the organism. Such dose effects are not typical of integral snRNP proteins because increasing the dose of the gene encoding the U1 protein U1-70k or that encoding the U2 protein SF3a⁶⁰ has no effect on Sxl autoregulation. This negative result is particularly meaningful in light of the demonstration that while lowering the level of SF3a⁶⁰ interferes with Sxl autoregulation, this does not eliminate the effects of increased snf⁺ dose (Cline, 1999).

Could the influence of increased snf⁺ dose reflect a quirk of fruit fly regulatory circuitry in which snRNP levels are tied to U1A/U2B" levels? A priori, this would seem a disadvantageous strategy for the fly to use. Because most RNA splicing involves a sensitive balance between competing potential splice sites that one might expect to be affected by changes in the levels of these two snRNPs, one would expect regulatory circuitry to insulate the general splicing system from perturbation, not tie it to a single gene product in this way. Moreover, because a maternal effect of increased snf⁺ dose is observed that is nearly as striking as the zygotic dose effect, such a sensitive regulatory connection would have to operate both maternally during oogenesis to govern subsequent snRNP levels in the embryo and zygotically to govern snRNP levels at later stages. Two experimental observations argue against such a tie to snf. (1) Although striking effects on Sxl by even a single extra copy of snf⁺ are seen in various sensitized situations, males and females wild-type for Sxl can carry as many as 10 extra copies of the same snf⁺ construct and be fully viable. (2) Most damaging for this unlikely hypothesis, increasing snf⁺ dose does not suppress the mutant phenotypes caused by decreasing the level of U1-70k or SF3a⁶⁰ (Cline, 1999).

In contrast, if Snf functions specifically in Sxl autoregulation not as an integral component of U1 or U2 snRNPs but as an individual protein, the snf⁺ dose effects would not be reflecting changes in functional snRNP levels, but simply the established tendency of metazoan gene product levels to be roughly proportional to structural gene dose. Dose effects in this case would be indicating Snf's key participation in the process by which Sxl protein inhibits the male Sxl pre-mRNA splice by binding to RNA, a process likely to directly involve relatively few proteins (Cline, 1999).

The fly's use of U1A/U2B'' as an alternative splicing factor in sex determination would not be the first case of an integral spliceosomal protein acting outside of the snRNPs. Non-snRNP mammalian U1A negatively regulates its level by binding to sites in U1A pre-mRNA to block polyadenylation. U1A may also function more generally to couple splicing and 3' end formation. Such pleiotropy raises the possibility of an undiscovered world of biological functions for integral snRNP proteins operating as free agents. Because these proteins also have essential housekeeping functions, their other roles might not be easily revealed in vivo. Positive autoregulation gives the Sxl assay used here an extremely nonlinear character that surely facilitated study of biochemical effects that might otherwise have been too small to detect (Cline, 1999).

How might Snf be involved in Sxl autoregulation? There is evidence that a small fraction of Snf is in proximity to Sxl on RNA. Previous models have assumed that any interaction between Snf and Sxl occur with Snf acting as part of U1 or U2 snRNPs; it is suggested that this interaction is preceded by Sxl binding to pre-mRNA between exons 3 and 4 to block the male splice. Through an interaction between Snf within the snRNPs and Sxl bound to RNA surrounding the male exon, an abortive presplicing complex for exon-3 has been proposed to form, allowing the alternative exon 2-4 female-specific splice to proceed by default (Cline, 1999 and references therein).

In light of the data reported here, it now appears that Snf may bind with Sxl to pre-mRNA flanking the male exon, perhaps each facilitating or stabilizing the other's binding. By this model, it would not be surprising if the consequences of such an association were most significant at low concentrations of Sxl, such as those which surely prevail in the sensitized situations describe here. In addition to stabilizing Sxl binding, or even as an alternative to it, non-snRNP Snf associating with Sxl may be necessary to inhibit further spliceosomal complex assembly around the male-specific exon 3. Perhaps independent Snf protein interacting with Sxl bound to the pre-mRNA interferes with an essential association that Snf in the snRNPs themselves would need to have with other splicing factors to define exon 3 splice sites (Cline, 1999).

The dose-sensitive involvement of snf in somatic Sxl autoregulation described here is one of the strongest similarities between the regulation of sex-specific gene expression in the soma and in the germ line. It was shown earlier that simply increasing the dose of snf⁺ in an otherwise wild-type fly can trigger female-specific splicing of Sxl transcripts in male germ cells. For the soma, increasing snf⁺ alone will not suffice to engage the autoregulatory splicing loop; however, somatic Sxl regulation can be made nearly as sensitive to increased snf⁺ dose as germline Sxl regulation by alleles such as Sxl^Mf1 that are so weak that by themselves they do not lower male viability or fertility. The ease with which Sxl splicing control in the soma can be made to respond to the dose of RNA splicing factors favors the idea that the ancestral system controlling the sex-specific expression of Sxl in both the germline and the soma might have been based entirely on dose effects of RNA splicing factors (Cline, 1999).

In view of the central and remarkably specific role snf plays in controlling sex-specific expression of Sxl, it is a curious coincidence that the only genus known to use Sxl as a master sex switch is also the only genus with a species known to use a single protein, Snf, for tasks that two proteins, U1A and U2B'', handle in species as diverse as potatoes and humans. Learning how closely the evolution of Sxl as the master sex-determination gene for Drosophila was paralleled by the evolution of this difference in integral U1 and U2 snRNP proteins might suggest what the driving forces were that led to both changes (Cline, 1999).

The Drosophila U1-70K protein is required for viability, but its arginine-rich domain is dispensable

The conserved spliceosomal U1-70K protein is thought to play a key role in RNA splicing by linking the U1 snRNP particle to regulatory RNA-binding proteins. Although these protein interactions are mediated by repeating units rich in arginines and serines (RS domains) in vitro, tests of this domain's importance in intact multicellular organisms have not been carried out. This paper reports a comprehensive genetic analysis of U1-70K function in Drosophila. Consistent with the idea that U1-70K is an essential splicing factor, it was found that loss of U1-70K function results in lethality during embryogenesis. Surprisingly, and contrary to the current view of U1-70K function, animals carrying a mutant U1-70K protein lacking the arginine-rich domain, which includes two embedded sets of RS dipeptide repeats, have no discernible mutant phenotype. Through double-mutant studies, however, it was shown that the U1-70K RS domain deletion no longer supports viability when combined with a viable mutation in another U1 snRNP component. Together these studies demonstrate that while the protein interactions mediated by the U1-70K RS domain are not essential for viability, they nevertheless contribute to an essential U1 snRNP function (Salz, 2004).

A striking outcome of this study is the finding that U1-70K can accomplish its vital function in the absence of an RS domain. The failure to detect a phenotype in these mutant animals challenges the prevailing view that the U1-70K RS motif provides a vital link between splicing regulators and the U1 snRNP. It is suggested instead that either the interactions detected in vitro are not essential in vivo or there are multiple means by which the U1 snRNP can interact with splicing regulators. Support for the latter view comes from the demonstration that U1 snRNP particles lacking both the U1-70K RS domain and SNF can no longer support viability. Synthetic lethality is attributable to the simultaneous loss of two functions that contribute to the same activity or pathway. Interestingly, in S. cerevisiae synthetic lethal interactions have been observed between nonlethal mutations in several different U1 snRNP components. Thus, the in vivo results argue that while disruption of the U1-70K RS-mediated protein links has no detectable consequence to the living organism, the simultaneous disruption of multiple connections causes U1 snRNP function to fall below the level needed to support development and viability (Salz, 2004).

In Drosophila, the female-specific Sex-lethal (Sxl) protein is required for oogenesis, but how Sxl interfaces with the genetic circuitry controlling oogenesis remains unknown. An allele of sans fille (snf) that specifically eliminates Sxl protein in germ cells was used to carry out a detailed genetic and cell biological analysis of the resulting ovarian tumor phenotype. It was found that tumor growth requires both Cyclin B and zero population growth, demonstrating that these mutant cells retain at least some of the essential growth-control mechanisms used by wild-type germ cells. Using a series of molecular markers, it was established that while the tumor often contains at least one apparently bona fide germline stem cell, the majority of cells exhibit an intermediate fate between a stem cell and its daughter cell fated to differentiate. In addition, snf tumors misexpress a select group of testis-enriched markers, which, remarkably, are also misexpressed in ovarian tumors that arise from the loss of bag of marbles (bam). Results of genetic epistasis experiments further reveal that bam's differentiation-promoting function depends on Sxl. Together these data demonstrate a novel role for Sxl in the lineage progression from stem cell to committed daughter cell and suggest a model in which Sxl partners with bam to facilitate this transition (Chau, 2009).

The observation that female germ cells lacking Sxl are tumorigenic was first published >20 years ago, yet the place of this female-specific RNA binding protein in the genetic circuitry controlling oogenesis has remained elusive. This study investigated Sxl's role in the germline by taking advantage of a snf mutant allele that specifically eliminates Sxl expression in the germline. Genetic and cell biological analysis established that Sxl is required for the transition from stem cell to committed daughter cell by showing that the majority of Sxl-deficient germ cells have acquired an intermediate fate. These findings are in contrast to the commonly held view, based on fusome morphology alone, that Sxl mutant germ cells arrest development later in the differentiation pathway. This study also offers new insight into the function of bam by demonstrating that its differentiation-promoting function depends on Sxl and, importantly, that Sxl and bam control the same sex-specific expression network (Chau, 2009).

In current models, maintenance of GSC identity requires contact with the niche to trigger the signal transduction cascade required for transcriptional repression of bam. This in turn provides a permissive environment that allows PUM, which forms a complex with its partner protein Nanos (NOS), to inhibit translation of a yet unidentified set of mRNAs required for differentiation. Differentiation begins when one of the daughter cells is displaced from the niche and can no longer receive the signals that silence bam transcription. Bam then initiates the differentiation program by antagonizing the translation-inhibitory functions of the PUM/NOS complex. This model predicts a strong negative correlation between the expression of bam and the GSC markers, and, while this is true in general, there have been reports of rare single cells that coexpress bam and one or more GSC-specific markers. These and other studies have suggested that cells fated to differentiate first pass through an intermediate stage that transitions, without dividing, to a mature cystoblast (Chau, 2009).

It was shown that Sxl is required to complete the transition from GSC to a mature cystoblast (CB) by demonstrating that the majority of germ cells lacking Sxl resemble an immature CB-like cell. Furthermore, genetic epistasis experiments suggest that the failure to progress beyond this intermediate stage is attributable to a lack of bam function. This conclusion is supported by studies showing that the tumors resulting from the lack of Sxl and bam are remarkably similar. Specifically, the loss of Sxl and bam results in germ cell tumors with the same unique molecular signature including expression of stem cell markers and with the same set of testis-enriched markers. Both types of germ cell tumors also require CycB and zpg for growth. This comparison reveals that snf and bam tumors both result from a failure to initiate the differentiation pathway in stem cell progeny. It will be interesting to determine what role the misregulated testis-enriched markers play in this process (Chau, 2009).

On the basis of these data, it is proposed that Sxl partners with bam to facilitate the transition between GSCs and the daughter cell that is fated to differentiate. In females, differentiation via control of bam transcription is initiated in response to position-dependent extrinsic cues from the somatic gonad. Extrinsic cues from the somatic gonad also provide essential sex-specific information, via control of Sxl expression. These findings suggest that the intrinsic Sxl/bam partnership serves to integrate these two different extrinsic signaling pathways. This proposal is particularly compelling because it explains how bam function is substantially different in males and females (Chau, 2009).

How might Sxl and bam function converge to promote female germ cell differentiation? Sxl acts post-transcriptionally to repress splicing and translation. The molecular function of Bam, on the other hand, is unknown but is also thought to act post-transcriptionally. At a genetic level, one function of bam is to antagonize the differentiation-inhibiting activity of PUM/NOS. The presence of putative high-affinity Sxl-binding sites in both the 5'-UTR and the 3'-UTR of the nos mRNA leads to the speculation that Sxl functions with Bam to promote differentiation by inhibiting the translation of nos. Although this model is consistent with the finding that Sxl and Bam are coexpressed in the appropriate cell type, biochemical studies to address this point have proved to be technically challenging (Chau, 2009).

In summary, these studies support a model in which the Sxl/bam pathway is required for germ cells to progress from a stem cell fate to a differentiation-competent CB fate. These studies also suggest that if this pathway is blocked, germ cells will continue to proliferate, forming a tumor. It is proposed that the block in the developmental progression from stem cell to fully committed daughter cell is the initial tumorigenic event. This model is consistent with the general view that adult stem cells are the source of some, and perhaps all, tumors. Not only do some human germ cell tumors display many of the same characteristics as the Drosophila tumors described in this study, including expression of stem cell markers, but also they occur frequently in individuals with intersex disorders. While true orthologs of Sxl and bam are not found in vertebrates, the processes that they regulate are likely to be conserved. Future studies aimed at understanding the functional connections between the failure to engage the Sxl/bam genetic programs, misexpression of testis-enriched markers, and tumorigenesis will likely provide mechanistic insight into the pathogenesis of germ cell tumors in humans (Chau, 2009).

eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs

In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors -- the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d--that have been directly implicated in Sxl splicing regulation (Graham, 2011).

Translation initiation is mediated by the binding of a pre-initiation complex to the 5' cap of the mRNA (reviewed in (Merrick, 1996 ; Gingras, 1999) that in turn recruits the small subunit of the 40S ribosome to the mRNA. The pre-initiation complex consists of the cap binding protein, eIF4E, and a scaffolding protein, eIF4G, which mediates interactions with various components of the 40S initiation complex. In many organisms there is also a third protein in the complex, eIF4A, an ATP dependent RNA helicase. Modulating eIF4E activity appears to be a key control point for regulating translation. One of the most common mechanisms of regulation is by controlling the association eIF4E with eIF4G. Factors such as poly-A binding protein that promote the association between eIF4E and eIF4G activate translation initiation, while factors such as the 4E-binding proteins (4E-BPs; see Drosophila 4E-BP) that block their association, inhibit initiation (Graham, 2011 and references therein).

Although eIF4E's primary function in the cell is in regulating translation initiation, studies over the past decade have revealed unexpected activities for eIF4E at steps prior to translation. Among the more surprising findings is that there are substantial amounts of eIF4E in eukaryotic nuclei. One role for eIF4E in the nucleus is the transport of specific mRNAs, like cyclin D1, to the cytoplasm (Rousseau, 1996). This eIF4E activity is distinct from translation initiation since an eIF4E mutation that prevents it from forming an active translation complex still allows cyclin D1 mRNA transport. The transport function of eIF4E is modulated by at least two other proteins, PML and PRH (Topisirovic, 2002; Topisirovic, 2003). While PML seems to be ubiquitously expressed, PRH is found only in specific tissues. In addition, the intracellular distribution of eIF4E exhibits dynamic changes during Xenopus development (Strudwick, 2002). These observation raise the possibility that eIF4E might have additional functions in the nucleus during development. Consistent with this idea, this study shows that eIF4E plays a novel role in the process of sex determination in Drosophila (Graham, 2011).

Sex determination in the fly is controlled by the master regulatory switch gene Sex-lethal (Sxl). The activity state of the Sxl gene is selected early in development by an X chromosome counting system. The target for the X/A signaling system is the Sxl establishment promoter, Sxl-Pe. When there are two X chromosomes, Sxl-Pe is turned on, while it remains off when there is a single X chromosome. Sxl-Pe mRNAs encode RRM type RNA binding proteins which mediate the transition from the initiation to the maintenance mode of Sxl regulation by directing the female-specific splicing of the first pre-mRNAs produced from a second, upstream promoter, the maintenance promoter, Sxl-Pm. Sxl-Pm is turned on before the blastoderm cellularizes, just as Sxl-Pe is being shut off. In the presence of Sxl-Pe proteins, the first Sxl-Pm transcripts are spliced in the female-specific pattern in which exon 2 is joined to exon 4 (see Model of the alternatively spliced region of Sxl ). The resulting Sxl-Pm mRNAs encode Sxl proteins that direct the female specific splicing of new Sxl-Pm pre-mRNAs and this establishes a positive autoregulatory feedback loop that maintains the Sxl gene in the 'on' state for the remainder of development. In male embryos, which lack the Sxl-Pe proteins, the Sxl-Pm pre-mRNAs are spliced in the default pattern, incorporating the male specific exon 3. This exon has several in-frame stop codons that prematurely truncate the open reading frame so that male specific Sxl-Pm mRNAs produce only small non-functional polypeptides. As a consequence the Sxl gene remains off throughout development in males (Graham, 2011).

In females, Sxl orchestrates sexual development by regulating the alternative splicing of transformer (tra) pre-mRNAs. Like Sxl, functional Tra protein is only produced by female-specific tra mRNAs, while mRNAs spliced in the default, male pattern encode non-functional polypeptides. Sxl also negatively regulates the dosage compensation system, which is responsible for hyperactivating X-linked transcription in males, by repressing male-specific lethal-2 (msl-2). Sxl represses msl-2 by first blocking the splicing of an intron in the 5' UTR of the msl-2 pre-mRNA, and then by inhibiting the translation of the mature mRNA. In addition, there are two other known targets for Sxl translational repression. One is the Sxl mRNA itself. Sxl binds to target sequences in the Sxl 5' and 3' UTRs and downregulates translation. It is thought that this negative autoregulatory activity provides a critical homeostasis mechanism that prevents the accumulation of excess Sxl protein. This is important as too much Sxl can disrupt development and have female lethal effects. The other known target is the Notch (N) mRNA (Penn, 2007). Sxl-dependent repression of N mRNA translation is important for the elaboration of sexually dimorphic traits in females. Like msl-2 and Sxl, translational repression appears to be mediated by Sxl binding to sites in the N UTRs (Graham, 2011).

Translational repression of msl-2 mRNA by Sxl is thought to involve two separate mechanisms acting coordinately. Binding sites for Sxl in the unspliced intron in the 5' UTR and in the 3'UTR of msl-2 are required for complete repression. Sxl binding to the 5'UTR blocks recruitment of the 40S pre-initiation complex (. While factors that act with Sxl at the 5'UTR of msl-2 have yet to be identified, repression by the 3'UTR requires Sxl, PABP and a co-repressor UNR. Somewhat unexpectedly, this complex does not affect recruitment of eIF4E or eIF4G to the 5' end. Instead it prevents ribosomes that do manage to attach to the msl-2 mRNA from scanning (Graham, 2011).

Although eIF4E does not appear to be a key player in the translational repression of msl-2 mRNAs, this study reports that it has an important role in the process of sex determination in Drosophila. eIF4E activity is required in females to stably activate and maintain the Sxl positive autoregulatory feedback loop and to efficiently repress msl-2. Surprisingly, this requirement for eIF4E activity in fly sex determination is in promoting the female-specific splicing of the Sxl and msl-2 transcripts, not in translational regulation (Graham, 2011).

The RNA binding protein Sxl orchestrates sexual development by controlling gene expression post-transcriptionally at the level of splicing and translation. To exert its different regulatory functions Sxl must collaborate with sex-non-specific components of the general splicing and translational machinery. In this study evidence is presented that one of the splicing co-factors is the cap binding protein eIF4E. eif4e was initially identified in a screen for mutations that dominantly suppress the male lethal effects induced by ectopic expression of a mutant Sxl protein, Sx-N, which lacks part of the N-terminal domain. The Sx-N protein is substantially compromised in its splicing activity, but appears to have closer to wild type function in blocking the translation of the Sxl targets msl-2 and Sxl-Pm. As the male lethal effects of Sx-N (in an Sxl^- background) are due to its inhibition of Msl-2 expression, it is anticipated that general translation factors needed to help Sxl repress msl-2 mRNA would be recovered as suppressors in the screen. Indeed, one of the suppressors identified was eif4e. However, consistent with in vitro experiments, which have shown that Sxl dependent repression of msl-2 mRNA translation is cap independent, this study found that eif4e does not function in Sxl mediated translational repression of at least one target mRNA in vivo. Instead, the results indicate that eif4e is needed for Sxl dependent alternative splicing, and it is argued that it is this splicing activity that accounts for the suppression of male lethality by eif4e mutations. In wild type females, Sxl protein blocks the splicing of a small intron in the 5' UTR of the msl-2 pre-mRNA. This is an important step in msl-2 regulation because the intron contains two Sxl binding sites that are needed by Sxl to efficiently repress translation of the processed msl-2 mRNA. When this intron is removed repression of msl-2 translation by Sxl is incomplete and this would enable eif4e/+ males to escape the lethal effects of the Sx-N transgene (Graham, 2011).

Several lines of evidence support the conclusion that eif4e is required for Sxl dependent alternative splicing. One comes from the analysis of the dominant maternal effect female lethal interactions between eif4e and Sxl. The initial activation of the Sxl positive autoregulatory feedback loop in early embryos can be compromised by a reduction in the activity of splicing factors like Snf, Fl(2)d, and U1-70K, and mutations in genes encoding these proteins often show dose sensitive maternal effect, female lethal interactions with Sxl. Like these splicing factors, maternal effect female lethal interactions with Sxl are observed for several eif4e alleles. Moreover, these female lethal interactions can be exacerbated when the mothers are trans-heterozygous for mutations in eif4e and the splicing factors snf or fl(2)d. Genetic and molecular experiments indicate that female lethality is due to a failure in the female specific splicing of Sxl-Pm mRNAs. First, female lethality can be rescued by gain-of-function Sxl mutations that are constitutively spliced in the female mode. Second, transcripts expressed from a Sxl-Pm splicing reporter in the female Sxl^-/+ progeny of eif4e/+ mothers are inappropriately spliced in a male pattern at the time when the Sxl positive autoregulatory loop is being activated by the Sxl-Pe proteins. While splicing defects are evident in these embryos at the blastoderm/early gastrula stage, obvious abnormalities in expression of Sxl protein are not observed until several hours later in development (Graham, 2011).

Though this difference in timing would favor the idea that eif4e is required for splicing of Sxl-Pm transcripts rather than for the export or translation of the processed Sxl-Pm mRNAs, the possibility cannot be excluded that there are subtle defects in the expression of Sxl protein at the blastoderm/early gastrula stage that are sufficient to disrupt splicing regulation during the critical activation phase yet aren't detectable in the antibody staining experiments. However, evidence from two different experimental paradigms using adult females indicates that this is likely not the case. In the first, it was found that reducing eif4e activity in a sensitized snf¹⁶²¹ Sxl^f1/++ background can compromise Sxl dependent alternative splicing even though there is no apparent reduction in Sxl protein accumulation. In this experiment advantage was taken of the fact that once the positive autoregulatory feedback loop is fully activated a homeostasis mechanism (in which Sxl negatively regulates the translation of Sxl-Pm mRNAs) ensures that Sxl protein is maintained at the same level even if there are fluctuations in the amount of female spliced mRNA. While only a small amount of male spliced Sxl-Pm mRNAs can be detected in snf¹⁶²¹ Sxl^f1/++ females, the level increases substantially when eif4e activity is reduced. Since these synergistic effects occur even though Sxl levels in the triply heterozygous mutant females are the same as in the control snf¹⁶²¹ Sxl^f1/++ females, it is concluded that the disruption in Sxl dependent alternative splicing of Sxl-Pm transcripts in this context (and presumably also in early embryos) can not be due to a requirement for eif4e in either the export of Sxl mRNAs or in their translation. Instead, eif4e activity must be needed specifically for Sxl dependent alternative splicing of Sxl-Pm pre-mRNAs. Consistent with a more general role in Sxl dependent alternative splicing, there is a substantial increase in msl-2 mRNAs lacking the first intron when eif4e activity is reduced in snf¹⁶²¹ Sxl^f1/++ females. In the second experiment the splicing was examined of pre-mRNAs from the endogenous Sxl gene and from a Sxl splicing reporter in females heterozygous for two hypomorphic eif4e alleles. Male spliced mRNAs from the endogenous gene and from the splicing reporter are detected the eif4e/+ females, but not in wild type females. Moreover, the effects on sex-specific alternative splicing seem to be specific for transcripts regulated by Sxl as no male spliced dsx mRNAs were seen in eif4e/+ females (Graham, 2011).

Two models could potentially explain why eif4e is needed for Sxl dependent alternative splicing. In the first, eif4e would be required for the translation of some critical and limiting splicing co-factor. When eif4e activity is reduced, insufficient quantities of this splicing factor would be produced and this, in turn, would compromise the fidelity of Sxl dependent alternative splicing. In the second, the critical splicing co-factor would be eif4e itself. It is not possible to conclusively test whether there is a dose sensitive requirement for eif4e in the synthesis of a limiting splicing co-factor. Besides the fact that the reduction in the level of this co-factor in flies heterozygous for hypomorphic eif4e alleles is likely to be rather small, only a subset of the Sxl co-factors have as yet been identified. For these reasons, the first model must remain a viable, but unlikely possibility. As for the second model, the involvement of a translation factor like eif4e in alternative splicing is unexpected if not unprecedented. For this to be a viable model, a direct role for eif4e must be consistent with what is known about the dynamics of Sxl pre-mRNA splicing and the functioning of the Sxl protein. The evidence that the second model is plausible is detailed below (Graham, 2011).

Critical to the second model is both the nuclear localization of eIF4E and an association with incompletely spliced Sxl pre-mRNAs. Nuclear eIF4E has been observed in other systems, and this was confirmed for Drosophila embryos. It was also found that eIF4E is bound to Sxl transcripts in which the regulated exon2-exon3-exon4 cassette has not yet been spliced. In contrast, it is not associated with incompletely processed transcripts from the tango gene that are constitutively spliced. With the caveat that only one negative control is available, it is not surprising that Sxl transcripts might be unusual in this respect. There is growing body of evidence that splicing of constitutively spliced introns is co-transcriptional. However, recent in vivo imaging experiments have shown that the splicing of the regulated Sxl exon2-exon3-exon4 cassette is delayed until after the Sxl transcript is released from the gene locus in female, but not in male cells. These in vivo imaging studies also show that, like bulk pre-mRNAs, the 1^st Sxl intron is spliced co-transcriptionally in both sexes. Consistent with a delay in the splicing of the regulated cassette, it has been previously reported that polyadenylated Sxl RNAs containing introns 2 and 3 can be readily detected by RNase protection, whereas other Sxl intron sequences are not observed. The delay in the splicing of the regulated Sxl cassette until after transcription is complete and the RNA polyadenylated could provide a window for exchanging eIF4E for the nuclear cap binding protein (Graham, 2011).

To function as an Sxl co-factor, eIF4E would have to be associated with the pre-mRNA-spliceosomal complex before or at the time of the Sxl dependent regulatory step. There is still a controversy as to exactly which step in the splicing pathway Sxl exerts its regulatory effects on Sxl-Pm pre-mRNAs and two very different scenarios have been suggested. The first is based on an in vitro analysis of Sxl-Pm splicing using a small hybrid substrate consisting of an Adenovirus 5' exon-intron fused to a short Sxl-Pm sequence spanning the male exon 3' splice site. These in vitro studies suggest that Sxl acts very late in the splicing pathway after the 1^st catalytic step, which is the formation of the lariat intermediate in the intron between exon 2 and the male exon. According to these experiments Sxl blocks the 2^nd catalytic step, the joining of the free exon 2 5' splice site (or Adeno 5' splice site) to the male exon 3' splice site. It is postulated that this forces the splicing machinery to skip the male exon altogether and instead join the free 5' splice site of exon 2 to the downstream 3' splice site of exon 4. Since this study has shown that eIF4E binds to Sxl-Pm pre-mRNAs that have not yet undergone the 1^st catalytic step, it would be in place to influence the splicing reaction if this scenario were correct (Graham, 2011).

The second scenario is more demanding in that it proposes that Sxl acts during the initial assembly of the spliceosome. Evidence for Sxl regulation early in the pathway comes from the finding that Sxl and the Sxl co-factor Fl(2)d show physical and genetic interactions with spliceosomal proteins like U1-70K, Snf, U2AF38 and U2AF50 that are present in the early E and A complexes and are important for selecting the 5' and 3' splice sites. In addition to these proteins, Sxl can also be specifically cross-linked in nuclear extracts to the U1 and U2 snRNAs. Formation of the E complex depends upon interactions of the U1 snRNP with the 5' splice site, and this is thought to be one of the first steps in splicing. The other end of the intron is recognized by U2AF, which recruits the U2 snRNP to the 3' splice site. After the base pairing of the U2 snRNP with the branch-point to generate the A complex the next step is the addition of the U4/U5/U6 snRNPs to form the B complex. However, Sxl and Fl(2)d are not found associated with components of the splicing apparatus like U5-40K, U5-116K or SKIP that are specific for complexes B and B*, or the catalytic C complex. Nor can Sxl be cross-linked to the U4, U5 or U6 snRNAs. If Sxl and Fl(2)d dissociated from the spliceosome before U4/U5/U6 are incorporated into the B complex, then they must influence splice site selection during the formation/functioning of the E and/or A complex. (Since the transition from the E to the A complex has been shown to coincide with an irreversible commitment to a specific 5'—3' splice site pairing, Sxl would likely exerts its effects in the E complex when splice site pairing interactions are known to still be dynamic. If this is scenario is correct, eIF4E would have to be associated with factors present in the earlier complexes in order to be able to promote Sxl regulation. This is the case. Thus, eIF4E is found in complexes containing the U1 snRNP protein U1-70K, the U1/U2 snRNP protein Snf, and the two U2AF proteins, U2AF38 and U2AF50. With the exception of the Snf protein bound to the U2 snRNP, all of these eIF4 associated factors are present in the early E or A complexes, but are displaced from the spliceosome together with the U1 and U4 snRNPs when the B complex is rearranged to form the activated B* complex. This would imply that eIF4E is already in place either before or at the time of B complex assembly. Arguing that eIF4E associates with these E/A components prior to the assembly of the B complex is the finding that eIF4E is also in complexes with both Sxl and Fl(2)d. Thus, even in this more demanding scenario for Sxl dependent splicing, eIF4E would be present at a time when it could directly impact the regulatory activities of Sxl and its co-factor Fl(2)d (Graham, 2011).

Taken together these observations would be consistent with a Sxl co-factor model. While further studies will be required to explain how eIF4E helps promote female specific processing, an intriguing possibility is suggested by the fact that hastening the nuclear export of msl-2 in females would favor the female splice (which is no splicing at all). Hence, one idea is that eIF4E binding to the pre-mRNA provides a mechanism for preventing the Sxl regulated splice sites from re-entering the splicing pathway, perhaps by constituting a 'signal' that blocks the assembly of new E/A complexes. A similar post-transcriptional mechanism could apply to female-specific splicing of the regulated Sxl exon2-exon3-exon4 cassette. The binding of eIF4E (and PABP) to incompletely processed Sxl transcripts after transcription has terminated in females would prevent the re-assembly of E/A complexes on the two male exon splice sites, and thus promote the formation of an A complex linking splicing factors assembled on the 5' splice sites of exons 2 and on the 3' splice site of exon 4 (Graham, 2011).

EVOLUTIONARY HOMOLOGS

A complete set of seven U1-related sequences have been cloned and characterized from Drosophila melanogaster. These sequences are located at the three cytogenetic loci 21D, 82E, and 95C. Three of these sequences have been previously studied: one U1 gene at 21D that encodes the prototype U1 sequence (U1a), one U1 gene at 82E that encodes a U1 variant with a single nucleotide substitution (U1b), and a pseudogene at 82E. The four previously uncharacterized genes comprise another U1b gene at 82E, two additional U1a genes at 95C, and a U1 gene at 95C that encodes a new variant (U1c) with a distinct single nucleotide change relative to U1a. Three blocks of 5' flanking sequence similarity are common to all six full length genes. The U1b RNA is expressed in Drosophila Kc cells and is associated with snRNP proteins, suggesting that the U1b-containing snRNP particles are able to participate in the process of pre-mRNA splicing. The expression throughout Drosophila development of the two U1 variants has been observed relative to the prototype sequence. The U1c variant is undetectable, while the U1b variant exhibits a primarily embryonic pattern reminiscent of the expression of certain U1 variants in sea urchin, Xenopus, and mouse (Lo, 1990).

Both experimental work and surveys of the lengths of internal exons in nature have suggested that vertebrate internal exons require a minimum size of approximately 50 nucleotides for efficient inclusion in mature mRNA. This phenomenon has been ascribed to steric interference between complexes involved in recognition of the splicing signals at the two ends of short internal exons. To determine whether U1 small nuclear ribonucleoprotein, a multicomponent splicing factor that is involved in the first recognition of splice sites, contributes to the lower size limit of vertebrate internal exons, advantage was taken of the observation that U1 small nuclear RNAs (snRNAs), which bind upstream or downstream of the 5' splice site (5'SS) stimulate splicing of the upstream intron. By varying the position of U1 binding relative to the 3'SS, it is shown that U1-dependent splicing of the upstream intron becomes inefficient when U1 is positioned 48 nucleotides or less downstream of the 3'SS, suggesting a minimal distance between U1 and the 3'SS of approximately 50 nucleotides. This distance corresponds well to the suggested minimum size of internal exons. The results of experiments in which the 3'SS region of the reporter is duplicated suggest an optimal distance of greater than 72 nucleotides. Inclusion of a 24-nucleotide miniexon is promoted by the binding of U1 to the downstream intron but not by binding to the 5'SS (Hwang, 1997).

U1 snRNP of yeast

The formation of mRNAs in the nuclei of eukaryotic cells involves several co- and post-transcriptional processing events. These include 5' end capping, 3' end formation, usually by cleavage and polyadenylation, and frequently the removal of intervening sequences by splicing. Pre-mRNA splicing can be conceptually divided into distinct stages. The initial step is recognition of conserved intronic sequences near the 5' splice site and branchpoint region by a subset of splicing factors. This is followed by assembly of multiple additional splicing factors to form the spliceosome. Rearrangements within the spliceosome then occur, accompanying the two chemical steps of intron removal. Spliced mRNA is released for export to the cytoplasm while intronic RNA is degraded and splicing factors are recycled (Fortes, 1999).

The first defined step of splicing consists of the formation of commitment complexes in yeast and E complex in mammals. In yeast, two forms of commitment complex are experimentally separable, CC1 and CC2. It is likely, though not definitively proven, that CC1 is a precursor of CC2. Both contain U1 snRNP, which interacts with the 5' splice site. CC2 additionally contains at least two proteins, BBP and Mud2p, that bind to the branchpoint sequence and an adjacent pyrimidine-rich tract, respectively. mBBP/SF1 and U2AF65 (see Drosophila U2 small nuclear riboprotein auxiliary factor 50), the mammalian homologs of these proteins, are present in E complex (Fortes, 1999 and references therein).

These facts about early steps in spliceosome formation point to a critical role for U1 snRNP in 5' splice site definition and choice, and lead to the question of how the choice is made between two alternative 5' splice sites that can both be spliced to a common 3' splice site. Examination of alternative splicing in vertebrates suggests that factors that are not components of U1 snRNP can influence the selection of splice sites. Recent work in yeast has shown, however, that at least one U1 snRNP protein can also influence 5' splice site choice (Fortes, 1999 and references therein).

Yeast U1 snRNA is significantly larger than vertebrate U1 snRNA. The yeast-specific regions of the RNA are not absolutely essential for survival, but nevertheless they play a role in splicing. Yeast U1 snRNP, as biochemically purified, is considerably more complex than vertebrate U1 snRNP. Both contain the Sm core proteins and three U1-specific proteins: U1 70K/Snp1p, U1A/Mud1p, and U1C/yU1-C. In addition, the yeast U1 snRNP contains at least six specific proteins (Snu71p, Snu65p, Snu56p, Prp39p, Prp40p, and Nam8p) that have no currently characterized vertebrate homologs. U1 snRNP interacts with the 5' splice site via base-pairing through U1 snRNA. Recent data indicate that the yeast U1 snRNP proteins also make extensive contact with the pre-mRNA both upstream and downstream of the 5' splice site. These interactions are likely to increase the stability of U1 snRNP-5' splice site binding. In addition, at least one U1 snRNP protein-pre-mRNA interaction, involving Nam8p, is affected by the sequence of the pre-mRNA to which the protein binds. The sequence specificity of this interaction can affect 5' splice site choice (Fortes, 1999 and references therein).

Other signals on a pre-mRNA can also influence binding of U1 snRNP to a 5' splice site or other steps that affect the efficiency of intron recognition and removal. Examples include the effects of adjacent introns or 3' end formation signals, exon enhancer sequences, and, in the case of the cap-proximal intron, the cap structure. The effect of the cap structure is mediated by the nuclear cap-binding complex (CBC), a conserved heterodimeric complex composed of CBP80 and CBP20. In both yeast and mammals, CBC appears to act by increasing the efficiency of recognition of the cap-proximal 5' splice site by U1 snRNP during commitment complex/E complex assembly. Much of the initial evidence for this mechanism comes from biochemical experiments but in yeast a considerable body of genetic data indicates that CBC plays an important role in commitment complex assembly. The gene encoding yCBP20, MUD13, was identified by a mutation that causes synthetic lethality in combination with a nonlethal deletion of part of U1 snRNA. A more extensive search for genes whose mutation led to synthetic lethality in the absence of CBC led to the identification of LUC genes (lethal unless CBC is produced). The LUC collection includes genes that encode several components of the commitment complex, including both Mud2p/Luc2p and several protein components of yeast U1 snRNP. Some of these genes encode proteins conserved between yeast and vertebrates, like SmD3/Luc6p or Mud1p/Luc1p, the yeast homolog of the human U1A protein, and others encode several of the recently identified yeast-specific U1 snRNP proteins, Nam8p/Luc3p, Snu56p/Luc4p, and Snu71p/Luc5p (Fortes, 1999 and references therein).

One functionally uncharacterized gene identified in the screen was named LUC7. Luc7p is an additional component of the yeast U1 snRNP. LUC7 is an essential gene, and Luc7p is required for commitment complex formation in vitro. In the presence of a temperature-sensitive form of Luc7p, the protein composition of U1 snRNP is altered. Although the defective U1 snRNP still appears to be partially active in vivo, splicing efficiency is reduced and 5' splice site selection is altered. The change in 5' splice site recognition is similar to that seen in the absence of CBC, suggesting that CBC-U1 snRNP interaction is affected by the absence of Luc7p. The LUC7 gene was identified by a mutation that causes lethality in a yeast strain lacking the nuclear cap-binding complex (CBC). Luc7p is similar in sequence to metazoan proteins that have arginine-serine and arginine-glutamic acid repeat sequences characteristic of a family of splicing factors. Although the in vivo defect in splicing wild-type reporter introns in a luc7 mutant strain is comparatively mild, splicing of introns with nonconsensus 5' splice site or branchpoint sequences is more defective in the mutant strain than in wild-type strains. By use of reporters that have two competing 5' splice sites, a loss of efficient splicing to the cap proximal splice site is observed in luc7 cells, analogous to the defect seen in strains lacking CBC. CBC can be coprecipitated with U1 snRNP from wild-type yeast strains (but not from luc7). These data suggest that the loss of Luc7p disrupts U1 snRNP-CBC interaction, and that this interaction contributes to normal 5' splice site recognition (Fortes, 1999).

Examination of protein sequence databases has revealed the existence of metazoan relatives of Luc7p, including three in human and C. elegans and others in Arabidopsis, Drosophila, and other eukaryotes. The regions encoding the zinc finger motifs are particularly highly conserved (57% similarity conserved across the whole family). LUC7 appears to have been duplicated early in evolution, leading to the Luc7A and Luc7B subfamilies in higher eukaryotes. Interestingly, all metazoan LUC7 family members contain carboxy-terminal extensions with multiple arginine-serine or arginine-glutamate repeats, characteristic of a large number of metazoan splicing factors (Fortes, 1999).

Analysis of U1A, the A protein component of U1 snRNP

Many RNA-associated proteins contain a ribonucleoprotein (RNP) consensus octamer encompassed by a conserved 80 amino acid sequence, termed an RNA recognition motif (RRM). RRM family members contain either one (class I) or multiple (class II) copies of this motif. A class II component of the U1 small nuclear RNP (snRNP), the A protein of U1 snRNP (U1snRNP-A), contains two RRMs (RRM1 and -2), yet has only one binding domain (RRM1) that interacts specifically with stem-loop II of U1 RNA. Quantitative analysis of binding affinities of fragments of U1snRNP-A demonstrates that an 86-amino acid polypeptide is competent to bind to U1 RNA with an affinity comparable to that of the full-length protein (Kd approximately 80 nM). The carboxyl-terminal RRM2 of U1snRNP-A does not bind to U1 RNA and may recognize an unidentified heterologous RNA. It is proposed that class II proteins may function as bridges between RNA components of RNP complexes, such as the spliceosome (Lutz-Freyermuth, 1990).

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups (on the single-stranded nucleotides) and residues (on the beta-sheet surface) are potentially common to RNP proteins with diverse specificity, and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to the current understanding of protein-protein recognition than that of DNA-protein recognition (Allain, 1997).

By the use of hybrids between a U1 small nuclear ribonucleoprotein (snRNP: U1A) and a U2 snRNP (U2B"), regions have been identified containing 29 U1A-specific amino acid residues scattered throughout the 117 N-terminal residues of the protein, which are involved in binding to U1 RNA. The U1A-specific amino acid residues have been arbitrarily divided into seven contiguous groups. None of these groups is sufficient for U1 binding when transferred singly into the U2B" context, and none of the groups is essential for U1 binding in U1A. Several different combinations of two or more groups can, however, confer the ability to bind U1 RNA to U2B", suggesting that most or all of the U1A-specific amino acid residues contribute incrementally to the strength of the specific binding interaction. Further evidence for the importance of the U1A-specific amino acid residues, some of which lie outside the region previously shown to be sufficient for U1 RNA binding, is obtained by comparison of the sequence of human and Xenopus laevis U1A cDNAs. These are extremely similar (94.4% identical) between amino acid residues 7 and 114 but much less conserved immediately upstream and downstream from this region (Scherly, 1991).

U1 snRNP-A protein (U1A) interacts with elements in SV40 late polyadenylation signal. This association increases polyadenylation efficiency. It is postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. Direct binding occurs between U1A and the 160-kD subunit of cleavage-polyadenylation specificity factor (CPSF). U1A copurifies with CPSF to a point but can be separated in the highly purified fractions. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. The addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, poly(A) polymerase, and a precleaved RNA substrate results in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilizes the interaction of CPSF with the AAUAAA-containing substrate RNA. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation (Lutz, 1996).

The human U1A protein-U1A pre-mRNA complex and the relationship between its structure and function in inhibition of polyadenylation in vitro has been investigated. Two molecules of U1A protein bind to a conserved region in the 3' untranslated region of U1A pre-mRNA. The secondary structure of this region was determined by a combination of theoretical prediction, phylogenetic sequence alignment, enzymatic structure probing and molecular genetics. The U1A binding sites form (part of) a complex secondary structure that is significantly different from the binding site of U1A protein on U1 snRNA. Studies with mutant pre-mRNAs show that the integrity of much of this structure is required for both high affinity binding to U1A protein and specific inhibition of polyadenylation in vitro. In particular, binding of a single molecule of U1A protein to U1A pre-mRNA is not sufficient to produce efficient inhibition of polyadenylation (van Gelder, 1993).

The human U1 snRNP-specific U1A protein autoregulates its production by binding its own pre-mRNA and inhibiting polyadenylation. The mechanism of this regulation has been elucidated by in vitro studies. U1A protein prevents neither the binding of cleavage and polyadenylation specificity factor (CPSF) to its recognition sequence (AUUAAA) nor the cleavage of U1A pre-mRNA. Instead, U1A protein bound to U1A pre-mRNA inhibits both specific and nonspecific polyadenylation by mammalian, but not by yeast, poly(A) polymerase (PAP). Domains are identified in both proteins whose removal uncouples the polyadenylation activity of mammalian PAP from its inhibition via RNA-bound U1A protein. U1A protein specifically interacts with mammalian PAP in vitro. This interaction may possibly reflect a broader role of the U1A protein in polyadenylation (Gunderson, 1994).

The inactivity of the 5' long terminal repeat (LTR) poly(A) site, immediately downstream of the cap site maximizes the production of HIV-1 transcripts. This inactivity has been found to be mediated by the interaction of the U1 snRNP with the major splice donor site (MSD). The inhibition of the HIV-1 poly(A) site by U1 snRNP relies on a series of delicately balanced RNA processing signals. These include the poly(A) site, the major splice donor site and the splice acceptor sites. The inherent efficiency of the HIV-1 poly(A) site allows maximal activity where there is no donor site (in the 3' LTR) but full inhibition by the downstream MSD (in the 5' LTR). The MSD must interact efficiently with U1 snRNP to completely inhibit the 5' LTR poly(A) site, whereas the splice acceptor sites are inefficient, allowing full-length genomic RNA production (Ashe, 1997).

The inhibition of poly(A) polymerase (PAP) by the U1 snRNP-specific U1A protein (a reaction whose function is to autoregulate U1A protein production) requires a substrate RNA to which at least two molecules of U1A protein can bind tightly, but the secondary structure of the RNA is not highly constrained. A mutational analysis reveals that the carboxy-terminal 20 amino acids of PAP are essential for its inhibition by the U1A-RNA complex. Remarkably, transfer of these amino acids to yeast PAP, which is otherwise not affected by U1A protein, is sufficient to confer U1A-mediated inhibition onto the yeast enzyme. A glutathione S-transferase fusion protein containing only these 20 PAP residues can interact in vitro with an RNA-U1A protein complex containing two U1A molecules, but not with one containing a single U1A protein. This, explains the requirement for two U1A-binding sites on the autoregulatory RNA element. A mutational analysis of the U1A protein demonstrates that amino acids 103-119 are required for PAP inhibition. A monomeric synthetic peptide consisting of the conserved U1A amino acids from this region has no detectable effect on PAP activity. However, the same U1A peptide, when conjugated to BSA, inhibits vertebrate PAP. In addition to this activity, the U1A peptide-BSA conjugate specifically uncouples splicing and 3'-end formation in vitro without affecting uncoupled splicing or 3'-end cleavage efficiencies. This suggests that the carboxy-terminal region of PAP with which it interacts is involved not only in U1A autoregulation but also in the coupling of splicing and 3'-end formation (Gunderson, 1997).

Human, mouse, and Xenopus mRNAs encoding the U1 snRNP-specific U1A protein contain a conserved 47 nt region in their 3' untranslated regions (UTRs). In vitro studies show that human U1A protein binds to two sites within the conserved region that resemble, in part, the previously characterized U1A-binding site on U1 snRNA. Overexpression of human U1A protein in mouse cells results in down-regulation of endogenous mouse U1A mRNA accumulation. In vitro and in vivo experiments demonstrate that excess U1A protein specifically inhibits polyadenylation of pre-mRNAs containing the conserved 3' UTR from human U1A mRNA. Thus, U1A protein regulates the production of its own mRNA via a mechanism that involves pre-mRNA binding and inhibition of polyadenylation (Boelens, 1993).

An in vitro genetic system was developed as a rapid means for studying the specificity determinants of RNA-binding proteins. This system was used to investigate the origin of the RNA-binding specificity of the mammalian spliceosomal protein U1A. The U1A domain responsible for binding to U1 small nuclear RNA was locally mutagenized and displayed as a combinatorial library on filamentous bacteriophage. Affinity selection identified four U1A residues in the mutagenized region that are important for specific binding to U1 hairpin II. One of these residues (Leu-49) disproportionately affects the rates of binding and release and appears to play a critical role in locking the protein onto the RNA. Interestingly, a protein variant that binds more tightly than U1A emerged during the selection, showing that the affinity of U1A for U1 RNA has not been optimized during evolution (Laird-Offringa, 1995).

Nuclear transport of the U1 snRNP-specific protein U1A has been examined. U1A moves to the nucleus by an active process that is independent of interaction with U1 snRNA. Nuclear localization requires an unusually large sequence element situated between amino acids 94 and 204 of the protein. U1A transport is not unidirectional. The protein shuttles between nucleus and cytoplasm. At equilibrium, the concentration of the protein in the nucleus and cytoplasm is not, however, determined solely by transport rates, but can be perturbed by introducing RNA sequences that can specifically bind U1A in either the nuclear or cytoplasmic compartment. Thus, U1A represents a novel class of protein that shuttles between cytoplasm and nucleus and whose intracellular distribution can be altered by the number of free binding sites for the protein present in the cytoplasm or the nucleus (Kambach, 1992).

Macromolecules that are imported into the nucleus can be divided into classes according to their nuclear import signals. The best characterized class consists of proteins that carry a basic nuclear localization signal (NLS), whose transport requires the importin alpha/beta heterodimer. U snRNP import depends on both the trimethylguanosine cap of the snRNA and a signal formed when the Sm core proteins bind the RNA. Here, factor requirements for U snRNP nuclear import are studied using an in vitro system. Depletion of importin alpha, the importin subunit that binds the NLS, is found to stimulate rather than inhibit U snRNP import. This stimulation is due to a common requirement for importin beta in both U snRNP and NLS protein import. Saturation of importin beta-mediated transport with the importin beta-binding domain of importin alpha blocks the import of U snRNP both in vitro and in vivo. Immunodepletion of importin beta inhibits protein import, whether NLS-mediated or U snRNP. While the former requires re-addition of both importin alpha and importin beta, re-addition of importin beta alone to immunodepleted extracts is sufficient to restore efficient U snRNP import. Thus importin beta is required for U snRNP import, and it functions in this process without the NLS-specific importin alpha (Palacios, 1997).

Precursors of U1 snRNA are associated with nuclear proteins prior to export to the cytoplasm. The approximately 15S complexes containing pre-U1 RNA, termed pre-export U1 snRNPs, can be identified in extracts of Xenopus laevis oocyte nuclei that are synthesizing U1 RNAs from injected U1 genes. The U1 snRNP-specific A protein is associated with nuclear pre-U1 RNA. The interaction of the U1-A protein with pre-U1 RNA required sequences in the loop II region although this region of U1 RNA was not necessary for the association of U1 A protein with mature U1 snRNPs. The U1 A protein helps protect pre-U1 RNA against degradation in the nucleus (Terns, 1993).

In an enhancer screen for yeast mutants that may interact with U1 small nuclear RNA (snRNA), a gene was identified that encodes the apparent yeast homolog of the well-studied human U1A protein. Both in vitro and in vivo, the absence of the protein has a dramatic effect on the activity of U1 snRNP containing the mutant U1 snRNA used in the screen. Surprisingly, the U1A gene is inessential in a wild-type U1 RNA background, as growth rate and the splicing of endogenous pre-mRNA transcripts are normal in these strains that lack the U1A protein. Even in vitro, the absence of the protein has little effect on splicing. On the basis of these observations, it is suggested that a principal role of the U1A protein is to help fold or maintain U1 RNA in an active configuration (Liao, 1993).

The interaction of the U1-specific proteins 70k, A and C with U1 snRNP was studied by gradually depleting U1 snRNPs of the U1-specific proteins. U1 snRNP species are obtained that are selectively depleted of either protein C, A, C and A, or of all three U1-specific proteins (C, A and 70k) while retaining the common proteins B' to G. These various types of U1 snRNP particles were used to study the differential accessibility of defined regions of U1 RNA towards nucleases V1 and S1 dependent on the U1 snRNP protein composition. U1 snRNP protein 70k interacts with stem/loop A of U1 RNA, and protein A interacts with stem/loop B of U1 RNA . The presence or absence of protein C does not affect the nuclease digestion patterns of U1 RNA. These results suggest further that the binding of protein A to the U1 snRNP particle should be independent of proteins 70k and C. Mouse cells contain two U1 RNA species, U1a and U1b, which differ in the structure of stem/loop B: U1a exhibits the same stem/loop B sequence as U1 RNA from HeLa cells. Protein A is always preferentially lost from U1b snRNP as compared to U1a snRNPs. This indicates that one consequence of the structural difference between U1a and U1b is a lowering of the binding strength of protein A to U1b snRNP. The possible functional significance of this finding is discussed with respect to the fact that U1b RNA is preferentially expressed in embryonal cells (Bach, 1990).

U1 small nuclear ribonucleoprotein (snRNP) may function during several steps of spliceosome assembly. However, most spliceosome assembly assays fail to detect the U1 snRNP. A new native gel electrophoretic assay was used to find the yeast U1 snRNP in three pre-splicing complexes (delta, beta1, alpha2) formed in vitro. The order of complex formation is deduced to be delta --> beta1 --> alpha2 --> alpha1 --> beta2, the active spliceosome. The delta complex is formed when U1 snRNP binds to pre-mRNA in the absence of ATP. There are two forms of delta: a major one, deltaun (unstable to competitor RNA), and a minor one, deltacommit (committed to the splicing pathway). The other complexes are formed in the presence of ATP and contain the following snRNPs: beta1 (the pre-spliceosome) has both U1 and U2; alpha2 has all five, however U1 is reduced compared with the others; and alpha1 and beta2 have U2, U5, and U6. Prior work by others suggests that U1 is "handing off" the 5' splice site region to the U5 and U6 snRNPs before splicing begins. The reduced levels of U1 snRNP in the alpha2 complex suggests that the handoff occurs during formation of this complex (Ruby, 1997).

Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the protein complex mediating pre-mRNA splicing. Association of U1 snRNP with the pre-mRNA is required for these early steps. The yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro and in vivo, Nam8p becomes indispensable for efficient 5' splice site recognition when this process is impaired as a result of the presence of noncanonical 5' splice sites or the absence of a cap structure. Nam8p stabilizes commitment complexes in the latter conditions. Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5' splice site, in a region of nonconserved sequence. Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p is involved in a novel mechanism by which an snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence. This supports a model where early 5' splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA (Puig, 1999).

Epitopes depending on three-dimensional folding of proteins have during recent years been acknowledged to be main targets for many autoantibodies. However, a detailed resolution of conformation-dependent epitopes has, to date, not been achieved in spite of its importance for understanding the complex interaction between an autoantigen and the immune system. In analysis of immunodominant epitopes of the U1-70K protein, the major autoantigen recognized by human ribonucleoprotein (RNP)-positive sera, diversely mutated recombinant Drosophila 70K proteins were used as antigens in assays for human anti-RNP antibodies. Thus, the contribution of individual amino acids to antigenicity could be assayed with the overall structure of the major antigenic domain preserved, and analysis of how antigenicity can be reconstituted rather than obliterated was enabled. Amino acid residue 125 is shown to be situated at a crucial position for recognition by human anti-RNP autoantibodies. Flanking residues at positions 119-126 also appear to be of utmost importance for recognition. These results are discussed in relation to structural models of RNA-binding domains. Tertiary structure modeling indicates that the residues 119-126 are situated at easily accessible positions in the end of an alpha-helix in the RNA binding region. This study identifies a major conformation-dependent epitope of the U1-70K protein and demonstrates the significance of individual amino acids in conformational epitopes. Using this model, it will be possible to analyze other immunodominant regions in which protein conformation has a strong impact (Welin Henriksson, 1999).

Attempts have been made to relate the conformational epitope to a three-dimensional position on the U1-70K protein. Using the closely related human hnRNP A1 and Drosophila Sex lethal proteins for modeling the three-dimensional structure of the U1-70K protein, one can observe that the region around residues 119-126 is situated in the end of alpha-helix 1. These residues face away from the residues in the RNA-binding beta1 and beta3 sheets, and, by using this model, the amino acid residue at position 125 should be an easily accessible B-cell epitope. Not only is the amino acid residue sequence important: in addition, the epitope has to be kept in place by the alpha-helix and beta-sheet. By now, having demonstrated that valine-125 is part of a major human epitope, it is not surprising that a switch to phenylalanine induces a disarrangement of the epitope surface. Both amino acids involved are hydrophobic and contain nonpolar side chains, but phenylalanine is larger and contains a bulky aromatic side chain. Valine, however, is one of the smallest amino acids containing a less complex carbon side chain. Thus, the tertiary structure derivation supports the theory of the region 119-126 as an autoantigenic, conformational epitope of the U1-70K protein. The positions of the key amino acids that constitute this major epitope also make it possible to understand why previous approaches have failed to identify smaller fragments than 56-67 amino acid residues as being antigenic. A truncation from either end would affect protein conformation and would cause the compressed helix-loop-beta-sheet structure to unfold, thus obscuring the originally prominent and crucial valine at position 125. These findings might have clinical implications because all tested sera recognized the identified epitope. This demonstrates a remarkable homogeneity of the otherwise heterogeneous autoantigenic B-cell response and might also instigate therapeutic approaches of inducing tolerance (Welin Henriksson, 1999).

Analysis of U1, the RNA component of U1 snRNP

Evidence for at least four U1 RNA variants of the snRNP has been obtained from a U1 cDNA library using U1 snRNA from Bombyx mori BmN cells in culture. Sequence analysis of thirty cDNA clones showed that: (1) the nucleotide changes are in the hairpin structures I, II and III; (2) the majority of the base changes in stem structures between a posterior silk gland (PSG) U1 RNA and the BmN U1 clones, as well as among the BmN U1 clones, are compensatory; (3) although the base differences between PSG U1 and BmN U1 clones, and among the BmN U1 clones, are not the same, they are located in similar positions in moderately conserved sites, frequently at the bases of loops; (4) when comparing the PSG U1 with the BmN U1 clones, twelve out of nineteen stem differences generate stronger pairing resulting in a more stable hairpin II in the BmN U1 clones; and (5) the Sm and 70K proteins binding site sequences are highly conserved among these U1 clones. Although a comparison of sequences changes associated with U1 isoforms from different species indicates that there are no common base changes with the B. mori U1 clones reported here, similarities in the multitude and location of base differences in hairpins I, II and III are observed in mouse and/or Xenopus. It is possible that U1 variants like the ones reported here play a role in alternative pre-mRNA splicing by way of different RNA-protein factor interactions (Gao, 1995).

To dissect U1 snRNA function, 14 single point mutations were analyzed in the six nucleotides complementary to the 5' splice site for their effects on growth and splicing in the fission yeast S. pombe. Three of the four alleles previously found to support growth of S. cerevisiae are lethal in S. pombe, implying a more critical role for the 5' end of U1 in fission yeast. Furthermore, a comparison of phenotypes for individual nucleotide substitutions suggests that the two yeasts use different strategies to modulate the extent of pairing between U1 and the 5' splice site. The importance of U1 function in S. pombe is further underscored by the lethality of several single point mutants not examined previously in S. cerevisiae. In total, only three alleles complement the U1 gene disruption, and these strains are temperature-sensitive for growth. Each viable mutant was tested for impaired splicing of three different S. pombe introns. Among these, only the second intron of the cdc2 gene (cdc2-I2) showed dramatic accumulation of linear precursor. Notably, cdc2-I2 is spliced inefficiently even in cells containing wild-type U1, at least in part due to the presence of a stable hairpin encompassing its 5' splice site. Although point mutations at the 5' end of U1 have no discernible affect on splicing of pre-U6, significant accumulation of unspliced RNA is observed in a metabolic depletion experiment. Taken together, these observations indicate that the repertoire of U1 activities is used to varying extents for splicing of different pre-mRNAs in fission yeast (Alvarez, 1996).

In the flagellated protozoon Euglena gracilis, characterized nuclear genes harbor atypical introns that usually are flanked by short repeats, adopt complex secondary structures in pre-mRNA, and do not obey the GT-AG rule of conventional cis-spliced introns. In the nuclear fibrillarin gene of E. gracilis, three spliceosomal-type introns have been identified that have GT-AG consensus borders. A small RNA has been isolated from E. gracilis and on the basis of primary and secondary structure comparisons, it is proposed to be a homolog of U1 small nuclear RNA, an essential component of the cis-spliceosome in higher eukaryotes. Conserved sequences at the 5' splice sites of the fibrillarin introns can potentially base pair with Euglena U1 small nuclear RNA. These observations demonstrate that spliceosomal GT-AG cis-splicing occurs in Euglena, in addition to the nonconventional cis-splicing and spliced leader trans-splicing previously recognized in this early diverging unicellular eukaryote (Breckenridge, 1999).

U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation

In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. This study used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. It was further shown that U1 snRNA-pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, a function that is proposed explains its overabundance (Kaida, 2010).

U1 snRNP bound to 5' splice sites may thus serve a dual purpose-in splicing and suppression of premature cleavage and polyadenylation. The perimeter of U1 snRNP’s protective zone is not known, but its binding to 5' splice site alone is unlikely to be able to protect the majority of introns, which in humans average ~3.4 kb in length. Furthermore, if suppression of actionable PASs was provided only via U1 snRNP bound to 5' splice sites, 5' splice-site mutations would be expected to cause premature termination, as opposed, for example, to exon skipping, which would be extremely deleterious and has not been observed. Additional U1 snRNP binding sites, including cryptic 5' splice sites, may function as tethering sites for its activity in suppression of cleavage and polyadenylation in introns. Viewed from this perspective, sequences referred to as cryptic 5' splice sites may serve a non-splicing purpose to recruit U1 snRNP to protect introns. It is also reasonable to consider that modulating U1 snRNP levels or its binding at sites that protect actionable PASs could be a mechanism for regulating gene expression, including downregulation of the mRNA or switching expression to a different mRNA produced from a prematurely terminated pre-mRNA. It is suggested that the vulnerability to premature cleavage and polyadenylation would be expected to increase with increasing intron size if U1 snRNP and cognate base-pairing sites are not available to protect it. It is proposed that the large excess of U1 snRNP over what is required for splicing in human cells serves an additional critical biological function, to suppress premature cleavage and polyadenylation in introns and protect the integrity of the transcriptome (Kaida, 2010).

Widespread recognition of 5' splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides

An established paradigm in pre-mRNA splicing is the recognition of the 5' splice site (5'ss) by canonical base-pairing to the 5' end of U1 small nuclear RNA (snRNA). A small subset of 5'ss base-pair to U1 in an alternate register that is shifted by 1 nucleotide. Using genetic suppression experiments in human cells, it was demonstrated that many other 5'ss are recognized via noncanonical base-pairing registers involving bulged nucleotides on either the 5'ss or U1 RNA strand, which were termed 'bulge registers.' By combining experimental evidence with transcriptome-wide free-energy calculations of 5'ss/U1 base-pairing, it is estimated that 10,248 5'ss (~5% of human 5'ss) in 6577 genes use bulge registers. Several of these 5'ss occur in genes with mutations causing genetic diseases and are often associated with alternative splicing. These results call for a redefinition of an essential element for gene expression that incorporates these registers, with important implications for the molecular classification of splicing mutations and for alternative splicing (Roca, 2012).

Splicing of >99% of pre-mRNA introns is catalyzed by the major spliceosome, a dynamic macromolecular machine composed of five small nuclear RNAs (snRNAs) and associated polypeptides, plus many other protein factors. The U1 small nuclear ribonucleoprotein particle (snRNP), comprising the U1 snRNA and 10 polypeptides, is the main component for early 5' splice site (5'ss) recognition by the major or U2-type spliceosome. The vast majority of such introns (>99%) belong to the GT-AG (or GU-AG) category, as defined by their intronic terminal dinucleotides. For more than 30 years, it has been firmly established that 5'ss are recognized by base-pairing to the 5' end of U1 snRNA in a canonical register, defined as +1G at the 5'ss (the first intronic nucleotide) base-pairing to C8 of U1 (the eighth nucleotide of U1). Thus, the 5'ss element spans the last 3 nucleotides (nt) of the exon and the first 8 nt of the intron, establishing a maximum of 11 base pairs (bp) to U1—although the contribution of the seventh and eighth nucleotides in the intron, which are much more variable, appears to depend on the species. Later in spliceosome assembly, U1 is replaced by U6 snRNA, which forms a few base pairs to the 5'ss and is likely involved in catalysis. In a handful of documented cases, U1 base-pairs at some distance from the 5'ss, and the cleavage site depends on subsequent U6 base-pairing. There is also an example of a natural human U2-type intron whose splicing appears to be U1 snRNA-independent (Roca, 2012 and references therein).

Two minor categories of U2-type splice sites have been known for a long time: GC-AG 5'ss (0.9%) and very rare AT-AC 5'ss (only 15 introns in the human genome). These 5'ss conform to consensus motifs very similar to the major U2-type GT-AG 5'ss and are recognized by analogous mechanisms. It has recently been shown that restoration of base-pairing to both U1 and U6 is essential to rescue recognition of a mutant AT 5'ss that causes aberrant splicing and myotonia. U12-type introns are spliced by the minor spliceosome and are very rare as well (0.36%) (Roca, 2012 and references therein).

It has recently been shown that a small subset of GT-AG 5'ss, which are here termed atypical 5'ss, is recognized by a base-pairing register with U1 that is shifted by 1 nt (+1G base-pairs to U1 C9 instead of C8) without changing the actual exon-intron boundary or the sequence of the spliced mRNA. In budding yeast, mutational analysis led to the suggestion that the noncanonical HOP2 5'ss is recognized by a base-pairing register involving a bulged nucleotide. A bulge in a strand of RNA (or DNA) duplex is defined as a nucleotide (or more) that is not opposed by any nucleotide on the other strand. This study presents extensive experimental evidence for multiple base-pairing registers between human 5'ss and U1, with bulged nucleotides on either RNA strand, and estimates that ~5% of all 5'ss (present in ~40% of human genes) use one of these noncanonical registers (Roca, 2012).

Interaction of U1 snRNP with SR proteins: the role of SR proteins in splicing

SR proteins are required for the first step of spliceosome assembly: the interaction of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) with the 5' splice site of the pre-mRNA. Individual SR proteins have been shown to be distinctly capable of promoting the interaction of U1 snRNP with alternative 5' splice junctions. These results suggest that SR proteins direct 5' splice site selection by regulation of U1 snRNP assembly onto the pre-mRNA (Zahler, 1995).

Addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble (respectively) on RNAs containing only 5' and 3' splice sites. It is concluded that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting factors for this function in HeLa nuclear extracts. Specific, splice site-dependent RNA-protein interactions of SR proteins can be demonstrated in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. A discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which has been designated the Enhancer complex, contains U1 small nuclear RNP and is associated with different SR protein family members, depending on the sequence of the enhancer. It is propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex (Staknis, 1994).

SR proteins are essential splicing factors that also influence 5' splice site choice. Addition of excess mixed SR proteins to a HeLa in vitro splicing system stimulates utilization of a novel 5' splice site (site 125) within the intron of the standard adenovirus pre-mRNA substrate. When U1 snRNPs are debilitated by sequestering the 5' end of U1 snRNA with a 2'-O-methyl oligoribonucleotide, excess SR proteins not only rescue splicing at the normal site and site 125 but also activate yet another 5' splice site (site 47) in the adenovirus intron. One SR protein, SC35, is sufficient to exhibit the above activities. The possibility that excess SR proteins recruit residual unblocked U1 snRNPs to participate in 5' splice site recognition has been ruled out by psoralen cross-linking studies, which demonstrate that the 2'-O-methyl oligoribonucleotide effectively blocks 5' splice site/U1 interaction. Native gel analysis reveals a nearly normal splicing complex profile in the 2'-O-methyl oligoribonucleotide pretreated, SR protein-supplemented extract. These results indicate that SR proteins can replace some functions of the U1 snRNP but underscore the contribution of U1 to the fidelity of 5' splice site selection (Tarn, 1994).

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). The U1 snRNP in HeLa cell nuclear extract is perturbed by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of the 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for the 5' splice site selection. Accordingly, it was found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. These results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection (Tarn, 1995).

A class of pre-mRNAs has been identified that are spliced in HeLa extracts depleted for U1 snRNP (delta U1 extracts). Previously, pre-mRNAs were described that can be spliced in delta U1 extracts only when high concentrations of SR splicing factors are added. In contrast, the substrates characterized here are efficiently processed in delta U1 extracts without the addition of excess SR proteins. The members of this class comprise both a naturally occurring pre-mRNA, from the Drosophila fushi tarazu gene, and a chimera containing sequences from two different pre-mRNAs that individually are dependent upon either U1 snRNP or excess SR proteins. Several sequence elements account for the variations in dependence on U1 snRNP and SR proteins for splicing. In one pre-mRNA, a single element was identified adjacent to the branch site. In the other, two elements flanking the 5' splice site were found to be critical. This U1-independent splicing reaction may provide a mechanism for cells to control the extent of processing of different classes of pre-mRNAs in response to altered activities of SR proteins, and furthermore suggests that U1 snRNP-independent splicing may not be uncommon (Crispino, 1996).

Members of the SR family of proteins, can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. Purified U1 snRNP and ASF/SF2 form a ternary complex with pre-mRNA, which is dependent on a functional 5' splice site. Sequences in the pre-mRNA, domains in ASF/SF2 and components of the U1 snRNP particle are shown to be required for complex formation. Sequences at the 5' splice site are necessary and sufficient for complex formation. One functional RNA binding domain and the RS domain are both required for ASF/SF2 to participate in complex formation. The RNA binding domains were redundant in this assay, suggesting that either domain can interact with the pre-messenger RNA. There is no function for the U1-specific A protein in complex formation, whereas a function for U1-specific C protein is strongly suggested (Jamison, 1995).

Exactly how specific splice sites are recognized during the processing of complex precursor messenger RNAs is not clear. Small nuclear ribonucleoprotein particles (snRNPs) are involved, but are not sufficient by themselves to define splice sites. Now a human protein essential for splicing in vitro, called alternative splicing factor/splicing factor 2, is shown to cooperate with the U1 snRNP particle in binding pre-mRNA. This cooperation is probably achieved by specific interactions between the arginine/serine-rich domain of the splicing factor and a similar region in a U1 snRNP-specific protein (Kohtz, 1994).

ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins. These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins. The RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the U1 snRNP-specific 70-kD protein (70K) in vitro. An apparent interaction of the isolated RS domain with 70K is observed if contaminating RNA is not removed, suggesting a nonspecific bridging among the basic RS domain, RNA, and 70K. In vitro phosphorylation of recombinant ASF/SF2 significantly enhances binding to 70K and also eliminates the RS domain-RNA interaction. Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to U1 snRNP in an RS domain-dependent, phosphorylation-enhanced manner. Conditions are described that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro (Xiao, 1997).

The 70K subunit of U1 snRNP

Transient transfection of the U1 snRNP 70K protein into COS cells induces nuclear reorganization and redistribution of the splicing factor SC-35; hnRNP proteins are not affected. Correspondingly, splicing and nucleocytoplasmic transport of a coexpressed mRNA substrate is reduced by overexpression of U1-70K. The carboxy-terminal portion of U1-70K-encompassing repeats of Arg/Ser, Arg/Glu, and Arg/Asp localizes to the nucleus independently of U1 RNA and is responsible for these inhibitory effects. This region of U1-70K contains amino acid residues similar to those found in splicing factors SC-35, U2AF, su(wa), and in other SR proteins, suggesting that U1-70K protein may serve as a focus of assembly for functional components of the splicing/transport machinery. These findings are compatible with models that propose that direct interaction between U1-70K and SR proteins plays a regulatory role in early events of spliceosome assembly (Romac, 1995).

A monoclonal antibody (mAb) to the U1 snRNP component U1 70K recognizes several proteins, in addition to U1 70K, in purified spliceosomal complexes and in total HeLa cell nuclear extract preparations. The novel mAb U1 70K antigens can also be specifically immunoprecipitated by the antibody. Similarly to U1 70K, many of the mAb U1 70K antigens can be phosphorylated by a co-purifying kinase activity. The epitope recognized by mAb U1 70K has been previously shown to be a repeating arginine/aspartate (RD) dipeptide. Thus the novel mAb U1 70K antigens have been designated as the RD family. Comparison of mAb U1 70K with a recently characterized antibody (mAb 16H3) whose epitope is a repeating R/D or R/E motif, shows that a large subset of the antigens are common. In contrast, most of the mAb U1 70K antigens are distinct from the proteins detected by mAb 104, an antibody to the SR family of splicing factors (Staknis, 1995).

The S. cerevisiae SNP1 gene encodes the U1 snRNP specific protein U1-70K. The RRM and glycine rich domains in U1-70K proteins are well conserved from yeast to metazoan, with over 80% amino acid similarity. Yeast strains in which the SNP1 gene is disrupted are viable, but exhibit greatly increased doubling rates and severe temperature sensitivities. In addition, snp1-null strains are defective in nuclear, pre-mRNA splicing. Deletion alleles of SNP1 have been tested for their ability to complement these phenotypes. The highly conserved RRM and glycine rich domains of Snp1 are not required for complementation of the snp1-null growth or splicing defects. However, the amino terminal domain of Snp1, which is not highly conserved, is necessary and sufficient for complementation (Hilleren, 1995).

Expression of the recombinant human U1-70K protein in COS cells results in its rapid transport to the nucleus, even when binding to U1 RNA is debilitated. Deletion analysis of the U1-70K protein reveals the existence of two segments of the protein that were independently capable of nuclear localization. One nuclear localization signal (NLS) was mapped within the U1 RNA-binding domain and consists of two typically separated but interdependent elements. The major element of this NLS resides in structural loop 5 between the beta 4 strand and the alpha 2 helix of the folded RNA recognition motif. The C-terminal half of the U1-70K protein that is capable of nuclear entry contains two arginine-rich regions, which suggests the existence of a second NLS. Site-directed mutagenesis of the RNA recognition motif associated NLS demonstrates that the U1-70K protein can be transported independently of U1 RNA and that its association with the U1 small nuclear ribonucleoprotein particle can occur in the nucleus (Romac, 1994).

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP have been studied. Both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrate that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lack the U1-70K RNP-80 motif and do not bind naked U1 RNA, associate stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain is specific for the U1 snRNP; stable binding is not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins is supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, are detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins (Nelissen, 1994).

Fas and the type I tumor necrosis factor receptor (TNF-R) are two cell surface receptors that trigger apoptotic cell death when stimulated with ligand or cross-linking antibody -- the mechanism involved has yet to be elucidated. The CrmA protein is a serpin family protease inhibitor than can inhibit interleukin-1 beta converting enzyme (ICE) and ICE-like proteases (See Drosophila Caspase-1). Expression of CrmA potently blocks apoptosis induced by activation of either Fas or TNF-R, implicating protease involvement in these death pathways. The 70-kDa component of the U1 small ribonucleoprotein (U1-70 kDa) is a proteolytic substrate rapidly cleaved during both Fas- and TNF-R-induced apoptosis. This cleavage is inhibited by expression of CrmA, but not by expression of an inactive point mutant of CrmA, confirming the involvement of an ICE-like protease. These data for the first time identify U1-70 kDa as a death substrate cleaved during Fas- and TNF-R-induced apoptosis and emphasize the importance of protease activation in the cell death pathway (Tewari, 1995).

Analysis of protein C, the C component of U1 snRNP

The U1 small nuclear ribonucleoprotein (snRNP) contains three specific proteins denoted 70K, A and C, in addition to the common proteins. Protein C is involved in the binding of U1 snRNP to the 5' splice site of a pre-mRNA. Unlike proteins A and 70K, U1-C lacks an RNA binding domain (RNP-80 motif) and does not appear to bind directly to U1 snRNA. At the amino terminal end, however, protein C contains a zinc finger-like structure of the CC-HH type found in transcription factor TF IIIA. Several lines of evidence indicate that the zinc finger-like structure is essential for the binding of protein C to U1 snRNP particles: (1) deletion analysis of protein C shows that the N-terminal 45 amino acids are sufficient for binding to U1 snRNPs; (2) there is modification of the cysteine residues in the N-terminal domain with N-ethylmaleimide and (3) single point mutations of the cysteines and histidines contributing to the putative zinc finger abolish the binding of protein C to U1 snRNPs. Interestingly, unlike the proteins U1-A and U1-70K the U1-C protein is unable to bind to naked U1 snRNA. It is shown however, that protein C does not bind to the known protein constituents of the U1 particle without the U1 snRNA being present. These data indicate that the binding of protein C to U1 snRNP is dependent on the presence of both the U1 snRNA and one or more of the U1 snRNP proteins (Nelissen, 1991).

The U1 snRNP-specific protein C contains an N-terminal zinc finger-like CH motif that is required for the binding of the U1C protein to the U1 snRNP particle. Recently a similar motif was reported to be essential for in vivo homodimerization of the yeast splicing factor PRP9. In the present study it is demonstrated that the human U1C protein is able to form homodimers as well. U1C homodimers are found in three cases: when the human U1C protein is expressed in Escherichia coli; when immunoprecipitations with anti-U1C antibodies are performed on in vitro translated U1C, and when the yeast two hybrid system is used. Analyses of mutant U1C proteins in an in vitro dimerization assay and the yeast two hybrid system reveal that amino acids within the CH motif, i.e. between positions 22 and 30, are required for homodimerization (Gunnewiek, 1995).

The nuclear localization signals of two of the three U1 snRNP-specific proteins, U1-70K and U1A, have been mapped. Both proteins are transported actively to the nucleus. The third U1 snRNP-specific protein, U1C, passively enters the nucleus. In both X. laevis oocytes and cultured HeLa cells, mutant U1C proteins that are not able to bind to the U1 snRNP do not accumulate in the nucleus, indicating that nuclear accumulation of U1C is due to incorporation of the protein into the U1 snRNP (Klein Gunnewiek, 1997).

To study the intranuclear localization of the U1-specific snRNP C protein and its assembly into U1 snRNPs, transcripts encoding a myc-tagged C protein were injected into amphibian oocytes. The distribution of protein translated from the injected RNA is essentially the same in continuous and pulse-label experiments. In both cases the C protein localizes within the germinal vesicle in those structures known to contain U1 snRNPs, namely the lampbrush chromosome loops and hundreds of extrachromosomal granules called snurposomes. Oocytes were also injected with an antisense oligodeoxynucleotide that causes truncation of U1 snRNA at the 5' end. In these oocytes, myc-tagged C protein localizes normally in the germinal vesicle and can be immunoprecipitated together with truncated U1 snRNA. These experiments suggest that the C protein can enter the germinal vesicle on its own, there to associate with previously assembled U1 snRNPs. In transfected tissue culture cells, the myc-tagged C protein localizes within the nucleus in a speckled pattern similar to that of endogenous U1 snRNPs (Jantsch, 1992).

The U1 small nuclear ribonucleoprotein particle (snRNP) has an important function in the early formation of the spliceosome, the multicomponent complex in which pre-mRNA splicing takes place. The nuclear localization signals of two of the three U1 snRNP-specific proteins, U1-70K and U1A, have been mapped. Both proteins are transported actively to the nucleus. The third U1 snRNP-specific protein, U1C, passively enters the nucleus. In both X. laevis oocytes and cultured HeLa cells, mutant U1C proteins that are not able to bind to the U1 snRNP do not accumulate in the nucleus, indicating that nuclear accumulation of U1C is due to incorporation of the protein into the U1 snRNP (Gunnewiek, 1997).

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals -- this engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 snRNP proteins also contribute to U1 snRNP activity. U1 snRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. Recombinant yeast U1C protein, a U1 snRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. It is proposed that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway (Du, 2002).

Interaction of u1snRNP with nuclear cap-binding complex

The mechanism by which intron-containing RNAs are recognized by the splicing machinery is as yet only partly understood. A nuclear cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts, has been shown to play a role in pre-mRNA splicing. CBC is required for efficient recognition of the 5' splice site by U1 snRNP during formation of E (early) complex on a pre-mRNA containing a single intron. However, in a pre-mRNA containing two introns, CBC is not required for splicing of the cap distal intron. In this case, the presence of an intact polypyrimidine tract in the cap-proximal intron renders splicing of the cap-distal intron independent of CBC. In summary, efficient recognition of the cap-proximal 5' splice site by U1 snRNP is facilitated by CBC in what may be one of the earliest steps in pre-mRNA recognition. This function of CBC is conserved in humans and yeast (Lewis, 1996).

U snRNP assembly in yeast involves the La protein

In all eukaryotic nuclei, the La autoantigen binds nascent RNA polymerase III transcripts, stabilizing these RNAs against exonucleases. The La protein also functions in the assembly of certain RNA polymerase II-transcribed RNAs into RNPs. A mutation in a core protein of the spliceosomal snRNPs, Smd1p, causes yeast cells to require the La protein Lhp1p for growth at low temperatures. Precursors to U1, U2, U4 and U5 RNAs are bound by Lhp1p in both wild-type and mutant cells. At the permissive temperature, smd1-1 cells contain higher levels of stable U1 and U5 snRNPs when Lhp1p is present. At low temperatures, Lhp1p becomes essential for the accumulation of U4/U6 snRNPs and for cell viability. When U4 RNA is added to extracts, the pre-U4 RNA, but not the mature RNA, is bound by Smd1p. These results suggest that, by stabilizing a 3'-extended form of U4 RNA, Lhp1p facilitates efficient Sm protein binding, thus assisting formation of the U4/U6 snRNP (Xue, 2000).

These results reveal that the role of the yeast La protein is not limited to the biogenesis of RNA polymerase III transcripts. Instead, Lhp1p plays a more general role in small RNA biogenesis. Consistent with the preference of La proteins for RNAs terminating in UUUOH, each of the pre-U RNAs ends in a run of uridylates. While the mechanism by which snRNA 3' ends are generated in S. cerevisiae is not fully understood, strains defective in the enzyme RNase III exhibit decreased levels of similar U1, U4 and U5 RNA precursors and reduced levels of mature U2 and U5L RNAs. Also, similar pre-U1, pre-U4 and pre-U5 RNAs accumulate in cells containing mutations in several 3' exonucleases. Thus, the pre-U RNAs bound by Lhp1p are most likely to be processing intermediates, generated by RNase III cleavage and subsequent exonuclease digestion (Xue, 2000).

These experiments reveal that the binding of Lhp1p to pre-U RNAs has important consequences for snRNP assembly. As only the pre-U4 RNA is an efficient substrate for Smd1p binding in extracts, the major role of Lhp1p in U4/U6 snRNP assembly may be to stabilize this RNA, thus facilitating Sm (the core proteins of snRNPs) protein binding. Since cells that contain wild-type SMD1 do not require Lhp1p, Sm protein binding may normally be sufficiently rapid such that prolonged stabilization of the precursor is unnecessary. Since addition of Lhp1p to lhp1::LEU2 extracts results in a small increase in Smd1p binding, Lhp1p may also directly facilitate assembly of pre-U4 RNAs into snRNPs by assisting RNA folding, stabilizing RNA structure or interacting with snRNP proteins. Moreover, as Lhp1p has a small effect on Smd1p binding in wild-type extracts, other situations that reduce the efficiency of U snRNP assembly could cause cells to require Lhp1p. In any case, the finding that Lhp1p facilitates U4/U6 snRNP biogenesis supports the hypothesis that Lhp1p functions as a molecular chaperone, i.e. a transiently binding protein, not found in the final assembly, that facilitates the correct fate of newly synthesized RNAs in vivo (Xue, 2000).

Does stabilization of pre-U RNAs by the La protein facilitate U snRNP assembly in higher cells? In vertebrates, binding by Sm proteins to pre-U RNAs occurs in the cytoplasm, and several snRNAs undergo 3' end maturation prior to reimport into the nucleus. As the human La protein binds a cytoplasmic population of U1 RNAs that are longer than mature U1 RNA, the vertebrate protein could function in the cytoplasm to facilitate assembly of pre-U1 RNA into snRNPs. However, the mammalian La protein has not been described as binding U2, U4 or U5 RNA precursors, making analogies difficult. Moreover, as a cytoplasmic phase in snRNP assembly has not been demonstrated in S.cerevisiae, U snRNPs could assemble entirely within the nucleus in this yeast. Consistent with nuclear assembly, pre-U4 RNAs (which are confined to the cytoplasm in mammalian cells) assemble into U4/U6·U5 tri-snRNPs in yeast. Interestingly, the SMN protein (the spinal muscular atrophy gene product), which binds Sm core proteins and is required for snRNP assembly in the vertebrate cytoplasm, has not been identified in S. cerevisiae. Thus, binding by Lhp1p to pre-U RNAs in the nucleus of budding yeast may substitute for the cytoplasmic role played by SMN in other organisms (Xue, 2000).

SMN complex and the assembly of the spliceosome

The small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U5 and U4/U6 are major components of the spliceosome. Each snRNP consists of one snRNA (U1, U2, U5 or U4/U6), an Sm protein core and a set of proteins that are specific to individual snRNAs. The Sm proteins B/B', D1, D2, D3, E, F and G are common to all spliceosomal snRNPs and are arranged into a seven-membered ring that assembles for each snRNA on a consensus sequence motif (PuAU4- 6GPu) called the Sm site, where Sm refers to a series of autoantigens defined using autoantibodies. This assembly process takes place in the cytoplasm shortly after the nuclear export of nascent snRNAs. After the formation of the Sm core, the 7-methyl guanosine (m7G) cap of these snRNAs is hypermethylated to become a 2,2,7-trimethyl guanosine (m3G or TMG). Properly assembled Sm core, cap hypermethylation and 3' end processing of the snRNAs are required for the subsequent nuclear import of the snRNPs, where they function in splicing (Yong, 2002 and references therein).

Important and unexpected insights into the process of snRNP assembly came from studies on the function of the survival of motor neurons (SMN) protein. SMN is the protein product of the spinal muscular atrophy (SMA) disease gene. SMA is a severe neuromuscular disease characterized by degeneration of motor neurons in the spinal cord. Over 98% of SMA patients have deletions or mutations of the telomeric copy of the gene (SMN1) and produce markedly reduced levels of the SMN protein. SMN is part of a large multiprotein complex that also contains Gemin2, the DEAD box RNA helicase Gemin3, Gemin4 and several additional as yet uncharacterized proteins. The SMN complex is present in both the nucleus and the cytoplasm of all metazoan cells, suggesting that it may have multiple functions in cells. Most lines of evidence indicate that the SMN complex functions in the assembly and metabolism of various RNPs, including snRNPs, snoRNPs, and the machineries that carry out transcription and pre-mRNA splicing (Yong, 2002 and references therein). The process of snRNP assembly can be most readily studied in Xenopus oocytes, where specific reagents and intermediates can be microinjected into either the nucleus or the cytoplasm and where dissection of nuclear and cytoplasmic fractions can be readily performed. Such experiments have revealed that the SMN complex associates with spliceosomal U1, U4 and U5 snRNAs in the cytoplasm. Antibodies against components of the SMN complex microinjected into Xenopus oocytes also inhibit the assembly of snRNPs, indicating that the SMN complex plays a crucial role in the biogenesis of snRNPs. In addition, overexpression of a dominant-negative SMN mutant blocks snRNP assembly in the cytoplasm of somatic cells, suggesting a general function for the SMN complex in the cytoplasmic phase of U snRNAs biogenesis. Recent studies have further demonstrated that the SMN complex is necessary for assembly of U1 snRNP in Xenopus egg extracts (Yong, 2002 and references therein).

The capacity of the SMN complex to associate with and mediate the assembly of snRNPs is probably due, at least in part, to interactions between the SMN complex and snRNP proteins. Several of the components of the SMN complex interact directly with Sm proteins. In particular, SMN binds avidly to RG-rich C-terminal domains that are found in the Sm proteins B, D1 and D3, whereas several SMN mutants found in SMA patients are defective in Sm protein binding. Importantly, SMN binds preferentially to the RG domains of D1 and D3 after arginines in specific positions are converted to symmetric dimethylarginines (sDMAs). Thus, arginine dimethylation has a key role in the protein substrate recognition by the SMN complex, and RNP assembly is likely to be regulated by arginine methylation (Yong, 2002 and references therein).

To serve in snRNP assembly, the SMN-Sm protein complex must also recruit the snRNAs. The binding of U1 snRNA, an abundant and high-avidity substrate, to the SMN complex has been investigated. The binding is sequence-specific and is mediated by the loop of stem- loop 1 domain (SL1) of U1 snRNA. SL1 is both necessary and sufficient for the interaction with the SMN complex in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. The interaction between the SMN complex and U1 snRNA is required for U1 snRNP assembly and thus mediates the function of the SMN complex in U1 snRNP biogenesis (Yong, 2002).

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