InteractiveFly: GeneBrief

half pint : Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Half pint (Hfp; FlyBase designation Poly-U-binding splicing factor) regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor (otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp. hfp is also required for efficient splicing of gurken mRNA and in the splicing of eukaryotic initiation factor 4E (eIF4E), which binds to the seven-methylguanosine cap at the 5' end of messenger RNAs and is a limiting factor in translation initiation (Van Buskirk, 2002).

Alternative exon selection is a means of producing multiple transcripts, often encoding distinct polypeptides, from a single gene. At least 35% of human genes are alternatively spliced, leading in some cases to a remarkable diversity of transcripts. Changes in splicing have been shown to alter the activities of transcription factors and cell death regulators, the ligand binding specificities of growth factor receptors and cell adhesion molecules, and the subcellular distribution of proteins. Despite the prevalence of alternative splicing, however, little is known about its regulation in vivo. One notable exception comes from the sex determination pathway of Drosophila, in which the Sex-lethal (Sxl) protein catalyzes a series of sex-specific alternative splicing events that lead to a mode of sexual differentiation and dosage compensation in keeping with the X:A ratio of the animal. Interestingly, the male-specific lethal-2 (msl-2) gene is regulated by Sxl at two levels: Sxl binds within and suppresses the removal of an intron in the 5'UTR of msl-2 and also represses translation of the unspliced, exported message. From this and other examples, it is becoming clear that several aspects of RNA metabolism, from RNA export to translation, are connected with splicing regulation (Van Buskirk, 2002 and references therein).

The gurken (grk) gene of Drosophila, a mammalian TGFalpha homolog, has been used to study many aspects of RNA regulation, including nuclear export, RNA localization, and translational control. During midoogenesis, grk RNA becomes restricted to one side of the oocyte, where it produces a localized source of protein that induces EGF receptor activation in the overlying follicle cells, establishing the dorsal side of the egg shell and embryo. The importance of grk RNA localization is evidenced by the defects in dorsoventral (DV) polarity that result when the grk transcript is not correctly localized. In egg chambers mutant for squid (sqd), grk RNA is not restricted to the dorsal-anterior corner of the stage 9 oocyte and instead forms an anterior ring, resulting in activation of the EGF receptor around the circumference of the oocyte and leading to the production of dorsalized eggs. The sqd gene encodes three isoforms of hrp40, a Drosophila hnRNP protein found within the nuclei and cytoplasm of all cells in the egg chamber, and has been proposed to function in the regulated nuclear export and translational repression of grk mRNA (Van Buskirk, 2002 and references therein).

The encore (enc) gene is also required for proper grk mRNA localization (Hawkins, 1997). However, in enc mutant egg chambers, Grk protein levels are reduced, and these females lay ventralized, not dorsalized, eggs. Thus it appears that enc is not only required for proper grk mRNA localization but for its translation as well. enc encodes a large, cytoplasmic protein of unknown function that becomes concentrated at the dorsal side of the oocyte in stage 9 egg chambers, where it colocalizes with grk mRNA (Van Buskirk, 2000). While the DV defects of enc are cold sensitive, enc females raised at higher temperatures show a highly penetrant extra mitosis in the germline, producing oocytes that are associated with 31 nurse cells instead of the normal 15 (Hawkins, 1996). However, the effectors that mediate this extra mitosis are not known (Van Buskirk, 2002 and references).

Half pint was isolated in a yeast two-hybrid screen with Enc; mutations in enc affect grk RNA localization and germline mitosis. Half pint is so named based on the mutant phenotype, which includes the production of short eggs and the formation of egg chambers that contain eight instead of sixteen germline cells. Surprisingly, half pint encodes the Drosophila homolog of human PUF60, an RNA binding protein characterized as a general splicing factor. Drosophila Hfp does indeed affect splicing: it specifically regulates the alternative splicing of a subset of genes within the ovary (Van Buskirk, 2002).

The encore (enc) gene is required for the regulation of germline mitosis, karyosome formation, and establishment of dorsoventral (DV) polarity of the Drosophila egg and embryo (Hawkins, 1996; Hawkins, 1997; Van Buskirk, 2000). enc encodes a large cytoplasmic protein with a single conserved region that contains an R3H motif (Van Buskirk, 2000), which is implicated in RNA binding. A portion of the Enc protein containing this conserved domain was used as bait in a two-hybrid screen for interacting proteins. A single factor that shows a specific interaction with Enc was recovered, and this interaction has been confirmed through coimmunoprecipitation. The gene encoding this interacting protein has been named half pint (Van Buskirk, 2002).

Multiple transcripts produced from alternative splicing can encode proteins with different or even opposing functions, and, hence, the regulation of alternative splicing can have profound biological consequences. However, few factors that regulate splice site selection in vivo have been identified, and much of the knowledge of splicing regulation comes from in vitro studies. The difficulty in isolating factors with effects on alternative splicing in vivo has, in part, led to the hypothesis that splice site choice is influenced by many factors that act combinatorially, each exerting a small influence on any one splicing event. Hfp, however, represents a factor with a significant role in alternative splicing regulation. Reductions in Hfp activity disrupt the developmentally regulated splicing of ovarian tumor, splice site usage within eIF4E and the efficiency of grk splicing. However, several other splicing events appear to be unaffected. The specificity of Hfp function is highlighted by the observation that, though both the otu and sqd splicing patterns shift between early and late oogenesis, otu, but not sqd, splicing is regulated by Hfp (Van Buskirk, 2002).

In vivo analysis of Hfp function along with in vitro studies on the human homolog, PUF60, demonstrate a role for this factor in splicing. However, the human protein has been implicated in a number of other processes. It has been identified as Ro-BP1, a component of RNP complexes of unknown function found in both nuclear and cytoplasmic compartments (Peek, 1993). It has also been isolated as FBP-interacting repressor (FIR), shown to block activated transcription by interacting with the TFIIH complex (Liu, 2000). PUF60/Ro-BP1/FIR therefore appears to represent an unusually multifunctional protein, participating directly in transcriptional repression and pre-mRNA splicing and also associating with RNA-protein complexes in the cytoplasm (Van Buskirk, 2002).

The observation that all of the ovarian phenotypes of hfp can be rescued by expression of the missing 104 kDa Otu splice form underscores the importance of otu splicing regulation and reveals previously undetected aspects of Otu function. While some otu egg chambers that have too few or too many germline cells have been observed, and the tumorous phenotype of strong otu mutations indicates a role in cell proliferation, none of the otu mutations specifically prevent the fourth mitosis of the germline cluster. The observations that Otu104 can rescue this defect in hfp and that the otu¹¹ mutation can suppress the enc mitotic defect point to a role for Otu in the precise regulation of the four rounds of germline mitosis. Similarly, while otu egg chambers have been shown to display defects in the localization of oskar RNA to the posterior pole of the oocyte, a defect also observed in hfp, a role for otu in the localization of grk mRNA has not been detected. These results suggest that otu does indeed play a role in grk RNA localization and that it is the 104 kDa isoform that performs this function. How does Otu participate in these diverse processes, from the regulation of germline mitosis to the dispersal of polytene chromosomes to the localization of RNAs? Mutations in otu are associated with aberrations in actin distribution, including ectopic accumulation of actin filaments within the fusome, a germline organelle implicated in the control of cystocyte division. Thus, otu may affect germline mitosis by regulating the interaction between fusome components and actin microfilaments. Likewise, otu may participate in mRNA localization by regulating the interaction of localized RNAs with the cortical actin cytoskeleton, which may act to anchor transcripts at their subcellular destinations. A direct role for otu in RNA localization is supported by its cofractionation with RNP complexes (Van Buskirk, 2002).

Both hfp and enc affect the regulation of germline mitosis. While hfp mutant egg chambers often contain eight germline cells, enc mutations result in an extra round of division, resulting in egg chambers with 32 germline cells. Overexpression of Otu104 can rescue the division defect of hfp and reduction of Otu104 activity can suppress the enc extra division. However, though Otu104 levels are strongly decreased in hfp, no converse overproduction of this isoform is detected in enc mutants, and, thus, enc does not appear to antagonize Hfp's splicing activity. It is possible that enc regulates germline mitosis through a pathway independent of hfp. However, the observed interaction between Hfp and Enc proteins raises the possibility that these genes function in a common pathway. If Enc does not antagonize Hfp's splicing activity, though, under what circumstances do these proteins interact? It may be useful to consider the Hfp-Enc interaction in terms of the roles these genes play in DV patterning. hfp and enc mutants both show defects in grk RNA localization, and enc is required for the accumulation of Grk protein (Hawkins, 1997). hfp plays a role in grk splicing. As has been observed for other splicing factors, it is possible that Hfp remains associated with its targets during nuclear export and perhaps delivers the spliced grk transcript to Enc in the cytoplasm. Enc may in turn be required for both anchoring and translation of the grk message. It is thus proposed that Hfp may have two roles: one in splicing regulation and one in the subcellular targeting of RNAs (Van Buskirk, 2002).

Since Otu104 overexpression can rescue the RNA localization defect of the hypomorphic allele hfp⁹, the delivery of grk RNA to the anchoring machinery must also be dependent on otu. Otu's tudor domain and observed cofractionation in RNP complexes suggest that it might associate with mRNAs during transport or anchoring. It is therefore proposed that Hfp may also be present within the RNP complexes with which Otu fractionates. In support of this, the vertebrate homolog of Hfp has been isolated as a component of nucleo-cytoplasmic RNP complexes (Peek, 1993), and Hfp has also been found to copurify with proteins found in cytoplasmic RNP particles (J. Wilhelm, personal communication to Van Buskirk, 2002). This putative cytoplasmic function of Hfp may involve an interaction with Enc. For instance, Enc may be required to release grk RNA from an intermediate Hfp-/Otu-dependent stage of transport to an anchored, translationally competent state (Van Buskirk, 2002).

Much of the knowledge of splicing regulation in vivo comes from the study of the Sex-lethal (Sxl) protein of Drosophila. This factor has been shown to control splice site usage by blocking U2snRNP auxiliary factor (U2AF) from binding to the polypyrimidine tract upstream of the 3' splice acceptor site. Thus, Sxl binding represses usage of the proximal 3' splice site, and a competing splice acceptor was used. How does Hfp function to regulate splice site choice? In the case of the otu transcript, does hfp promote splicing to exon 6a or repress selection of exon 7? In vitro studies with the human homolog of hfp, PUF60, point to a stimulatory role (Page-McCaw, 1999). In these studies, PUF60 [60 kDa poly(U) binding factor] was isolated as part of a complex that could stimulate efficient splicing of several introns in Hela nuclear extracts. This complex, along with U2AF, was found to facilitate the association of U2snRNP with the pre-mRNA, thus promoting spliceosome assembly (Van Buskirk, 2002).

The rat homolog of Hfp, Siah-BP, was isolated in a three-hybrid screen for factors that could bind to a splicing enhancer, a short RNA sequence required for neuron-specific alternative splicing of the amyloid precursor protein (APP) gene (Poleev, 2000). Siah-BP was also found to directly bind the large subunit of U2AF, consistent with studies on PUF60 suggesting that it acts in conjunction with U2AF in the early steps of splicing (Page-McCaw, 1999). The ability of the human PUF complex to stimulate splicing of all introns assayed suggests that it acts constitutively, facilitating spliceosome assembly at all polypyrimidine tracts. Half pint, however, appears to possess sequence specificity, since it is capable of influencing splice site choice and affects only a subset of genes analyzed. This apparent discrepancy between the human and Drosophila homologs may come from the artificial nature of the in vitro splicing assay, in which addition of perhaps nonphysiological levels of splicing factors may stimulate the splicing of targets that would not normally be affected in vivo. Alternatively, the orthologs may indeed differ in their target range, a point that could be addressed through in vitro analysis of Hfp activity (Van Buskirk, 2002).

Little is known about the cis-acting regulatory elements that control alternative splicing, posing a challenge for genomic protein prediction models. The recognition element for Hfp is likely to be somewhat degenerate, since the known Hfp targets do not share an obvious binding site. RNA-protein crosslinking studies and/or the identification of several more Hfp target genes may define this binding site, providing insight into the elusive cis-acting sequences that control alternate splice site selection during development (Van Buskirk, 2002).

Hfp inhibits Drosophila myc transcription and cell growth in a TFIIH/Hay-dependent manner

An unresolved question regarding the RNA-recognition motif (RRM) protein Half pint (Hfp) has been whether its tumour suppressor behaviour occurs by a transcriptional mechanism or via effects on splicing. The data presented in this study demonstrate that Hfp achieves cell cycle inhibition via an essential role in the repression of Drosophila myc (dmyc) transcription. Regulation of dmyc requires interaction between the transcriptional repressor Hfp and the DNA helicase subunit of TFIIH, Haywire (Hay). In vivo studies show that Hfp binds to the dmyc promoter and that repression of dmyc transcription requires Hfp. In addition, loss of Hfp results in enhanced cell growth, which depends on the presence of dMyc. This is consistent with Hfp being essential for inhibition of dmyc transcription and cell growth. Further support for Hfp controlling dmyc transcriptionally comes from the demonstration that Hfp physically and genetically interacts with the XPB helicase component of the TFIIH transcription factor complex, Hay, which is required for normal levels of dmyc expression, cell growth and cell cycle progression. Together, these data demonstrate that Hfp is crucial for repression of dmyc, suggesting that a transcriptional, rather than splicing, mechanism underlies the regulation of dMyc and the tumour suppressor behaviour of Hfp (Mitchell, 2010).

Tight control of c-myc transcription is essential as upregulation of c-MYC expression is associated with most human cancers. In vitro mammalian studies have suggested that one mechanism for c-myc promoter regulation involves the presence of a paused, but transcriptionally engaged, Pol II at the c-myc start site. The paused polymerase can allow a rapid response to developmental/mitogenic signals and protect the c-myc promoter from unwanted activation. This study provides strong evidence that the FIR homologue Hfp is crucial for transcriptional repression of dmyc and cell growth, suggesting that a transcriptional, rather than a splicing, mechanism underlies the tumour suppressor behaviour of Hfp. In addition, these data show that the mechanism proposed for repression of c-myc transcription by the mammalian RRM protein FIR is conserved in Drosophila (Mitchell, 2010).

First, FIR negatively regulates c-myc transcription (Liu, 2006), and this study has shown that Hfp can bind the dmyc promoter and is essential for repression of dmyc transcription. Although FIR mutations correlate with colorectal cancer incidence (Matsushita, 2006), whether dysregulated FIR is the cause of the increased c-myc expression and/or the overgrowth phenotypes associated with these cancers is unknown. This study has demonstrated that loss of Hfp results in a cell growth phenotype, which occurs in a dMyc-dependent manner. These data strongly suggest that dysregulated FIR in the human context might be causative in cancer initiation and progression. Further support for conservation of the proposed FIR and XPB mechanism for c-myc control is provided by the finding that the repression of dmyc by Hfp occurs in a manner dependent on the XPB helicase homologue Hay, as the increases in dmyc transcription and cell growth associated with loss of Hfp are dependent on the presence of Hay. Thus, these studies provide novel insights into the molecular mechanisms required for controlling c-myc transcription, which are likely to be important for understanding FIR- and XPB-related cancers (Mitchell, 2010).

Although in vitro mammalian studies have shown that the response of c-myc to serum is defective in FIR loss-of-function and XPB-related cancer cells (Liu, 2006), the upstream factors in the pathway by which serum mediates c-myc repression via XPB and FIR have not been identified. In Drosophila, this study has shown that Hfp protein levels are regulated, in part, by Wg. As Hfp is responsive to the Wg pathway, and promoter occupancy by FIR responds to factors in serum, it is hypothesized that Hfp levels and/or activity will be controlled by developmental/growth signals. It is predicted that cross-talk between a specific complement of growth signals, including Wg, will tightly regulate dmyc transcription and growth via Hfp and Hay, which are likely to be relevant to the processes involved in the dysregulation of c-MYC during human malignancy. Thus, a current working model for repression of dmyc by Hfp is presented. In response to negative growth signals Hfp binds to inhibit dmyc transcription, but upon mitogenic stimulation dmyc transcription results from the prevention of promoter occupancy by Hfp. The possibility cannot be ruled out that Hfp might in some instances provide a repressive effect that must be overcome by the presence of activators. Thus, the mechanism(s) regulating Hfp levels and/or occupancy of the dmyc promoter is the subject of ongoing studies (Mitchell, 2010).

In conclusion, this work suggests analogous systems are required for transcriptional regulation of the c-myc oncogene and dmyc. The knowledge gained from future studies on the developmental regulation of these proteins in Drosophila will be informative in understanding the regulation of c-myc by the homologous proteins in mammals (Mitchell, 2010).

DEVELOPMENTAL BIOLOGY

In order to determine the subcellular localization of Hfp during oogenesis, monoclonal antibodies to Hfp were generated. Using these, Hfp protein was detected within the nuclei of all germline cells and also within the follicle cell nuclei. In hfp ovaries, the expression of Hfp is decreased within the germline of early stage egg chambers, while expression in the surrounding somatic follicle cells appears normal. The reduction in Hfp expression specifically within the germline of hfp mutants suggests that different cis-acting sequences control germline and somatic expression of hfp; also, the observed hfp phenotypes are due to loss of hfp function in the germline. Consistent with this, germline clones of the strong allele hfp³⁸ result in developmental arrest early in oogenesis. Interestingly, the polyclonal serum obtained from the immunized animal prior to hybridoma production also detects cytoplasmic aggregates in the germline, which are particularly abundant within the oocyte. This polyclonal serum, like the monoclonal antibodies, recognizes one major band that is less abundant in hfp mutant ovaries on Western blots. However, since none of the monoclonal antibodies show this nucleo-cytoplasmic distribution and the rescuing Hfp-GFP fusion protein is detectable only in nuclei, the significance of the cytoplasmic staining is unknown (Van Buskirk, 2002).

EFFECTS OF MUTATION

The P element insertion line EP(3)3058 harbors a recessive lethal insertion in the 5'UTR of hfp, and the deficiency Df(3L)Ar14-8 (61C7;62A8) fails to complement this lethality. The EP(3)3058/Df animals are slower to develop than their siblings and die shortly after pupariation, displaying melanized patches of tissue. To determine the requirement for hfp during oogenesis, it was necessary to circumvent this pupal lethality. To this end, several P element excision lines were isolated, with the aim of generating small excisions within the 5'UTR hfp that might function as hypomorphic alleles. The majority of imprecise excisions were lethal but several were semi-viable and sterile. Of these, the hypomorphic alleles hfp¹³ and hfp⁹ were the most amenable to study. While hfp¹³ homozygotes are fertile, hfp¹³/Df males and females are sterile. The hfp⁹ allele is homozygous male and female sterile and hemizygous lethal. PCR analysis shows that the excisions do not delete neighboring genes and, in fact, are internal to the P element itself, leaving behind an insertion of 74 nt in the case of hfp¹³ and 481 nt in the case of hfp⁹ (Van Buskirk, 2002).

The Drosophila egg chamber is composed of a cluster of 16 interconnected germline cells (15 nurse cells and an oocyte) surrounded by a layer of somatic follicle cells. The ovary contains several strings of developing egg chambers, or ovarioles, that can be divided into two regions: the germarium, where egg chambers are formed, and the vitellarium, in which egg chambers mature. Within the germarium, a germline cystoblast undergoes four rounds of mitosis with incomplete cytokinesis to produce a cluster of 16 cystocytes that are linked by actin-rich connections called ring canals. One of the two cells with four ring canals becomes the oocyte, while the other 15 differentiate into polyploid nurse cells. hfp egg chambers commonly contain eight germline cells: seven nurse cells and an oocyte or, in some cases, eight nurse cells. At room temperature, 24% of hfp⁹ egg chambers and 45% of hfp¹³/Df egg chambers contain eight germline cells. Viable females of stronger hfp alleles have rudimentary ovaries that contain few, undifferentiated egg chambers. To determine whether the eight-cell hfp egg chambers arise from a defect in germline mitosis, the number of oocyte-associated ring canals was examined. An oocyte possesses one ring canal for each round of cell division within the cyst and hence is normally associated with four ring canals. However, in hfp mutant egg chambers that contain seven nurse cells and an oocyte, the oocyte invariably has three ring canals, revealing that these cysts have undergone only three rounds of mitosis (Van Buskirk, 2002).

In addition to having fewer cells per egg chamber, hfp mutants also display defects in germline nuclear morphology. During the early endocycles of wild-type nurse cells, the replicated chromosomes are polytene but are dispersed by midoogenesis. In hfp mutant egg chambers, nurse cell chromosomes remain polytene throughout oogenesis, giving them a striking banded appearance. In addition, less penetrant defects are observed in the morphology of the oocyte nucleus. The DNA of a wild-type oocyte nucleus forms a compact sphere, or karyosome, which persists from shortly after egg chamber formation until the late stages of oogenesis. In a fraction of hfp mutant oocytes, however, the karyosome appears abnormally shaped, diffuse, or fragmented (Van Buskirk, 2002).

hfp mutant females lay few eggs, most of which are shorter than wild-type eggs and display dorsal appendage defects ranging from an expansion to a reduction of dorsal appendages. A wild-type egg is approximately 500 µm in length, while the eggs produced by hfp mutant females (hereafter referred to as hfp mutant eggs) range from 300 to 500 µm. An examination of late stage hfp egg chambers reveals that, in many cases, the nurse cells fail to transfer all of their cytoplasm into the oocyte prior to egg shell deposition. This defect in nurse cell to oocyte transport may explain the shortness of hfp mutant eggs (Van Buskirk, 2002).

The dorsal appendage phenotypes of hfp mutant eggs are suggestive of perturbations in DV patterning of the egg chamber. Since Hfp interacts with Enc, which is required for grk mRNA localization and Grk protein accumulation (Hawkins, 1997), it was asked whether grk expression is also affected in hfp mutants. In wild-type stage 9 egg chambers, grk mRNA is localized to a region overlying the oocyte nucleus, and Grk protein is thus restricted to the adjacent plasma membrane, where it induces dorsal follicle cell fates. In a small fraction of hfp mutant egg chambers, grk RNA appears to be poorly expressed or undetectable, which would account for the production of eggs with reduced dorsal egg shell structures. More striking, however, is that in 52% of the stage 9/10 hfp⁹ mutant egg chambers in which grk mRNA can be detected, the transcript is present in an anterior ring. Thus, hfp appears to be required for both the production of wild-type levels of grk transcript and, like enc, for proper grk mRNA localization. However, unlike enc, this mislocalized grk mRNA in hfp mutants gives rise to detectable Grk protein. This ring of Grk protein would be predicted to induce dorsal fates in all anterior follicle cells, accounting for the observed dorsalized eggs of hfp (Van Buskirk, 2002).

Since hfp encodes a predicted splicing factor, experiments were carried out to determine whether the grk RNA mislocalization in hfp is due to a splicing defect that leads to the production of a grk transcript lacking proper localization signals. The grk gene encodes a single identified transcript, and, in RT-PCR analysis of poly(A) RNA from wild-type ovaries, a single grk product is detected. In hfp mutants, a larger product is also detected, suggestive of inefficient removal of one of the grk introns. Using a series of primers, maintenance specifically of the third intron in a fraction of grk transcripts was detected in hfp mutants. This type of splicing defect is unlikely to account for the mislocalized grk RNA observed in hfp oocytes, since the unspliced transcript does not lack any sequences found in the wild-type transcript. Furthermore, previous studies have shown that insertion of a fragment of the lacZ gene into the grk cDNA at a position analogous to the third intron does not disrupt normal RNA localization. Thus, it appears that, while Hfp is required for efficient grk splicing, this is not the cause of the RNA localization defect (Van Buskirk, 2002).

The dorsalized eggs laid by hfp mutants are similar to those produced by females mutant for squid (sqd), which encodes an RRM-containing protein that has been shown to directly bind grk RNA. The sqd gene encodes three splice forms that contain the same N-terminal RNA binding domain but have divergent C termini and display distinct activities with respect to grk RNA localization and translational control.Are the dorsalized eggs of hfp caused by defects in the splicing of sqd? Using RT-PCR analysis of stage-dissected ovaries, it was found that, in wild-type oogenesis, sqd splicing is developmentally regulated. While the sqdA and sqdB transcripts are produced constitutively, the sqdS transcript is detected only in later stages. No alterations were detected in sqd splicing in hfp⁹ or in the stronger allelic combination hfp¹³/Df. Furthermore, no differences were detected in Sqd protein localization or levels in hfp mutants, and, thus, the defects in grk RNA localization seen in hfp are not likely due to alterations in sqd expression (Van Buskirk, 2002).

The polytene nurse cell chromosome morphology of hfp mutants is strikingly similar to that observed in females mutant for certain alleles of ovarian tumor (otu). otu also plays a role in germline cell division, as otu egg chambers can have too few or too many germline cells. The otu transcript is subject to alternative splicing, resulting in the production of a 98 kDa protein and a less abundant 104 kDa protein (Otu104), depending on the incorporation of a 126 bp alternatively spliced exon. The biochemical functions of the Otu isoforms are unknown, but they share an N terminus that contains a cysteine protease-like domain and a C terminus that possesses weak similarity to microtubule-associated proteins. Intriguingly, the alternatively spliced exon encodes a tudor domain, a sequence element found multiply repeated in the Drosophila Tudor protein and also present in proteins with putative RNA binding functions in several species. Genetic and molecular analysis reveal distinct requirements and expression patterns for the two Otu isoforms, suggesting that the splicing of the otu transcript may be developmentally regulated. Indeed, RT-PCR shows that expression of the transcript encoding the 104 kDa isoform is restricted to the early stages of oogenesis (Van Buskirk, 2002).

In order to determine whether the alternative splicing of otu is dependent on hfp function, the expression of otu was examined in hfp mutants. RT-PCR analysis of RNA isolated from early stage egg chambers shows that the relative abundance of the larger otu transcript is decreased in hfp, and Western analysis shows that the levels of the corresponding 104 kDa Otu isoform are significantly decreased. Severe loss of Otu104 activity results in the production of tumorous egg chambers, which are not observed in hfp. Therefore, some residual Otu104 protein must be present, possibly because the hfp mutants represent partial loss-of-function alleles. To determine which, if any, of the hfp phenotypes are due to an effect on otu splicing regulation, a hybrid genomic-cDNA transgene encoding exclusively the 104 kDa Otu isoform was introduced into an hfp mutant background. hfp mutants expressing two copies of the Otu104 transgene produce egg chambers with predominantly wild-type nurse cell nuclear morphology and expression of four copies of the transgene completely rescues the nurse cell and oocyte nuclear morphology as well as the germline division defect. Expression of Otu104 also restores normal grk mRNA localization. This was unexpected, since defects in grk RNA localization have not been previously described in otu mutants, and no grk RNA localization defects are observed in otu⁷ homozygotes or otu⁷/otu¹¹ mutants, which give rise to differentiated egg chambers. However, otu¹¹ females, though often producing tumorous germaria, will under certain conditions produce rare eggs, and these show defects, including the expansion of dorsal appendages associated with mislocalization of grk RNA, very similar to those of hfp⁹ mutant eggs. Interestingly, otu¹¹ is a point mutation in the alternatively spliced exon and hence specifically affects the activity of the 104 kDa Otu isoform (Van Buskirk, 2002).

Since Half pint was isolated based on its ability to interact with Encore and these two genes have opposite effects on germline mitosis, it was hypothesized that Enc may act to antagonize Hfp's role in splicing and that the extra cell division seen in enc mutants may be a result of an overproduction of Otu104. However, otu expression in enc appears to be normal by RT-PCR and Western analysis, and it is therefore unlikely that Enc acts to regulate the splicing activity of Hfp. However, suppression of the enc extra division phenotype is observed upon reduction of the dose of otu. Of the otu alleles tested, the suppression is most significant with the 104 kDa isoform-specific mutation otu¹¹. Thus, while Otu expression is not affected in enc, Otu104 activity is critical in mediating the enc extra division (Van Buskirk, 2002).

The expression of several other alternatively spliced ovarian transcripts was examined in hfp mutants. One potential target that was examined was eukaryotic initiation factor 4E (eIF4E), which binds to the seven-methylguanosine cap at the 5' end of messenger RNAs and is a limiting factor in translation initiation. The Drosophila eIF4E gene produces three ovarian transcripts through alternative splicing. Two of these give rise to the eIF4E-I isoform, while the other gives rise to eIF4E-II. Both isoforms have been shown to have cap binding activity in vitro, and transgenic expression of eIF4E-I is sufficient to rescue the phenotype of several loss-of-function eIF4E alleles; hence, the requirement for the different splice forms is unclear. The major splice form in wild-type utilizes the 1b splice acceptor site, producing eIF4E-I. In hfp mutants, the bias for the 1b acceptor is lost and all three splice variants are produced roughly equally, with a slight preference for the more proximal 1a splice acceptor site. Using antibodies specific for eIF4E-I, it has been found that the levels of this isoform are decreased in hfp. It was surprising to find that the levels of eIF4E-I were drastically affected, since the 1a transcript also gives rise to this isoform. This reduction in eIF4E-I protein levels may reflect either that the combined levels of eIF4E-I-encoding transcripts are lower in hfp than in wild-type or that the 1a transcript is not efficiently translated. The latter possibility would point to alternative splicing as a means of regulating eIF4E protein levels. Since the misregulation of otu splicing appears to account for all of the known ovarian defects of hfp, the significance of the regulation of eIF4E splicing during oogenesis is unclear. However, it is possible that Hfp's role in eIF4E splicing is important at other stages of development and that decreased eIF4E-I levels may contribute to the reduced viability of hfp mutants (Van Buskirk, 2002).

Thus, Hfp acts to regulate alternative splice site choice within at least two genes in the ovary. Hfp does not appear to participate in all cases of splice site selection, however, because several alternatively spliced genes are not affected by hfp mutations. Among these are par-1 and sqd. Defects were observed in the splicing of the sex-determining gene Sex-lethal (Sxl), detecting some of the male-specific transcript in hfp¹³ hemizygous females, but, since otu is known to affect Sxl splicing, hfp's role in Sxl expression may be indirect (Van Buskirk, 2002).

Drosophila Hfp negatively regulates dmyc and stg to inhibit cell proliferation

Mammalian FIR has dual roles in pre-mRNA splicing and in negative transcriptional control of Myc. Half pint (Hfp), the Drosophila ortholog of FIR, inhibits cell proliferation in Drosophila. Hfp overexpression potently inhibits G1/S progression, while hfp mutants display ectopic cell cycles. Hfp negatively regulates dmyc expression and function: reducing the dose of hfp increases levels of dmyc mRNA and rescues defective oogenesis in dmyc hypomorphic flies. The G2-delay in dmyc-overexpressing cells is suppressed by halving the dosage of hfp, indicating that Hfp is also rate-limiting for G2-M progression. Consistent with this, the cycle 14 G2-arrest of stg mutant embryos is rescued by the hfp mutant. Analysis of hfp mutant clones revealed elevated levels of Stg protein, but no change in the level of stg mRNA, suggesting that hfp negatively regulates Stg via a post-transcriptional mechanism. Finally, ectopic activation of the wingless pathway, which is known to negatively regulate dmyc expression in the wing, results in an accumulation of Hfp protein. These findings indicate that Hfp provides a critical molecular link between the developmental patterning signals induced by the wingless pathway and dMyc-regulated cell growth and proliferation (Quinn, 2004).

The Drosophila stock EP(3)3058 (hfp^EP) harbors a recessive lethal P element insertion in the 5' UTR of hfp, 94 bp upstream of the initiating methionine codon (Van Buskirk, 2002). Homozygous hfp^EP larvae were of similar size to age-matched wild type third instar larvae. However, the pupariation of hfp^EP larvae was consistently delayed by approximately 2 days, and continued growth during this period resulted in wandering larvae and pupae ~20% larger than wild-type third instar larvae. The duration of the pupal stage was normal for hfp^EP mutant animals; however, they failed to eclose and died as pharate adults that were larger than wild type. The hfp^EP/hfp^EP terminal phenotype included duplication of superior scutellar macrochaete, and malformation of legs, wings and sex combs (Quinn, 2004).

The pleiotropic phenotype of hfp mutant animals indicated that Hfp might be involved in several stages of development. In Drosophila, maternal transcripts are transferred during oogenesis and serve to sustain early embryonic development until stage 5, after which zygotic transcription commences. Northern analysis revealed that hfp mRNA is maternally deposited in the early embryo; however, zygotic hfp expression is low during late embryonic and early larval stages. hfp transcripts are also detected in third instar larvae, pupae and adults. A marked decrease in hfp mRNA occurs in hfp^EP/hfp^EP and hfp^EP/Df(3L)Ar14-8 larvae, when compared with age-matched wild-type third instar larvae. However, hfp transcript is still detectable, consistent with the notion that hfp^EP is not a null allele (Van Buskirk, 2002). In wild-type animals, expression of hfp during third instar coincides with the onset of differentiation in imaginal discs. Hfp protein expression was examined in wing discs using an antibody recognizing Hfp (Van Buskirk, 2002) and an antibody to Geminin, which is abundant in late S phase and G2 but absent in G1 cells, was used to visualize the dorsoventral compartment boundary of the wing (the ZNC). Hfp protein is detected in the nucleus of most wing disc cells, with higher staining in cells in the ZNC. Consistent with Northern analysis, Hfp protein level is significantly reduced in wing discs from hfp^EP/hfp^EP larvae (Quinn, 2004).

In order to investigate whether Hfp regulates cell proliferation during Drosophila development, BrdU incorporation was measured in wing discs from wandering hfp^EP/hfp^EP larvae. In wild-type wing discs the ZNC is clearly marked by the absence of BrdU labelling. The number of S-phase cells is markedly increased in hfp^EP mutant wing discs: BrdU incorporation is uniform across the disc and cell cycle arrest is not evident in the ZNC region. Strikingly, anti-phosphohistone H3 antibody staining of mitotic cells, is also elevated, indicating an overall increase in cell proliferation in hfp wing discs (Quinn, 2004).

Hfp is a negative regulator of cell cycle entry in Drosophila as evidenced by (1) ectopic S phases in the ZNC of hfp mutant wing discs and increased S phase in the second mitotic wave in the eye disc; (2) inhibition of S phases in larval imaginal tissues by overexpression of the UAS-hfp transgene; and (3) dominant suppression of the GMR- driven human p21 or dacapo rough eye phenotypes and rescue of the posterior band of S phases in GMR-p21 eye discs by reducing the level of hfp. These data suggest that Hfp normally has a role in preventing S-phase entry in cells destined to differentiate in the eye and wing imaginal discs. Furthermore, this negative regulation of the cell cycle by Hfp is partly a consequence of inhibitory affects on dmyc, since (1) an increased level of dmyc mRNA transcript occurs in hfp-/- clones, and (2) reduced levels of Hfp can rescue the dmyc mutant ovary phenotype, by restoring levels of dmyc mRNA to more wild-type levels. Indeed, upregulation of dmyc expression in Hfp mutants may explain the rescue of S phases in eye discs overexpressing p21 or Dacapo, consistent with the observation that dmyc mutants dominantly enhance the GMR-p21 and GMR-driven dacapo rough eye phenotypes. Mammalian Myc stimulates cyclin E expression, activation of Cdks, antagonizes the action of Cdk inhibitors, including p27, and can downregulate p21 transcription and p21 activity via direct c-Myc-p21 protein-protein interaction. In Drosophila, dMyc has been shown to lead to an increase in Cyclin E protein levels by a post-transcriptional mechanism, which by itself could explain the suppression of the GMR-p21 eye phenotype by a reduction in the dose of hfp. Whether dMyc can also inhibit p21 or Dacapo activity in Drosophila is unknown (Quinn, 2004).

Increased levels of dmyc transcript are observed in hfp mutant clones, consistent with Hfp acting to repress dmyc transcript accumulation in Drosophila imaginal tissues. The upregulation of dmyc mRNA in hfp mutant tissue could occur through alterations in dmyc transcription (initiation or elongation), pre-mRNA splicing, mRNA message stability or a combination of these processes. Mammalian FIR was first shown to regulate pre-mRNA splicing by binding to RNA polypyrimidine tracts and cooperating with the essential splicing factor U2AF. Consistent with this, recent studies in Drosophila show that the FIR ortholog Hfp is required for correct splicing of several genes in the developing ovary (Van Buskirk, 2002). Mammalian FIR has been shown to have a second role as transcriptional repressor of Myc, through first forming a complex with the Myc activator FBP and interfering with the basal transcription apparatus by then binding TFIIH, thereby disrupting helicase function. The data described in this study suggest that the cell cycle inhibitory function of Hfp is partly a consequence of negatively regulating dmyc expression. Therefore, the dual roles of transcription regulation and mRNA splicing appear to have been evolutionarily conserved between Drosophila Hfp and mammalian FIR. It remains to be determined whether Hfp inhibits dmyc expression by a mechanism analogous to the mammalian FIR/FBP/FUSE interaction. A FUSE element has not been identified upstream of the dmyc promoter, and although the Drosophila splicing factor PSI is a highly conserved ortholog of FBP, it has not been reported whether PSI can activate dmyc expression (Quinn, 2004).

The finding that hfp mutants do not phenocopy dmyc overexpression suggests that inhibition of dmyc expression is not the only role of Hfp. Although increased S phases are observed in hfp mutant wing discs, this is not associated with increased cell size, as occurs with dmyc overexpression in the wing disc. Rather, in hfp mutant wing discs the ZNC, which normally contains domains of G1- and G2-arrested cells, has ectopic S-phase and M-phase cells. Since cells in hfp mutant wing discs are of normal size and ectopically enter S phase, it is possible that progression through G2 may also be accelerated. Indeed, the increased number of mitotic cells observed in eye imaginal discs when the level of Hfp is reduced in a dmyc overexpression background, suggests that Hfp normally negatively regulates G2-M phase progression. Furthermore, the abnormal mitotic figures observed in hfp^EP mutant embryos are consistent with accelerated cell cycle progression. Most importantly, the hfp mutant rescues the cycle 14 G2-arrest that normally occurs in stg mutant embryos, and hfp mutant clones have increased levels of Stg protein, suggesting that Hfp normally exerts an inhibitory affect on G2-M progression via negatively regulating Stg translation or protein stability. Thus, Hfp may be required for negatively regulating both the G1-S phase transition by downregulating dmyc and the G2-M transition by negatively regulating stg (Quinn, 2004).

The Wg pathway is required to downregulate both dmyc and stg expression in order to limit cell proliferation in the ZNC during wing development. Activation of the Wg pathway, using either dominant negative Shaggy or by generation of axin clones, results in a strong and specific increase in Hfp protein, demonstrating that Wg pathway activation is sufficient to cause Hfp induction. These findings support a model in which Wg signalling causes induction of Hfp in the wing disc ZNC, which in turn inhibits dmyc expression (to elicit the posterior, G1 arrest) and stg expression or activity (to provide the anterior, G2-arrested domains). The involvement of Achaete and Scute in this process, which play a role in the negative regulation of stg remains to be elucidated. Previous studies have shown that Ras signalling through Raf/MAPK upregulates dmyc post-transcriptionally in wing disc cells and is required to maintain normal dMyc protein levels in the wing disc. In contrast, since hfp clones have increased dmyc mRNA, Hfp must normally inhibit dmyc mRNA accumulation. Furthermore, overexpression of Hfp inhibits cell proliferation in all wing and eye imaginal discs, suggesting that Hfp may normally override mitogenic signals and lead to cell cycle arrest during particular stages of development (Quinn, 2004).

In summary, these results suggested that Hfp negatively regulates cell proliferation by inhibiting dmyc transcription and Stg protein accumulation. Hfp is required for the developmentally regulated cell cycle arrest within the ZNC and is responsive to the Wg signalling pathway that regulates this arrest, suggesting that Hfp links patterning signals to cell proliferation during Drosophila development (Quinn, 2004).

EVOLUTIONARY HOMOLOGS

PUF60/Ro-BP1/FIR/Siah-BP: a homolog of poly-U-binding splicing factor/half pint

Ro ribonucleoprotein particles (Ro RNPs) are complexes of several proteins with a small RNA polymerase III-transcribed Ro RNA. Despite their relative abundance and evolutionary conservation no function has as yet been ascribed to these complexes. Also their subcellular distribution is still largely unknown since immunofluorescence studies concerning their localization have produced conflicting data. Cell enucleation has been used to fractionate cells into cytoplasmic and nuclear fractions. Analysis of these fractions has revealed an exclusively cytoplasmic localization for the Ro RNPs. The majority of the Ro RNAs have been shown to be stably associated with all three known Ro RNP proteins. Although no Ro RNAs could be detected in the nuclear fraction, the Ro RNP-specific proteins were abundantly present. These nuclear non-Ro RNA-associated proteins have been shown to be capable of binding Ro RNAs (Peek, 1993).

A new pyrimidine-tract binding factor, PUF, has been identified that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions (Page-McCaw, 1999).

FUSE-binding protein (FBP) binds the single-stranded far upstream element of active c-myc genes, possesses potent transcription activation and repression domains, and is necessary for c-myc expression. A novel 60 kDa protein, the FBP interacting repressor (FIR), blocks activator-dependent (but not basal) transcription through TFIIH. Recruited through FBP's nucleic acid-binding domain, FIR forms a ternary complex with FBP and FUSE. FIR repressed a c-myc reporter via the FUSE. The amino terminus of FIR contains an activator-selective repression domain capable of acting in cis or even in trans in vivo and in vitro. The repression domain of FIR targets only TFIIH's p89/XPB helicase, required at several stages in transcription, but not factors required for promoter selection. Thus, FIR locks TFIIH in an activation-resistant configuration that still supports basal transcription (Liu, 2000).

Two clones were isolated in a three-hybrid screen of a rat fetal brain P5 cDNA library with an intronic splicing enhancer of the amyloid precursor protein (APP) gene as RNA bait. These clones represent the rat homologs of the previously described genes CUG-binding protein (CUG-BP) and Siah-binding protein (Siah-BP). Both interact in a sequence-specific manner with the RNA bait used for library screening as well as with the CUG repeat. In contrast, no interactions were observed in the three-hybrid assay with other baits tested. In two-hybrid assays, Siah-BP interacts with U2AF65 as well as with itself. EWS, an RGG-type RNA-binding protein associated with Ewing sarcoma, was identified as an interacting partner for the CUG-BP homolog in a two-hybrid assay for protein-protein interactions performed with various factors involved in RNA metabolism. Splicing assays performed by RT-PCR from cells cotransfected with certain cDNAs and an APP minigene, used as a reporter, indicate exclusion of exon 8 if the CUG-BP homolog is present. It is concluded that clone AF169013 and its counterpart in human CUG-BP could be the trans-acting factors that interact with the splicing enhancer downstream of exon 8, and in this way influence alternative splicing of the APP minigene (Poleev, 2000).

REFERENCES

Search PubMed for articles about Drosophila poly-U-binding splicing factor/half pint

Hawkins, N. C., Thorpe, J. and Schüpbach, T. (1996). encore, a gene required for the regulation of germ line mitosis and oocyte differentiation during Drosophila oogenesis. Development 122: 281-290. 8565840

Hawkins, N. C., et al. (1997). Post-transcriptional regulation of gurken by encore is required for axis determination in Drosophila. Development 124(23): 4801-4810. 9428416

Liu, J., He, L., Collins, I., Ge, H., Libuttti, D., Li, J., Egly, J.-M. and Levens, D. (2000). The FBP interacting repressor targets TFIIH to inhibit activated transcription. Mol. Cell 5: 331-341. 10882074

Liu J., et al. (2006). The FUSE/FBP/FIR/TFIIH system is a molecular machine programming a pulse of c-myc expression. EMBO J. 25: 2119-2130. PubMed Citation: 16628215

Matsushita, K., et al. (2006). An essential role of alternative splicing of c-myc suppressor FUSE-binding protein-interacting repressor in carcinogenesis. Cancer Res. 66: 1409-1417. PubMed Citation: 16452196

Mitchell, N. C., et al. (2010). Hfp inhibits Drosophila myc transcription and cell growth in a TFIIH/Hay-dependent manner. Development 137(17): 2875-84. PubMed Citation: 20667914

Page-McCaw, P. S., Amonlirdviman, K. and Sharp, P. A. (1999) PUF60: a novel U2AF65-related splicing activity. RNA 5: 1548-1560. 10606266

Peek, R., Prujin, J. M., van der Kemp, A. J. W. and van Venrooij, W. J. (1993). Subcellular distribution of Ro ribonucleoprotein complexes and their constituents. J. Cell Sci. 106: 929-935. 7508449

Poleev, A., Hartmann, A. and Stamm, S. (2000). A trans-acting factor, isolated by the three-hybrid system, that influences alternative splicing of the amyloid precursor protein minigene. Eur. J. Biochem. 267: 4002-4010. 10866799

Quinn, L. M., Dickins, R. A., Coombe, M., Hime, G. R., Bowtell, D. D. and Richardson, H. (2004). Drosophila Hfp negatively regulates dmyc and stg to inhibit cell proliferation. Development 131(6): 1411-23. 14993190

Van Buskirk C., Hawkins N. and Schüpbach T. (2000). encore encodes a member of a novel class of proteins and affects multiple processes during Drosophila oogenesis. Development 127: 4753-4762. 11044391

Van Buskirk, C. V. and Schüpbach, T. (2002). half pint regulates alternative splice site selection in Drosophila. Developmental Cell 2: 343-353. 11879639