InteractiveFly: GeneBrief

Upstream of N-ras: Biological Overview | References

Dosage compensation in Drosophila involves the assembly of the MSL-2-containing dosage compensation complex (DCC) on the single X chromosome of male flies. Translational repression of msl-2 mRNA blocks this process in females. The ubiquitous protein Upstream of N-ras (Unr) is a necessary co-factor for msl-2 repression in vitro. In mammals Unr interacts with PABP (see Drosophila Pabp) within complexes that bind to distinct regions in the target transcripts. This study explored the function of Drosophila Unr in vivo. Hypomorphic Unr mutant flies show DCC assembly on high-affinity sites in the female X chromosomes, confirming that Unr inhibits dosage compensation in female flies. Unexpectedly, male mutant flies and Unr-depleted SL2 cells show decreased DCC binding to the X chromosome, suggesting a role for Unr in DCC assembly or targeting. Consistent with this possibility, Unr overexpression results in moderate loss of DCC from the male X chromosome and predominant male lethality. Immunoprecipitation experiments revealed that Unr binds to roX1 and roX2, the non-coding RNA components of the DCC, providing possible targets for Unr function in males. These results uncover dual sex-specific functions of Unr in dosage compensation: to repress DCC formation in female flies and to promote DCC assembly on the male X chromosome (Patalano, 2009).

Dosage compensation is the process that equalizes the level of X-linked gene expression between males (XY) and females (XX). In Drosophila, dosage compensation occurs by increasing transcription of the single male X chromosome by ~2-fold. Hyper-transcription requires the binding of the dosage compensation complex (DCC) to hundreds of sites along the male X chromosome. The DCC is composed of five proteins (MSL-1, MSL-2, MSL-3, MLE and MOF), the mutation of which causes male-specific lethality, and for this reason the DCC is also known as the male-specific lethal (MSL) complex. The DCC also contains two non-coding RNAs (roX1 and roX2) that appear to have redundant functions. MSL-2 is a limiting RING finger protein that, together with MSL-1, nucleates the assembly of the DCC. MLE (Maleless) is a helicase thought to be required for stable integration of roX RNAs into the DCC, whereas MSL-3 is a chromodomain protein, and MOF (Males absent on the first) is an acetyl-transferase that promotes the acetylation of histone H4 on lysine 16 (H4K16), a modification that specifically marks the compensated X chromosome. Other proteins, in addition to the DCC components, have been implicated in dosage compensation, including the H3S10 kinase JIL-1, the DNA supercoiling factor (SCF), the chromatin-binding protein SU(VAR)3-7, and the nuclear pore components Mtor and NUP153 (Patalano, 2009 and references therein).

In female flies, dosage compensation is inhibited because the expression of msl-2 is repressed by the female-specific RNA-binding protein Sex lethal (Sxl). Enforced expression of MSL-2 leads to the assembly of the DCC on both female X chromosomes and to lethality. Sxl binds to both untranslated regions (UTRs) of msl-2 pre-mRNA and inhibits first the splicing of a facultative intron in the 5' UTR of the transcript, and then its translation in the cytoplasm. Translational repression of msl-2 by Sxl occurs by a double-block mechanism whereby Sxl bound to the 3' UTR inhibits the recruitment of the small ribosomal subunit, and Sxl bound to the 5' UTR inhibits the scanning of those subunits that presumably have escaped the 3'-mediated control. Studies performed in cell-free translation extracts and cultured cells have shown that translational repression requires the recruitment of the co-repressor Upstream of N-ras (Unr) to sequences adjacent to the Sxl binding sites in the 3' UTR (Abaza, 2006; Duncan, 2006). Unr is an evolutionarily conserved RNA-binding protein that contains five cold-shock domains (CSDs) and two glutamine (Q)-rich regions. The first CSD (CSD1) mediates interactions with Sxl and msl-2 mRNA, whereas the N-terminal third of the protein carries most of the translational repression function in vitro (Abaza, 2008). Although Unr is a ubiquitous, primarily cytoplasmic protein that is present in both males and females, it binds to msl-2 only in females because its association depends on Sxl. Thus, Sxl provides a sex-specific function to Unr (Patalano, 2009).

To gain insight into the roles of Unr in development, hypomorphic mutant flies that lack the C-terminal half of Unr were analyzed, as well as flies that overexpress full-length Unr or a fragment containing CSDs 1 and 2. In Unr hypomorphic mutant females, the DCC was detected on a limited set of high-affinity sites on the X chromosomes, indicating that, as predicted from translation studies, Unr represses DCC formation in females. Unexpectedly, Unr mutant males showed decreased DCC recruitment to the X chromosome. Consistent with this, Unr knockdown in male Drosophila SL2 cells abrogated DCC binding without affecting the levels of DCC components or their nucleocytoplasmic distribution. In addition, flies overexpressing Unr showed preferential male lethality and DCC recruitment defects, and the X chromosome of both mutant and transgenic Unr males exhibited an altered morphology. Importantly, roX1 and roX2 RNAs co-immunoprecipitated with Unr in males, suggesting that Unr might function by targeting these non-coding RNAs. These results uncover new roles for Unr in the regulation of dosage compensation in males by a mechanism that is independent of msl-2 translation (Patalano, 2009).

Specific recruitment of Unr to the 3' UTR of msl-2 mRNA by Sxl is required for repression of msl-2 translation both in vitro and in cell culture (Abaza, 2006; Duncan, 2006). A prediction from these results is that Unr represses dosage compensation in female flies. Indeed, in hypomorphic mutant females lacking the C-terminal half of Unr, the DCC assembles on a set of X chromosomal sites. These sites map closely with positions previously described as being high-affinity sites, which are occupied by the DCC in conditions of low complex concentration. These observations suggest partial derepression of msl-2 translation in mutant females. Two of the high-affinity sites correspond to the loci for roX1 and roX2 RNAs (cytological positions 3F and 10C, respectively). Expression of these RNAs requires MSL-2 and their stability depends on their association to the DCC complex. The fact that roX levels were similarly low in mutant and wild-type females supports the notion that msl-2 translational derepression in the mutant is only partial. These results indicate that the N-terminal half of Unr exerts strong translational inhibition in vivo, and are consistent with in vitro data showing that amino acids 1-397 of Unr are sufficient for translational repression in functional tethering assays (Abaza, 2008). Appropriate Unr levels are essential for viability and development because moderate (~2-fold) overexpression of Unr results in complete lethality early in development for both males and females. Accordingly, keeping the correct stoichiometry between Unr and Sxl is important for translational control of msl-2, and might be necessary for the regulation of other substrates (Patalano, 2009).

Unexpectedly, Unr mutant males showed decreased MSL-2 staining on the X chromosome, and Unr-depleted SL2 cells showed MSL-2 delocalization from the X chromosome and redistribution in the nucleoplasm. Reduced MSL-2 targeting to the X chromosome correlated with defective recruitment of other DCC components. These effects were independent of variations in MSL-2 levels, consistent with the observation that Unr does not bind to msl-2 mRNA in males (Abaza, 2006). Because DCC targeting defects have been observed under conditions of unbalanced concentrations of MSL proteins or disturbed MSL/roX ratios, it was reasoned that Unr might regulate the levels of other DCC constituents in males. Strikingly, however, the levels and nucleocytoplasmic distribution of all DCC protein components remained unaltered in Unr-depleted cells. Similarly, the levels of roX RNAs in Unr mutant flies or Unr-depleted cells were indistinguishable from those in the wild type. It is concluded that Unr does not interfere with the expression or localization of DCC components (Patalano, 2009).

In principle, Unr could affect DCC recruitment in males either directly or indirectly. A direct effect could be mediated by MLE and roX. Compared with other DCC proteins, binding of MLE to the X chromosome was more severely affected by Unr mutation or overexpression. MLE is loosely associated with the DCC: the presence of MLE in purified DCC complexes requires protection from RNA degradation and low salt conditions. In addition, RNase treatment of polytene chromosomes removes MLE from the DCC, suggesting that MLE recruitment to the X chromosome requires roX RNAs. Conversely, MLE is an RNA helicase necessary for roX incorporation into the DCC and its helicase activity is necessary for spreading of the DCC along the X chromosome. Thus, the binding of MLE and of roX RNAs to the X chromosome appear to be interdependent. A possible explanation for the role of Unr in males is that Unr affects the function of these DCC components. Unr is a CSD-containing protein and, in bacteria, CSD proteins associate with RNA helicases to modify the structure of RNA and regulate gene expression (reviewed by Horn, 2007). Indeed, mammalian Unr binds to the IRES of Apaf1 mRNA and modifies its conformation (Mitchell, 2003). Therefore, Unr might associate with MLE in order to promote the appropriate structure of the roX RNAs for incorporation into the DCC or for subsequent spreading along the X chromosome. In support of this hypothesis, Unr specifically binds to both roX1 and roX2 RNAs in males. In addition, as previously observed in blastoderm embryos, a fraction of Unr localizes to the nucleus of SL2 and salivary gland cells, where both MLE and roX concentrate (Patalano, 2009).

Unr could also function indirectly, via the regulation of chromatin structure, to promote DCC recruitment to the X chromosome. The Unr hypomorphic mutant and the transgenic Unr flies show abnormal packaging of the male X chromosome, consisting of bloated or knotted chromatin. The observation that staining of histone H3 appears normal suggests that the first level of chromatin compaction remains unaltered in Unr mutants. In order to regulate chromatin structure, Unr could interact with chromatin remodeling factors. For example, a member of the trithorax group, ALL-1 (MLL -- Human Gene Nomenclature Database), was found to interact with human Unr (CSDE1) in a yeast two-hybrid assay (Leshkowtz, 1996). Alternatively, Unr could control the expression of chromatin regulators that influence X chromosome morphology, such as ISWI, NURF, JIL-1 or SU(VAR)3-7. It is interesting to note that although mutations of most of these factors do not concur with loss of DCC binding, null mutations of Su(var)3-7 result in both a bloated X chromosome and depletion of the DCC from the X chromosome (Spierer, 2008). Thus, Unr could regulate the expression of SU(VAR)3-7 -- or of other regulators with similar functions -- in order to modulate DCC recruitment. In summary, at this point the results do not allow conclusion of whether the chromatin-packaging and DCC-binding defects observed in males are dissociable events. Nevertheless, the fact that Unr binds to roX RNAs implicates a direct role of Unr in DCC recruitment. Further studies are necessary to clarify the relationship between the multiple nuclear functions of Unr (Patalano, 2009).

The results show that Unr performs opposing functions in the regulation of dosage compensation in males and females. Dosage compensation is evolutionarily linked to sex determination. In D. melanogaster, a single master protein regulates both processes: Sxl determines the female sexual fate and represses dosage compensation. However, Sxl is not sex-specifically expressed in other distant species of Diptera, raising the possibility that the use of Sxl for sex determination is a recent adaptation of the Drosophila genus (Pomiankowski, 2004). Perhaps, Sxl made use of an existing regulator of dosage compensation, namely Unr, and adapted its function to a new role in females. Further genetic studies and biochemical analyses will help to identify the interactors and substrates that mediate the diverse roles of Unr (Patalano, 2009).

Functional domains of Drosophila UNR in translational control

Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (Sxl) is an essential regulatory step of X chromosome dosage compensation in Drosophila. Translation inhibition requires that Sxl recruits the protein upstream of N-ras (Unr) to the 3' UTR of msl-2 mRNA. Unr is a conserved, ubiquitous protein that contains five cold-shock domains (CSDs). This study dissected the domains of Unr required for translational repression and complex formation with Sxl and msl-2 mRNA. Using gel-mobility shift assays, the domain involved in interactions with Sxl and msl-2 was mapped specifically to the first CSD (CSD1). Indeed, excess of a peptide containing this domain derepressed msl-2 translation in vitro. The CSD1 of human Unr can also form a complex with Sxl and msl-2. Comparative analyses of the CSDs of the Drosophila and human proteins together with site-directed mutagenesis experiments revealed that three exposed residues within CSD1 are required for complex formation. Tethering assays showed that CSD1 is not sufficient for translational repression, indicating that Unr binding to Sxl and msl-2 can be distinguished from translation inhibition. Repression by tethered Unr requires residues from both the amino-terminal Q-rich stretch and the two first CSDs, indicating that the translational effector domain of Unr resides within the first 397 amino acids of the protein. These results identify domains and residues required for Unr function in translational control (Abaza, 2008).

Translational control is widely used in development to regulate processes such as embryonic patterning, cell differentiation, synaptic plasticity, sex determination, and dosage compensation. Dosage compensation is the process that equalizes the expression of X-linked genes in those organisms in which sex determination relies on highly dimorphic sex chromosomes. In Drosophila, dosage compensation is achieved by increasing the transcriptional output of the single male X chromosome by approximately twofold, as a result of the activity of a ribonucleoprotein assembly known as the dosage compensation complex (DCC) or male-specific-lethal (MSL) complex. The DCC fails to assemble in females because the expression of one of its subunits, the protein MSL2, is blocked. The female-specific RNA-binding protein Sex-lethal (Sxl) prevents msl-2 expression via a dual mechanism that includes the inhibition of the splicing of a facultative intron in the 5' UTR of msl-2 pre-mRNA, and the subsequent translational repression of the unspliced message. Translational repression requires Sxl binding to specific U-rich sequences in both the 5' and 3' UTRs of msl-2 mRNA. Sxl binding to the 3' UTR is thought to inhibit the recruitment of the small ribosomal subunit to the mRNA, while Sxl binding to the 5' UTR blocks the scanning toward the AUG initiation codon of those subunits that presumably have escaped control through the 3' UTR. How Sxl inhibits these steps of translation initiation is unknown. Recently, a factor necessary for Sxl-mediated translational repression has been identified as the protein upstream of N-ras (Unr) (Abaza, 2006; Duncan, 2006). Unr is a conserved, ubiquitous protein that is recruited to the 3' UTR of msl-2 by Sxl, but its mechanism of action remains obscure (Abaza, 2008).

Most of the current knowledge about Unr derives from mammalian systems. Human Unr (hUnr) is involved in c-fos mRNA destabilization and the translational repression of pabp mRNA (Chang, 2004; Patel, 2005). In both cases, Unr interacts with PABP within complexes that bind to distinct regions in the target transcripts. Mammalian Unr also regulates translation driven by the internal ribosome entry sites (IRESs) of a number of viral and cellular transcripts, including rhinovirus, poliovirus, c-myc, PITSLRE protein kinase, the pro-apoptotic factor Apaf-1, and Unr itself. At least in the case of Apaf-1, hUnr acts as an RNA chaperone, changing the conformation of the IRES to make it accessible to the activator PTB and, ultimately, the ribosome (Mitchell, 2003). RNA binding by hUnr is mediated by its five cold-shock domains (CSDs), an ancient β-barrel fold containing RNP1 and RNP2 motifs (Brown, 2004). Drosophila Unr (dUnr) contains an additional Q-rich amino terminus that is absent in its mammalian counterpart (Abaza, 2008).

The CSD is a domain highly conserved in evolution used to bind single stranded nucleic acids (Ermolenko, 2002). In addition, the CSD can support protein-protein interactions. Indeed, the CSD1 of Drosophila Unr sustains both binding to msl-2 mRNA and Sxl. Three specific residues within dCSD1 are responsible for these interactions: a tyrosine (Y) that is part of the RNP1 motif, and a lysine (K), and aspartic acid (D), which lay outside the RNP motifs. Although the assay used does not allow distinction between mRNA and protein binding, the location of these amino acids suggests that Y likely mediates msl-2 binding, while K and D may be involved in Sxl interaction. The data do not formally rule out that other domains of dUnr contribute separately to bind either Sxl or msl-2. However, this possibility is unlikely because the efficiency of binding of dCSD1 alone is identical to that of the full-length protein. The use of a dedicated CSD for RNA binding contrasts with the known properties of mammalian Unr. All five CSDs of hUnr are required to bind to the rhinovirus IRES (Brown, 2004). The fact that hUnr can bind to msl-2 mRNA in isolation while dUnr cannot, indeed suggests that the two proteins have different modes of RNA binding (Abaza, 2008).

In order to map the translational effector domain of Unr, tethering analysis was performed. Translational repression by tethered dUnr was less efficient than that observed for Sxl in its natural context, suggesting that Sxl function in 3' UTR-mediated repression is not limited to the recruitment of dUnr. Alternatively, the lesser efficiency of dUnr in repression could be due to aberrant conformation of the recombinant protein or to geometry constraints imposed on the tethered complex. In support for the latter, even though Sxl is critical for msl-2 translational repression, it does not function when tethered to the 3' UTR (Abaza, 2008).

Tethering assays show that dCSD1 is not sufficient for translational repression, indicating that elements in addition to Sxl and msl-2 binding are required for inhibition. These could include the interaction with other corepressors or with components of the translational apparatus. Similar to dCSD1, tethered hUnr could not support translational repression, implying that the translational effector domain is lacking from hUnr. An obvious domain absent in hUnr but present in its Drosophila counterpart is the N-terminal Q-rich domain. This domain contains 52 glutamines interrupted mainly by histidines, resulting in a highly polar stretch suitable for interactions. Certainly, Q-rich domains are present in proteins with diverse roles in gene expression and serve as protein-protein interaction and multimerisation modules. To test whether the Q-rich domain could confer translational repression, it was deleted from dUnr and fused to hUnr. dUnr lacking the Q-rich domain repressed translation less efficiently than the intact protein, indicating that the Q-rich domain was necessary for optimal repression. However, the Q-rich domain did not confer a significant translational repression activity to hUnr, suggesting that residues within the CSDs specific to the Drosophila protein were also relevant. Importantly, the fragment containing the Q-rich domain fused to dCSDs 1 and 2 showed a strong translational repression activity, indicating that the translational effector domain of dUnr is embedded within the first 397 amino acids of the protein. Consistent with these results, analysis of Unr mutant flies indicates that the N-terminal half of Unr exerts robust repression of dosage compensation in females (Abaza, 2008).

TIA-1, a splicing and translation regulator, contains a Q-rich C-terminal domain that interacts with the protein U1C facilitating the recruitment of the U1 snRNP to the 5' splice site (Forch, 2002). By analogy, the Q-rich domain of dUnr could facilitate the recruitment of corepressors, or components of the translation machinery that are so sequestered, to the 3' UTR of msl-2. One such component could be PABP. This translation factor has been shown to interact with hUnr in complexes binding to the coding region of c-fos mRNA and the 5' UTR of pabp mRNA, which are involved in destabilization and translational repression, respectively (Chang, 2004; Patel, 2005). However, it is not immediately obvious how PABP recruitment to the 3' UTR of msl-2 would result in repression, because PABP stimulates translation when tethered to the 3' as it does when it binds to the poly(A) tail. Furthermore, substantial translational repression by the Unr:Sxl complex occurs on nonadenylated msl-2 mRNA. Thus, even though PABP could play a role, additional factors are involved in translational repression by dUnr (Abaza, 2008).

In summary, these data delimit the functional domains of dUnr in msl-2 translational repression. Finding out which factors interact with the translational effector domain will help gain insight into the molecular mechanism of translation inhibition by this essential protein (Abaza, 2008).

Structural basis for the assembly of the Sxl-Unr translation regulatory complex

Genetic equality between males and females is established by chromosome-wide dosage-compensation mechanisms. In the fruitfly Drosophila melanogaster, the dosage-compensation complex promotes twofold hypertranscription of the single male X-chromosome and is silenced in females by inhibition of the translation of msl2, which codes for the limiting component of the dosage-compensation complex. The female-specific protein Sex-lethal (Sxl) recruits Upstream-of-N-ras (Unr) to the 3' untranslated region of msl2 messenger RNA, preventing the engagement of the small ribosomal subunit3. This study reports the 2.8 Å crystal structure, NMR and small-angle X-ray and neutron scattering data of the ternary Sxl-Unr-msl2 ribonucleoprotein complex featuring unprecedented intertwined interactions of two Sxl RNA recognition motifs, a Unr cold-shock domain and RNA. Cooperative complex formation is associated with a 1,000-fold increase of RNA binding affinity for the Unr cold-shock domain and involves novel ternary interactions, as well as non-canonical RNA contacts by the α1 helix of Sxl RNA recognition motif 1. These results suggest that repression of dosage compensation, necessary for female viability, is triggered by specific, cooperative molecular interactions that lock a ribonucleoprotein switch to regulate translation. The structure serves as a paradigm for how a combination of general and widespread RNA binding domains expands the code for specific single-stranded RNA recognition in the regulation of gene expression (Henning, 2014).

Translational repression of msl2 mRNA is coordinated by Sxl binding to uridine-rich stretches in both untranslated regions (UTRs): binding to the 3' UTR inhibits the recruitment of the small ribosomal subunit whereas binding to the 5' UTR inhibits the scanning of those subunits that presumably have escaped the 3' UTR-mediated control. At the 3' UTR, the recruitment of Unr by Sxl to bind in close spatial proximity is critical for translational repression. The region of Sxl containing residues 122-301 (Drosophila RNA binding domain 4, dRBD4) shows full translational repression activity, while the RNA recognition motifs (RRMs) alone (residues 123-294, dRBD3) are necessary and sufficient for RNA binding. Only the first cold-shock domain (CSD1) of Unr is required for complex formation with Sxl and msl2 mRNA. Notably, CSD1 and Sxl do not interact in the absence of RNA, suggesting a cooperative binding mechanism. A 46-nucleotide region in the msl2 3' UTR containing two uridine-rich Sxl-binding sites is sufficient for complex formation and translational repression. To identify the minimal region required for Unr and Sxl binding, ternary complex formation was analysed by electrophoretic mobility shift assays (EMSA) using wild-type and variant RNAs. Binding of dRBD4 and Unr to the wild-type RNA indicated the presence of two complexes. The number of complexes was reduced to one by mutation of either Sxl-binding site, and site F supported complex formation with a higher affinity than site E. Mutation of the sequences surrounding site F affected Unr binding, while more distal mutations did not impair complex formation. These data indicate the formation of 2:2:1 and 1:1:1 dRBD4-Unr-RNA complexes representing the two bands of slower mobility, which was further confirmed by static light scattering measurements (Henning, 2014).

Taken together these data demonstrate that the triple zipper and the non-canonical RNA contacts mediated by Sxl RRM1 are critical for translational regulation by Sxl and Unr. It is important to note that these interactions are essential for msl2 translational repression, but are dispensable for the regulation of transformer pre-mRNA splicing19, as recognition of the uridine-rich 5' region of msl2 RNA by Sxl dRBD3 in the ternary complex is virtually identical to that previously observed for transformer pre-mRNA11. Therefore, recognition of a uridine-rich RNA sequence by Sxl can play distinct roles in regulating splicing and translation depending on the binding of Unr in close proximity (Henning, 2014).

The data also explain why human Unr can form a complex with Drosophila Sxl and msl2 RNA as all residues involved are conserved (His 213, Asp 237 and Arg 239). In contrast, CSD1 alone can bind a variety of distinct RNA sequences with similar affinity in the absence of Sxl. This indicates that strong and specific RNA recognition for the GCACG motif in msl2 RNA depends on the presence of Sxl in the ternary complex. Interestingly, C11 does not conform to the previously reported consensus sequence for human Unr CSD1, but is nevertheless strictly conserved in the msl2 mRNA of organisms that may employ D. melanogaster-like dosage compensatio. Consistent with this, CSD1 Asp 237 and Arg 239, which recognize C11, are conserved in CSD1 but not in CSD2-5 of Unr proteins or in cold shock domains of other proteins (Henning, 2014).

Although the Drosophila dosage-compensation mechanism is not conserved in mammals, it is expected that ternary interactions involving RRM and CSD domains with RNA may be important for other biological functions. For example, human orthologues of the proteins examined in this study, such as the Sxl orthologue HuR or the RNA binding protein RBM6, share triple-zipper and α1-helix residues, which could mediate similar interactions (Henning, 2014).

Sandwiching of single-stranded RNA by multiple proteins has been observed previously, for example in small nuclear ribonucleic particles or the exon junction complex, but the intertwined recognition observed in this study is particularly intriguing. Moreover, the combination of these two general and abundant RNA binding domains (RRM and CSD), which are also involved in other RNA binding events, generates a new and unique binding specificity for single-stranded RNA. The intertwined cooperative binding of Sxl and Unr establishes a functionally active arrangement of multiple RNA binding domains from two distinct proteins, thus extending principles recently observed for multi-domain RNA binding proteins (Henning, 2014).

These results show that repression of a biological process with dramatic consequences for viability depends on the establishment of a specific set of novel molecular interactions. This is of particular significance considering that a limited set of RNA binding modules has been identified in the mRNA interactome. The Unr-Sxl-msl2 complex illustrates how the combinatorial activity of general RNA binding domains expands the code for RNA recognition by establishing unique and distinct ribonucleoprotein architectures and thus greatly amplifying the opportunities for regulation of gene expression (Henning, 2014).

UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation

Dosage compensation is a regulatory process that balances the expression of X-chromosomal genes between males (XY) and females (XX). In Drosophila, this requires non-coding RNAs and RNA-binding proteins (RBPs) whose specific functions remain elusive. This study shows that the Drosophila RBP UNR promotes the targeting of the activating male-specific-lethal complex to the X-chromosome by facilitating the interaction of two crucial subunits: the RNA helicase MLE and the long non-coding RNA roX2 (Militti, 2014).

In Drosophila, dosage compensation involves the binding of the MSL dosage compensation complex (MSL-DCC) to hundreds of sites on the single male X-chromosome and the subsequent twofold hypertranscription of active genes. The MSL-DCC contains five proteins: MSL1, MSL2, MSL3, Maleless (MLE) and Males-absent-on-first (MOF), and two long non-coding RNAs, RNA on X (roX) 1 and 2, which differ in size and sequence but display redundant functions. MSL2 is the limiting subunit of the MSL-DCC and, together with MSL1, nucleates complex formation at specific X-chromosomal sites known as high-affinity sites (HAS). The RNA helicase MLE colocalizes with MSL2 at HAS and facilitates the incorporation of roX into the complex. The RNA is important for the distribution of the MSL-DCC along the X-chromosome, where it activates target genes (Militti, 2014).

Upstream of N-Ras (UNR) is a conserved RNA-binding protein (RBP) containing five cold-shock domains (CSD) that regulates mRNA translation and stability by interacting with single-stranded RNA. It has been shown previously that Drosophila UNR performs sex-specific opposing roles in dosage compensation. In females, UNR inhibits MSL-DCC assembly by repressing the synthesis of MSL2. In males, UNR promotes the targeting of the MSL-DCC to the X-chromosome by a poorly understood mechanism that does not involve translational regulation of MSL proteins . UNR-dependent regulation can be recapitulated in male S2 cells, which express only roX2. This work describes efforts to determine whether UNR interacts with the DCC assembly and targeting machinery in more specific ways. Primed by the finding of a preferred RNA-binding element for UNR close to the known roX remodelling site of the helicase MLE, this study systematically explored the relationship between UNR and MLE. UNR was found to facilitates the binding of MLE to its target at limiting MLE concentrations. These biochemical analyses approximate the physiological conditions to a good extent, since depletion of UNR also diminishes the MLE-roXinteraction and reduces the association of MLE with HAS in dosage-compensating cells. These results identify UNR as a general RBP with specific roles in dosage compensation (Militti, 2014).

To assess whether recombinant UNR could directly bind to roX2, an electrophoretic mobility shift assay (EMSA) was used. UNR binds full-length roX2 RNA and several roX2-derived fragments. UNR interacted most strongly with a roX2 fragment spanning nucleotides 316-379. Interestingly, this purine-rich region is located within stem-loop 6 (SL6) just upstream of a prominent, conserved stem-loop structure (SLroX2 or SL7) that, when multimerized, is sufficient to restore the X-chromosomal targeting defects of a roX null mutant. Pull-down assays using MS2-tagged RNA as bait confirmed that UNR bound with highest affinity to roX2 fragments containing SL6 (Militti, 2014).

Because of the roles of UNR in RNA metabolism, whether UNR binding affected roX2 levels, nucleocytoplasmic distribution or splicing of its major isoforms in S2 cells was assessed. UNR depletion showed no effect on any of these features. To gain insight into UNR-roX2 interactions, enzymatic and chemical footprinting was performed. Addition of UNR protected unpaired nucleotides of SL6, in particular the terminal loop (nts 367-374) and the internal bulge (nts 352-357), suggesting UNR binding to these regions. UNR also protected to a lower extent the terminal loop of SL7 and the single-stranded region between SL7 and SL8 but did not protect efficiently the terminal loop of SL8. These results confirmed that UNR interacts preferentially with SL6. Mutational analysis showed that UNR recognizes the purine-rich stretches in the loops. Interestingly, nucleotides 417-419 and 497 were rendered more reactive upon the addition of UNR. These positions form a 7bp-extended version of SL7, which maybe disrupted by UNR. These results suggest a role for UNR as a roX2 chaperone (Militti, 2014).

In bacteria, CSD-containing RNA chaperones associate with RNA helicases to promote RNA remodelling . It was thus, asked whether UNR interacts with the RNA helicase MLE. Co-immunoprecipitation experiments with recombinant proteins demonstrated a weak interaction between MLE and UNR. Interestingly, this interaction was strongly stimulated by roX2 but not by an unrelated control RNA of similar length. The interaction persisted after efficient RNase treatment, monitored by measuring the presence of trace-labelled roX2 in the pellet, indicating that roX2 promotes strong direct interactions between MLE and UNR, or that any connecting RNA within the complex is protected from RNase digestion. To test whether these interactions occurred in vivo, UNR was immunoprecipitated from nuclear extracts of S2 cells. Although UNR is primarily cytoplasmic, a small amount can be found in the nucleus that interacts with MLE, but not with MSL3, indicating that the interaction is specific. Consistent with the in vitro data and with the presence of roX2 in the nucleus, the interaction of endogenous MLE and UNR is resistant to RNase treatment after formaldehyde crosslinking, suggesting that interactions involve protein-protein crosslinks and not merely protein-RNA crosslinks. Altogether, the data suggest that UNR, MLEand roX2 form a complex. Indeed, a ternary complex is detected by EMSA when roX2 or its 3' half fragment (SL678) is incubated with recombinant UNR and MLE (Militti, 2014).

Intriguingly, the base of the extended SL7 of roX2 that is potentially remodelled by UNR serves as a binding site for MLE, which disrupts SL7 thereby promoting subsequent interactions with MSL2. Thus, melting of the base of SL7 by UNR and exposure of the corresponding nucleotides could facilitate MLE interaction with roX2. To test this hypothesis, binding of recombinant UNR and MLE to roX2 derivatives was monotored using RNA pull-down assays. MLE on SL67 was titrated in the presence or absence of UNR. At high concentrations, MLE was able to bind SL67. MLE, however, was unable to efficiently bind the target RNA at reduced concentrations. Remarkably, under those conditions the interaction of MLE with SL67 was strongly facilitated by adjacent UNR binding. To test the correlation between MLE and UNR binding, the roX2 derivatives SL2345 and SL67 were employed. UNR bound to both RNAs in the absence of MLE and, consistent with the EMSA, it bound with higher affinity to SL67. MLE bound to roX2 fragments after UNR addition in a manner that correlated with the strength of UNR binding. In addition, MLE bound strongly to full-length roX2 in the presence of UNR, and this binding was reduced upon deletion of either SL6 or SL7. These results indicate that UNR promotes the association of MLE with roX2 in vitro (Militti, 2014).

To test whether UNR promotes the association of MLE with roX2 in vivo, UNR from was depleted from S2 cells. Ablation of UNR diminished the amounts of roX2 associated with the endogenous helicase. This effect could neither be attributed to variations in the amounts of MLE or roX2 upon UNR depletion nor to differences in the efficiency of MLE immunoprecipitation. To explore whether the reduced MLE-roX2 interaction affects the association of MLE with its chromosomal targets, the HAS, chromatin immunoprecipitation (ChIP) was used. Notably, it was found that UNR depletion decreased the MLE association to four different HAS. As controls, UNR depletion did not affect the association of MLE to promoters within the same genes, or the association of MOF to HAS or to an autosomal gene. Altogether, these results demonstrate a role for UNR in facilitating the binding of the RNA helicase MLE with its target sites on roX2 RNA in vitro and in vivo (Militti, 2014).

It is concluded that in the absence of MLE (and roX), MSL2 and MSL1 can associate with HAS but no functional DCC is assembled that distributes the activating histone acetylation to the target genes. ChIP experiments show prominent, specific interaction of MLE at HAS in S2 cells. The current data now suggest that the general RBP UNR facilitates this interaction. Remarkably, UNR also binds and regulates the activity of msl2 mRNA together with the sex determination switch SXL , suggesting that sex determination and dosage compensation have co-evolved to employ similar factors from the large portfolio of RBPs. UNR, thus, extends the small list of RBPs involved in dosage compensation. Such factors include hnRNPU and YY1, which tether the lncRNA Xist to the inactive X-chromosome during mammalian dosage compensation. Rather than a molecular tether, however, the role of UNR seems more transient and directed to facilitate initial steps of MSL-DCC assembly through the modulation of RNA conformation. UNR, therefore, acts as a 'catalyst' of the MLE-roX interaction. RNA structural transitions are at the basis of many fundamental post-transcriptional processes . The current results illustrate the emerging concept that lncRNA structural dynamics may contribute to chromatin organization, and indicate that general RBPs, such as UNR, can be harnessed to contribute key molecular events in the assembly of specialized machineries (Militti, 2014).

Sex-lethal imparts a sex-specific function to Unr by recruiting it to the msl-2 mRNA 3' UTR: translational repression for dosage compensation

MSL-2 (male-specific lethal 2) is the limiting component of the Drosophila dosage compensation complex (DCC) that specifically increases transcription from the male X chromosome. Ectopic expression of MSL-2 protein in females causes DCC assembly on both X chromosomes and lethality. Inhibition of MSL-2 synthesis requires the female-specific protein sex-lethal (Sxl), which binds to the msl-2 mRNA 5' and 3' untranslated regions (UTRs) and blocks translation through distinct UTR-specific mechanisms. Translationally silenced msl-2 mRNPs has been purified and Unr (upstream of N-ras) has been identified as a protein recruited to the 3' UTR by Sxl. Sxl requires Unr as a corepressor for 3'-UTR-mediated regulation, imparting a female-specific function to the ubiquitously expressed Unr protein. These results reveal a novel functional role for Unr as a translational repressor and indicate that Unr is a key component of a 'fail-safe' dosage compensation regulatory system that prevents toxic MSL-2 synthesis in female cells (Duncan, 2006).

Sequence analysis revealed that the protein specifically associated with translationally silenced msl-2 mRNA exhibited significant similarity to the previously characterized mammalian protein Unr. This was surprising for a putative translational corepressor, because mammalian Unr, a cytoplasmically localized RNA-binding protein, stimulates translation of both viral and cellular internal ribosome entry site (IRES) containing mRNAs. Unr is also a major regulator of translationally coupled mRNA turnover mediated by the c-fos mCRD RNA element (Duncan, 2006).

Unr has five cold-shock nucleic acid-binding domains, each with the unique substitution of the sequence FFH for the canonical FVH in part of the RNA-binding surface. CG7015, coding for the identified protein, also has five cold-shock domains (CSDs) with the signature FFH motif. Overall sequence identity between CG7015 and human Unr is ~45%, and this is higher within the CSDs (70%, 56%, 51%, 53%, and 66% identity for CSD-1-CSD-5, respectively). The Drosophila genome encodes no other protein with similarly high sequence identity to mammalian Unr, and it is therefore concluded that ORF CG7015 is Drosophila Unr and it is referred to as 'Unr' hereafter (Duncan, 2006).

This study has identified a novel component of the dosage-compensation regulatory machinery that has eluded genetic methods. Using an mRNP purification approach and functional analysis, the Drosophila Unr protein has been demonstrated to be recruited to msl-2 mRNA 3' UTR by Sxl for translational inhibition of msl-2 mRNA specifically in female cells. These data indicate that Sxl imparts a female-specific translational repressor function to Unr, and imply that this novel function of Unr is critical for negative regulation of the dosage compensation machinery to prevent toxic effects in female cells (Duncan, 2006).

Previous results implied that region 2456 of the msl-2 mRNA, 3'-UTR sequences adjacent to the Sxl-binding sites, is important for translational regulation via the 3' UTR (Grskovic, 2003). Since this region flanks the Sxl-binding sites, it was hypothesized to bind a putative corepressor that acts in conjunction with Sxl. Glutathione RNA (GRNA) chromatography in combination with sucrose-density gradient centrifugation was used to purify this factor, identifying Drosophila Unr. Functional analyses of msl-2 reporter genes and endogenous msl-2 expression in female and male cell lines demonstrate that Unr is necessary for translational repression of msl-2 mRNA by Sxl via the 3' UTR, but does not affect msl-2 mRNA translation in the absence of Sxl. Taken together, these results show that Unr is a cofactor for translational repression of MSL-2 protein synthesis, specifically in female cells. These conclusions are strongly supported by the results of Abaza, (2006), who independently isolated Unr using a different approach and could demonstrate that direct interaction of Unr with Sxl helps recruit Unr to the msl-2 3' UTR and is critical for translational inhibition of msl-2 reporter mRNAs by Sxl in vitro. This is the first time that a translational corepressor has been identified by a combined strategy of gradient and specific mRNP purification, and it is anticipated that this method will prove useful as a general strategy (Duncan, 2006).

Analysis of msl-2-ß-gal reporters further supports the recently proposed dual-mechanism model for msl-2 mRNA translational inhibition, which predicts that 3'-UTR corepressors should be required exclusively for 3'-UTR-mediated inhibition. Indeed, Unr depletion significantly affects only msl-2 reporters with wild-type 3' UTRs, and the strongest effect is on the 5'mut reporter, where all regulation must occur through the 3' UTR. In this case, the quantitative effect of Unr depletion approaches the effect of Sxl depletion. Since RNAi produces a 'knockdown' effect that likely reflects a partial-loss-of-function rather than true null phenotype, differences in RNAi efficiency and/or differences in relative concentrations of Unr and Sxl necessary for inhibition may explain why the 5'mut reporter is still slightly repressed after Unr knockdown. In any case, the in vivo analysis presented here directly supports the concept of independent regulatory contributions of 5'- and 3'-UTR Sxl complexes, and implies that Unr is a critical component for 3'-UTR-mediated inhibition (Duncan, 2006).

How does Unr recruited by Sxl to the 3' UTR interfere with translation initiation at the mRNA 5' end? Presumably the Sxl/Unr corepressor complex interacts with factors that affect small ribosomal subunit recruitment. This interaction might require direct participation of Sxl, or Sxl might serve only to recruit Unr to the 3' UTR. Similarly, Unr might directly contact factors affecting small subunit recruitment, or may do so through additional bridging factors as part of a larger 'corepressor assembly'. The biochemical approach identified factors in addition to Unr that specifically copurify with the repressed mRNP. Interestingly, Unr is the only one of the copurified proteins that displays significant corepressor activity when assayed by RNAi in Kc cells (Duncan, 2006).

Since msl-2 mRNA repression functions in the absence of a 5' m⁷GpppN cap structure, translational regulatory proteins like Cup or d4EHP are unlikely to be the molecular targets of repression by the 3'-UTR complex. A candidate target is Drosophila PABP, since mammalian PABP interacts with Unr, and promotes small ribosomal subunit binding to the mRNA. Although msl-2 translational inhibition does not require a poly(A) tail, PABP appears to have a critical function in initiation that is independent of the poly(A) tail, raising the possibility that Unr might nevertheless interfere with PABP-mediated recruitment of the small ribosomal subunit to msl-2 mRNA. Future studies will aim to determine the mechanism by which Sxl and Unr bound at the 3' end of msl-2 mRNA block translation initiation at the 5' end (Duncan, 2006 and references therein).

Consistent with a general role as a regulator of dosage compensation, Unr mRNA expression is ubiquitous throughout Drosophila development. Interestingly, Unr protein is expressed at similar levels in both male and female cells in culture and in flies (Abaza, 2006), but interacts with msl-2 mRNA to modulate its translation only when Sxl is present. Thus, Sxl imparts a sex-specific, mRNA-specific translational repressor function to Unr. Sex-specific modulation of Unr function by Sxl is presumably crucial for dosage compensation, which would be compromised if the abundant Unr protein in males were able to inhibit msl-2 mRNA translation (Duncan, 2006).

Sex-specific function at the cellular and organismal level can also be viewed as context-specific function at the molecular level, with Sxl acting as a context-specific modulator of Unr function. The hypothesis that Unr function is modulated by molecular context is supported by the previously determined functions of mammalian Unr, which involve different protein-interaction partners, in the context of different RNA sequence elements. Indeed, the previously reported role for mammalian Unr as a translational activator of cellular and viral IRESes made it a rather unexpected candidate for a translational corepressor. These data identify the first function for Unr in Drosophila, and demonstrate the surprising finding that Unr can also be a critical component of translational repression complexes, underscoring the importance of both protein and RNA context in modulation of Unr function in post-transcriptional control of gene expression (Duncan, 2006).

Another notable difference between Unr-mediated translational repression and the Unr functions described previously is that in the former case Unr is the recruited protein, whereas in the latter cases, high-affinity interaction of Unr with an RNA element underlies subsequent recruitment of additional proteins by Unr. This distinction has two important implications for Unr function. First, Unr's potential regulatory targets are not confined to mRNAs with high-affinity binding sites for Unr. Second, context-specific modulators such as Sxl can be expected to be key determinants of how Unr affects regulation of a particular mRNA. Detailed mechanistic and structural analysis will be essential to answer the intriguing question of how Unr can function as a translational activator in one molecular context, and a repressor in another (Duncan, 2006).

Unr depletion in Kc cells causes a significant increase in MSL-2 protein to ~20% of that in male SL-2 cells or Sxl-depleted Kc cells. Clearly, Sxl-dependent, Unr-independent inhibition mediated by the msl-2 5' UTR contributes to repression of MSL-2 protein synthesis. It was also observed that Sxl promotes reduced endogenous msl-2 mRNA levels, but Unr does not. The results warrant interpretation in the context of previous studies of transgenic flies; females expressing msl-2 transgenes lacking the 3'-UTR regulatory sequences produce detectable MSL-2 protein, but at a significantly lower level than males or females expressing transgenes with both 5'- and 3'-UTR Sxl-binding sites deleted. The lower level of MSL-2 protein made in 3'-UTR mutant females is nevertheless sufficient to promote DCC loading onto female X chromosomes. Therefore, at the organismal level, Unr, acting through the msl-2 mRNA 3' UTR, can be expected to make a significant contribution to robust repression of MSL-2 protein synthesis and prevention of deleterious activation of the X-chromosome dosage-compensation machinery in females (Duncan, 2006).

Drosophila Unr is required for translational repression of male-specific-lethal 2 mRNA during regulation of X-chromosome dosage compensation

The inhibition of male-specific lethal 2 (msl-2) mRNA translation by the RNA-binding protein sex-lethal (Sxl) is an essential regulatory step for X-chromosome dosage compensation in Drosophila. The mammalian upstream of N-ras (Unr) protein has been implicated in the regulation of mRNA stability and internal ribosome entry site (IRES)-dependent mRNA translation. The Drosophila homolog of mammalian Unr has been identified as a cofactor required for Sxl-mediated repression of msl-2 translation. Unr interacts with Sxl, a female-specific protein. Although Unr is present in both male and female flies, binding of Sxl to uridine-rich sequences in the 3' untranslated region (UTR) of msl-2 mRNA recruits Unr to adjacent regulatory sequences, thereby conferring a sex-specific function to Unr. These data identify a novel regulator of dosage compensation in Drosophila that acts coordinately with Sxl in translational control (Abaza, 2006).

Inhibition of msl-2 expression is essential for development of female flies; forced expression of MSL-2 causes the assembly of the DCC on both X chromosomes and lethality. The Drosophila homolog of mammalian Unr is necessary to inhibit msl-2 expression. Drosophila Unr is recruited to the 3' UTR of msl-2 mRNA by dSxl, a female-specific protein, and plays an essential role in repressing its translation. Unr associates to the 3' UTR of msl-2 mRNA in female cells and is necessary to repress msl-2 translation in vivo. Together, these data identify Unr as a regulator of dosage compensation (Abaza, 2006).

In vitro selection experiments (SELEX) indicate that human Unr binds to purine-rich regions in the mRNA, with the consensus sequences (A/G)₅AAGUA/G or (A/G)₈AACG and an apparent dissociation constant (Kd) of ~10 nM. Although Drosophila Unr also recognizes purine-rich sequences in the 3' UTR of msl-2 mRNA that fall within these consensus, it does so with a very poor affinity, a situation reminiscent to that of the bacterial cold-shock proteins. Binding of Unr to msl-2 mRNA requires the binding of Sxl. The observation that msl-2 RNA fragments containing mutated Sxl-binding sites, but wild-type Unr-binding sites, do not bind to either of the two proteins suggests that Sxl does not simply induce a conformational change in Unr that allows it to bind RNA. Rather, Sxl recruits Unr to bind in close proximity in the 3' UTR of msl-2 mRNA. Stable recruitment of Unr requires the interaction of Unr with both Sxl and msl-2 mRNA, as supported by the following evidence: (1) Unr is not retained in the dRBD4 column unless this column is saturated with msl-2 mRNA; (2) dUnr is not retained in the mRBD column despite the presence of msl-2 mRNA; (3) no complex formation is observed in a gel mobility-shift assay when the Unr-binding sites are mutated (mut2456); (4) msl-2 mRNA and Unr do not interact in male flies, which lack Sxl. Nevertheless, Unr and Sxl can interact directly in vitro. Addition of EF RNA does not improve this interaction, and addition of embryo extract actually competes it. These results suggest that, although the interaction of Sxl and Unr can occur directly, the interaction with msl-2 mRNA stabilizes the complex in the competitive conditions of the extract (Abaza, 2006).

Unr protein and msl-2 mRNA do not interact in male flies despite their relative abundance. In addition, supplementing cytoplasmic embryo extracts with Sxl -- a primarily nuclear protein -- promotes Unr association with msl-2 mRNA, and translational repression by Unr is only observed in Sxl-containing cells. These data suggest that the interaction of Unr with msl-2 mRNA is mediated by Sxl in vivo, and imply that Sxl is the critical determinant for the formation of a repressive complex on the 3' UTR of msl-2 mRNA. In this scenario, Sxl conveys a sex-specific function to Unr. The stepwise assembly of a translation inhibitory complex on msl-2 mRNA is reminiscent of Drosophila hunchback. The 3' UTR of maternal hunchback mRNA is bound by Pumilio (Pum), and this event triggers the sequential recruitment of Nanos (Nos) and Brain tumor (Brat), which ultimately results in the translational repression of hunchback mRNA. Sequential binding of Sxl and Unr to msl-2 mRNA could result from their respective subcellular locations: While Sxl is nuclear and associates with msl-2 pre-mRNA, Unr is primarily, if not exclusively, cytoplasmic. Interestingly, Unr accumulates at the nuclear periphery, which perhaps reflects or ensures the rapid formation of repressive complexes as msl-2 and probably other mRNAs are exported to the cytoplasm. Additionally, accumulation around the nucleus could reflect the association of Unr with the endoplasmic reticulum, as reported for mammalian Unr (Abaza, 2006).

Dosage compensation is believed to function from the blastoderm stage. As expected for a protein involved in the regulation of dosage compensation, Unr is present throughout development. Curiously, although Unr mRNA is dramatically more abundant in female flies, this difference is compensated at the protein level, suggesting the existence of sex-specific mechanisms to modulate Unr expression. Indeed, the amount of Unr might need to be tightly controlled. Overexpression of mammalian Unr leads to cell death, and preliminary data suggest that substantial overexpression of Unr results in lethality of both male and female flies. Three forms of Unr mRNA can be detected in Drosophila. Several mRNAs have also been detected in mammals, consistent with the observation of three alternative polyadenylation sites of the hUnr gene and alternative splicing of the hUnr pre-mRNA. These data suggest that the different Unr mRNAs arise by alternative processing, although the significance of this observation remains to be explored (Abaza, 2006).

The role of Unr in Drosophila contrasts with the known functions of Unr in mammals. hUnr is part of a complex assembled on the coding region of c-fos mRNA that is involved in the deadenylation-dependent destabilization of this transcript. The interaction of hUnr with PABP within this complex is believed to bridge the complex to the poly(A) tail, although the mechanism by which the complex influences deadenylation is unknown. In Drosophila, the steady-state levels of msl-2 mRNA are, indeed, lower in females. However, no effect of Unr and Sxl on msl-2 mRNA stability was detected in translation assays. hUnr also binds to the IRES elements in the 5' UTRs of several transcripts and activates their translation. In the best understood example, that of Apaf-1 mRNA, hUnr induces a conformational change in the IRES that makes it accessible for binding of PTB, a positive regulator of Apaf-1 translation. Contrary to hUnr, Drosophila Unr binds to the 3' UTR of msl-2 mRNA and represses its translation. Nonetheless, the underlying effects of Unr binding may be similar if Unr acts as an RNA or RNP chaperone to facilitate an RNA conformation, or the assembly of repressive factors, that inhibit translation (Abaza, 2006).

Translation of msl-2 occurs via a cap-dependent mechanism. Cap-dependent translation initiation involves the recruitment of 43S ribosomal complexes (molecular assemblies of the 40S ribosomal subunit with a set of translation initiation factors and the initiator tRNA) to the cap structure at the 5' end of the mRNA. Translation inhibition mediated by the 3' UTR of msl-2 results from a block of 43S ribosomal recruitment. However, translational repression of msl-2 mRNA by Sxl can occur in the absence of a cap structure and a poly(A) tail. Understanding how 43S recruitment is affected by Sxl without the involvement of the cap is, indeed, intriguing. A possibility is that, as with mammalian Unr, Unr interacts with PABP. PABP could, in turn, exert a poly(A)- and cap-independent effect on translation. Certainly, the mapping of Unr domains relevant for translational control and the identification of dedicated factors that interact with Unr are likely to provide insights into this mechanism of translation regulation that is key to control dosage compensation in Drosophila (Abaza, 2006).

Search PubMed for articles about Drosophila N-ras

Abaza, I., Coll, O., Patalano, S. and Gebauer, F. (2006). Drosophila Unr is required for translational repression of male-specific-lethal 2 mRNA during regulation of X-chromosome dosage compensation. Genes Dev. 20: 380-389. PubMed ID: 16452509

Abaza, I. and Gebauer, F. (2008). Functional domains of Drosophila Unr in translational control. RNA 14: 482-490. PubMed ID: 18203923

Brown, E. C. and Jackson, R. J. (2004). All five cold-shock domains of UNR (upstream of N-ras) are required for stimulation of human rhinovirus RNA translation. J. Gen. Virol. 85: 2279-2287. PubMed ID: 15269369

Chang, T.C. et al. (2004). UNR, a new partner of poly(A)-binding protein, plays a key role in translationally coupled mRNA turnover mediated by the c-fos major coding-region determinant. Genes Dev. 18: 2010-2023. PubMed ID: 15314026

Duncan, K., et al. (2006). Sex-lethal imparts a sex-specific function to Unr by recruiting it to the msl-2 mRNA 3' UTR: translational repression for dosage compensation. Genes Dev. 20: 368-379. PubMed ID: 16452508

Ermolenko, D. N. and Makhatadze, G. I. (2002). Bacterial cold-shock proteins. Cell. Mol. Life Sci. 59: 1902-1913. PubMed ID: 12530521

Forch, P., et al. (2002). The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5' splice sites. EMBO J. 21: 6882-6892. PubMed ID: 12486009

Hennig, J., Militti, C., Popowicz, G. M., Wang, I., Sonntag, M., Geerlof, A., Gabel, F., Gebauer, F., Sattler, M. (2014) Structural basis for the assembly of the Sxl-Unr translation regulatory complex. Nature. PubMed ID: 25209665

Horn, G., Hofweber, R., Kremer, W. and Kalbitzer, H. R. (2007). Structure and function of bacterial cold shock proteins. Cell Mol. Life Sci. 64: 1457-1470. PubMed ID: 17437059

Leshkowitz, D., Rozenblatt, O., Nakamura, T., Yano, T., Dautry, F., Croce, C. M. and Canaani, E. (1996). ALL-1 interacts with unr, a protein containing multiple cold shock domains. Oncogene 13: 2027-2031. PubMed ID: 8934551

Militti, C., Maenner, S., Becker, P. B. and Gebauer, F. (2014). UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation. Nat Commun 5: 4762. PubMed ID: 25158899

Mitchell, S. A., Spriggs, K. A., Coldwell, M. J., Jackson, R. J. and Willis, A. E. (2003). The Apaf-1 internal ribosome entry segment attains the correct structural conformation for function via interactions with PTB and unr. Mol. Cell 11: 757-771. PubMed ID: 12667457

Patalano, S., Mihailovich, M., Belacortu, Y., Paricio, N. and Gebauer, F. (2009). Dual sex-specific functions of Drosophila Upstream of N-ras in the control of X chromosome dosage compensation. Development 136(4):689-98. PubMed ID: 19168682

Patel, G. P., Ma, S. and Bag, J. (2005). The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex. Nucleic Acids Res. 33: 7074-7089. PubMed ID: 16356927

Pomiankowski, A., Nöthiger, R. and Wilkins, A. (2004). The evolution of the Drosophila sex determination pathway. Genetics 166: 1761-1773. PubMed ID: 15126396

Spierer, A., Begeot, F., Spierer, P. and Delattre, M. (2008). SU(VAR)3-7 links heterochromatin and dosage compensation in Drosophila. PLoS Genet. 4: e1000066. PubMed ID: 18451980

date revised: 25 October 2014

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