sans fille


REGULATION

Protein Interactions

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Since Fl(2)d is expressed in both sexes and has functions in alternative splicing that are not connected to Sxl, it could be argued that the physical interactions detected between Fl(2)d and different components of the splicing apparatus do not reflect its functioning in Sxl-dependent alternative splicing. While this is a potential concern, there are a number of reasons why this is believed to be unlikely. For one, these complexes appear to be physiologically relevant to Sxl-dependent alternative splicing; female-specific genetic interactions were observed between fl(2)d and the genes encoding several of these proteins. In the case of snf, not only a null allele was tested, but also two mutations, snf148 and snf5mer, which differentially affect Snf protein interactions with the U1 or the U2 snRNPs, respectively. When mothers heterozygous for the U1-deficient snf148 are mated to Sxl- fathers, there is a marked reduction in the viability of female offspring. This female lethality is enhanced by the antimorphic allele fl(2)d1, but not by the null allele fl(2)d2. In contrast to snf148, there is little, if any, female-specific lethality in the progeny of snf5mer/+ females and Sxl- fathers; however, the snf5mer allele shows a very strong synergistic female lethal interaction with fl(2)d1. Likewise, a strong synergistic interaction was observed between fl(2)d1 and U2AF38δE18 (Penn, 2008).

Another reason to believe that the association of Fl(2)d with early splicing regulators is relevant to how it promotes Sxl-dependent alternative splicing is the fact that Sxl is found in complexes with the same set of splicing factors in nuclear extracts as Fl(2)d. As noted above, these factors include Snf, U170k, and the two U2AF subunits U2AF38 and U2AF50. In the case of Snf, it has been shown that Sxl is in an immunoprecipitable complex with Snf in nuclear extracts. Although this is also true for Fl(2)d, there are some differences in how Fl(2)d and Sxl interact with Snf. For one, Fl(2)d:Snf interactions in nuclear extracts are insensitive to RNase, while Sxl:Snf interactions are RNase sensitive. While it was not possible to test whether Fl(2)d:Snf interactions involve direct protein contacts, both Fl(2)d and Snf can interact directly with Sxl in vitro. Fl(2)d and Sxl also differ in their interactions with the Snf mutant proteins 148 and 5mer. Sxl can associate with the Snf5mer protein, but does not form a complex with Snf148. By contrast, Fl(2)d complex formation with the Snf148 mutant protein appears to be equivalent to that observed for wild-type Snf, while complexes with Snf5mer appear to be destabilized and are present in reduced yield. In addition, it was also found that Sxl resembles Fl(2)d in that it is not stably associated either with the U5 snRNP protein U5-40k or with SKIP (Penn, 2008).

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

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

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


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

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