The pre-mRNA splicing factor U2AF plays a critical role in 3' splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3' splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. hU2AF65 contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF35 has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has been assigned to hU2AF65. While the requirement for the three RRMs on hU2AF65 is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. The RNA binding activity of the U2AF heterodimer was examined in vitro. When the Drosophila small-subunit homolog (dU2AF38) is complexed with the large-subunit (dU2AF50), pyrimidine tract RNA binding activity increased 20-fold over that of free dU2AF50. A similar increase in RNA binding activity is detected when the human U2AF heterodimer and hU2AF65 are compared. Surprisingly, the RS domain on dU2AF38 was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF50DeltaRS) severely impairs its binding activity. However, if the dU2AF38 RS domain is supplied in a complex with dU2AF50DeltaRS, high-affinity binding is restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo (Rudner 1998a).
The pre-mRNA splicing factor U2AF (U2 snRNP auxiliary factor) has an essential role in 3' splice site selection. U2AF binds the intron pyrimidine tract between the branchpoint and the 3' splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. Both subunits contain a domain enriched in arginine-serine dipeptide repeats termed an RS domain. The two U2AF RS domains have been assigned essential and independent roles in spliceosome assembly in vitro -- the hU2AF65 RS domain is required to target U2 snRNP to the branch site and the hU2AF35 RS domain is necessary for protein-protein interactions with constitutive and alternative splicing factors. The functional requirements for the RS domains on the Drosophila U2AF homolog have been examined in vivo. In sharp contrast to its essential role in U2 snRNP recruitment in vitro, the RS domain on the Drosophila large subunit homolog (dU2AF50) is completely dispensable in vivo. Prompted by this unexpected result, the RS domain on the Drosophila small subunit homolog (dU2AF38) was examined. Despite its requirement for enhancer-dependent splicing activity in vitro, the dU2AF38 RS domain was also inessential in vivo. Finally, whether the Drosophila U2AF heterodimer requires any RS domain was investigated. Flies mutant for both the small and large subunits could not be rescued by dU2AF50deltaRS and dU2AF38deltaRS transgenes. Therefore, in contrast to the separate roles assigned to the U2AF RS domains in vitro, genetic data suggest that they may have redundant functions in vivo (Rudner 1998b).
The heterodimeric pre-mRNA splicing factor, U2AF, plays a critical role in 3' splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. A genetic approach was taken to assess the requirement for the Drosophila U2AF heterodimer in vivo. A novel Escherichia coli copurification assay was developed to map the domain on the Drosophila U2AF large subunit (dU2AF50) that interacts with the Drosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, this 28-amino-acid interaction domain was scanned for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolishes interaction with dU2AF38 is incapable of complementation, whereas dU2AF50 mutations that do not affect heterodimer formation rescue the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines reveal a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro (Rudner 1998c).
The protein Sex-lethal activates a female-specific 3' splice site in the first intron of Transformer pre-mRNA while repressing an alternative non-sex-specific site. Using an in vitro system, the molecular basis of the splice site switch has been determined. Sxl inhibits splicing to the non-sex-specific (default) site by specifically binding to its polypyrimidine tract, blocking the binding of the essential splicing factor U2AF. This enables U2AF to activate the lower-affinity female-specific site. A splicing 'effector' domain present in U2AF but absent from Sxl accounts for the different activities of these two polypyrimidine-tract-binding proteins: addition of the U2AF effector domain to Sxl converts it from a splicing repressor to an activator and renders it unable to mediate splice-site switching (Valcarcel, 1993).
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 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).
In higher eukaryotes, the polypyrimidine-tract (Py-tract) adjacent to the 3' splice site is recognized by several proteins, including the essential splicing factor U2AF65, the splicing regulator Sex-lethal (Sxl), and polypyrimidine tract-binding protein (PTB), whose function is unknown. Iterative in vitro genetic selection was used to show that these proteins have distinct sequence preferences. The uridine-rich degenerate sequences selected by U2AF65 are similar to those present in the diverse array of natural metazoan Py-tracts. In contrast, the Sxl-consensus is a highly specific sequence, which can help explain the ability of Sxl to regulate splicing of transformer pre-mRNA and autoregulate splicing of its own pre-mRNA. The PTB-consensus is not a typical Py-tract; it can be found in certain alternatively spliced pre-mRNAs that undergo negative regulation. This study shows that PTB can regulate alternative splicing by selectively repressing 3' splice sites that contain a PTB-binding site (Singh, 1995).
The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3' splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5' splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5' splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3' splice site requires accessory pyrimidine sequences in the short region between the 5' splice site and branch point (Kennedy, 1997).
One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. A 59-nucleotide Drosophila melanogaster intron from the Drosophila mle gene contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. It is suggested that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly (Kennedy, 1998).
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 which 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 by analyzing the composition of splicing complexes assembled on an ESE-dependent pre-mRNA derived from the doublesex gene of Drosophila. 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 the presence and absence of the ESE. Time course experiments revealed differences in the levels and kinetics of association of individual SR proteins with the ESE-containing pre-mRNA, whereas U2AF-65 kDa binds prior to most SR proteins and hTra2beta 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).
Sex lethal antagonizes the general splicing factor U2AF65 to regulate splicing of Tra. Transgenic flies expressing chimeric proteins between Sxl and the effector domain of U2AF65 were used to study the mechanisms of splicing regulation by Sxl in vivo. Conferring U2AF activity to Sxl relieves its inhibitory activity on Tra splicing but not on Sxl splicing. Therefore, antagonizing U2AF65 can explain Tra splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of Sxl pre-mRNA. These results are a direct proof that Sxl, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing (Granadino, 1997).
The protein Sex-lethal (Sxl) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts. Splicing inhibition of msl-2 requires a binding site for Sxl at the polypyrimidine (poly(Y)) tract associated with the 3' splice site, and an unusually long distance between the poly(Y) tract and the conserved AG dinucleotide at the 3' end of the intron. Only this combination allows efficient blockage of U2 small nuclear ribonucleoprotein particle binding and displacement of the large subunit of the U2 auxiliary factor (U2AF65) from the poly(Y) tract by Sxl. Crosslinking experiments with ultraviolet light indicate that the small subunit of U2AF (U2AF35) contacts the AG dinucleotide only when located in proximity to the poly(Y) tract. This interaction stabilizes U2AF65 binding such that Sxl can no longer displace it from the poly(Y) tract. These results reveal a novel function for U2AF35, a critical role for the 3' splice site AG at the earliest steps of spliceosome assembly and the need for a weakened U2AF35-AG interaction to regulate intron removal (Merendino, 1999).
The protein Sex-lethal (Sxl) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 transcripts. Sxl blocks the binding of U2 auxiliary factor (U2AF) to the polypyrimidine (Py)-tract associated with the 3' splice site of the regulated intron. This study reports that a second pyrimidine-rich sequence containing 11 consecutive uridines immediately downstream from the 5' splice site is required for efficient splicing inhibition by Sxl. Psoralen-mediated crosslinking experiments suggest that Sxl binding to this uridine-rich sequence inhibits recognition of the 5' splice site by U1 snRNP in HeLa nuclear extracts. Sxl interferes with the binding of the protein TIA-1 to the uridine-rich stretch. Because TIA-1 binding to this sequence is necessary for U1 snRNP recruitment to msl-2 5' splice site and for splicing of this pre-mRNA, it is proposed that Sxl antagonizes TIA-1 activity and thus prevents 5' splice site recognition by U1 snRNP. Taken together with previous data, it is concluded that efficient retention of msl-2 intron involves inhibition of early recognition of both splice sites by Sxl (Forch, 2001).
Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, the Drosophila homolog of Dbp5 has been shown to be nonessential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [35S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). It is concluded that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export (Gatfield, 2001).
NXF1, p15 and UAP56 are essential nuclear mRNA export factors. The fraction of mRNAs exported by these proteins or via alternative pathways is unknown. The relative abundance was examined of nearly half of the Drosophila transcriptome in the cytoplasm of cells treated with the CRM1 inhibitor leptomycin-B (LMB) or depleted of export factors by RNA interference. While the vast majority of mRNAs were unaffected by LMB, the levels of most mRNAs were significantly reduced in cells depleted of NXF1, p15 or UAP56. The striking similarities of the mRNA expression profiles in NXF1, p15 and UAP56 knockdowns show that these proteins act in the same pathway. The broad effect on mRNA levels observed in these cells indicates that the functioning of this pathway is required for export of most mRNAs. Nonetheless, a set of mRNAs was identified whose export was unaffected by the depletions; some requiring NXF1:p15 but not UAP56. In addition, this analysis revealed a feedback loop by which a block to mRNA export triggers the upregulation of genes involved in this process (Herold, 2003).
The polypyrimidine-tract (Py-tract) adjacent to 3' splice sites is an essential splicing signal and is recognized by several proteins, including the general splicing factor U2AF65 and the highly specific splicing repressor Sex-lethal (Sxl). They both contain ribonucleoprotein-consensus RNA-binding motifs. However, U2AF65 recognizes a wide variety of Py-tracts, whereas Sxl recognizes specific Py-tracts such as the nonsex-specific Py-tract of the transformer pre-mRNA. It is not understood how these seemingly similar proteins differentially recognize the Py-tract. To define these interactions, chemical interference and protection assays, saturation mutagenesis, and RNAs containing modified nucleotides were used. These proteins recognize distinct features of the RNA. (1) Although uracils within the Py-tract are protected from chemical modification by both of these proteins, modification of any one of seven uracils by hydrazine, or any of eight phosphates by ethylnitrosourea strongly interfered with the binding of Sxk only. (2) The 2' hydroxyl groups or backbone conformation appear important for the binding of Sxl, but not U2AF65. (3) Although any of the bases (cytosine >> adenine > guanine) can substitute for uracils for U2AF65 binding, only guanine partially substitutes for certain uracils for Szl binding. The different dependence on individual contacts and nucleotide preference may provide a basis for the different RNA-binding specificities and therefore the functions of U2AF65 and Sxl in 3' splice site choice (Singh, 2000).
Editorial note: The Nagengast study, reported below, and the study by Chaouki and Salz, 2006 suggests that model proposed in the the Lallena study, described here, that concludes that Sxl blocks splicing after spliceosome assembly, at the second catalytic step of the reaction, is not correct.
The Drosophila protein Sex-lethal (Sxl) promotes skipping of exon 3 from its own pre-mRNA. An unusual sequence arrangement of two AG dinucleotides and an intervening polypyrimidine (Py)-tract at the 3' end of intron 2 is important for Sxl autoregulation. U2AF interacts with the Py-tract and downstream AG, whereas the spliceosomal protein SPF45 interacts with the upstream AG and activates it for the second catalytic step of the splicing reaction. SPF45 represents a new class of second step factors, and its interaction with Sxl blocks splicing at the second step. These results are in contrast with other known mechanisms of splicing regulation, which target early events of spliceosome assembly. A similar role for SPF45 is demonstrated in the activation of a cryptic 3' splice site generated by a mutation that causes human beta-thalassemia (Lallena, 2002; full text of article).
Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific Sex lethal RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is Sans-fille (Snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether Snf acts as part of an intact snRNP or a free protein. Evidence is provided that Sex lethal interacts with Sans-Fille in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with Sex lethal. Moreover, Sex lethal associates with other integral U1 snRNP components, and genetic evidence is provided to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link Sex lethal with the heterodimeric splicing factor, U2AF. These studies point specifically to a mechanism by which Sex lethal represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, these studies are difficult to reconcile with the current model that proposes that the Sex lethal blocks splicing at the second catalytic step, and instead argue that the Sex lethal protein acts after splice site recognition, but before catalysis begins (Nagengast, 2003).
The Sxl male exon is unusual in that it contains two 3' AG dinucleotides separated by a short polypyrimidine tract. Interestingly, although the upstream 3' splice site is used almost exclusively for exon ligation in tissue-culture cells, both 3' splice sites are required for Sxl-mediated male-exon skipping. Moreover, crosslinking studies in HeLa cell extracts have shown that the U2AF heterodimer binds to the downstream 3' splice site and the intervening polypyrimidine tract, suggesting that U2AF may play an active role in Sxl regulation. These biochemical data have been validated by demonstrating that the Sxl protein can associate with the Drosophila U2AF orthologs. More importantly, genetic data provide compelling support for the biological relevance of these interactions by demonstrating that in females, the small subunit is important for both Sxl male-exon skipping and female viability. In addition to demonstrating a role for U2AF in Sxl autoregulation, this genetic result is notable because previous studies have failed to find RNA splicing defects associated with small subunit mutations. Whether this success reflects substrate-specificity or sensitivity of the assay remains to be determined (Nagengast, 2003).
In addition to controlling the use of the male exon 3' splice site, these studies suggest that Sxl controls the use of the male-exon 5' splice site by interacting with the U1 snRNP. This connection was established in three ways: (1) it was found that mutation of a single residue in the N-terminal RRM of SNF compromises both complex formation with Sxl and assembly into the U1 snRNP, thus suggesting that the two events are linked; (2) it has been demonstrated that, in addition to SNF, Sxl can associate with other integral U1 snRNP components, including the U1-70K protein and the U1 snRNA in whole cell extracts; (3) genetic interaction data provide evidence that U1-70K, like SNF, is important for the successful establishment of the Sxl autoregulatory splicing loop in females (Nagengast, 2003).
Although the discovery that SNF is an snRNP protein was the first clue that Sxl might act by associating with components of the general splicing machinery, the role of SNF has remained enigmatic. The role of SNF has been clarified by demonstrating that its contribution to the function of the U1 snRNP is not absolutely essential for viability of either sex, and that Sxl can associate with the U1 snRNP through a SNF-independent mechanism. Nevertheless, in vivo analysis continues to support a role for snf in Sxl splicing autoregulation by demonstrating that Sxl splicing defects are detectable under specific conditions. Interestingly, the phenotypic consequences of these Sxl splicing defects are more severe in the germline than in the soma. One possible explanation for this difference is that the requirements for Sxl splicing autoregulation are fundamentally different in the two tissue types. It is thought more likely that the mechanism is the same, but that the additional interaction with the U1 snRNP provided by SNF becomes critical when Sxl protein levels are low. This hypothesis is based on the fact that, in the germline, the majority of Sxl protein is cytoplasmic, and thus low levels of nuclear Sxl protein are the norm. By contrast, in other tissues, the Sxl protein accumulates in the nucleus, enabling the Sxl-U1 snRNP complex to form even when SNF is not stably associated with the U1 snRNP. The finding that these snf mutant females rarely survive if they are also heterozygous for Sxl, provides additional support for the idea that SNF function is only critical when Sxl protein levels are low (Nagengast, 2003).
Together, these studies argue that interactions between Sxl, the U1 snRNP and U2AF underlie the mechanism by which Sxl promotes skipping of the male exon. Based on these studies, a model is proposed in which Sxl acts not by preventing assembly of the U1 snRNP or U2AF onto the pre-mRNA, but instead interacts with the U1 snRNP bound to the male-exon 5' splice site, and U2AF at the male-exon 3' splice site, to form complexes that block these general splicing factors from assembling into a functional spliceosome. These 5' and 3' Sxl blocking complexes might function independently or they might interact across the exon to form a larger inhibitory complex. Furthermore, because it has not been possible to demonstrate that Sxl interacts directly with either U1-70K or U2AF, it is speculated that one or more bridging proteins are required to link Sxl to the general splicing machinery (Nagengast, 2003).
Although the in vivo approach cannot directly address when in the pathway of spliceosome assembly Sxl acts, biochemical studies have shown that during the course of spliceosome assembly, U2AF and the U1 snRNP are only transiently associated with splicing substrates, and are released before the formation of a functional spliceosome. Therefore, based on these studies, it seems reasonable to propose that Sxl acts by blocking splicing after splice site recognition but before catalysis begins. The data are therefore difficult to reconcile with the recent model, which proposes that Sxl blocks splicing after spliceosome assembly, at the second catalytic step of the reaction. Using RNA interference in Drosophila tissue culture cells it has been demonstrated that efficient male exon skipping depends on the presence of SPF45, a protein that is known to be required for the second step of splicing. Together with studies that show that SPF45 can bind to the upstream 3' splice site of the Sxl male exon and physically interact with Sxl, these data point to a role for SPF45 in Sxl splicing regulation. However, the primary evidence that Sxl blocks the splicing reaction during the second step rests on the results of in vitro splicing assays in which Sxl was shown to inhibit splicing of a chimeric splicing substrate that contains only a small region of the intronic region required for successful autoregulation in vivo. It is suspected that by looking at this 48 bp region, which contains a dispensable Sxl-binding site in addition to the two potential 3' splice sites, out of context, a failsafe mechanism was uncovered that comes into play when Sxl-mediated splicing regulation is otherwise compromised. Additional studies investigating the function of SPF45 in vivo will be required to determine the importance of this second step blocking mechanism and should provide insight into whether multiple mechanisms are needed to drive efficient regulated exon skipping (Nagengast, 2003).
The molecular basis for specific recognition of simple homopolymeric sequences like the polypyrimidine tract (Py tract) by multiple RNA recognition motifs (RRMs) is not well understood. The Drosophila splicing repressor Sex lethal (Sxl), which has two RRMs, can directly compete with the essential splicing factor U2AF65, which has three RRMs, for binding to specific Py tracts. Site-specific photocross-linking and chemical cleavage of the proteins were combined to biochemically map cross-linking of each of the uracils within the Py tract to specific RRMs. For both proteins, RRM1 and RRM2 together constitute the minimal Py-tract recognition domain. The RRM3 of U2AF65 shows no cross-linking to the Py tract. Both RRM1 and RRM2 of U2AF65 and Sxl can be cross-linked to certain residues, with RRM2 showing a surprisingly high number of residues cross-linked. The cross-linking data eliminate the possibility that shorter Py tracts are bound by fewer RRMs. A model is presented to explain how the binding affinity can nonetheless change as a function of the length of the Py tract. The results indicate that multiple modes of binding result in an ensemble of RNA-protein complexes, which could allow tuning of the binding affinity without changing sequence specificity (Banerjee, 2003).
This systematic biochemical analysis with two proteins and three natural Py tracts revealed new information on Py-tract recognition by RRMs. Although it is possible that the nature of 5-IU cross-linking, which is efficient with only certain amino acids, may have influenced the result for a particular position, taken together, the large data set presented in this study supports a compelling trend. RRM1 is bound near the 3'-end of the Py tract, and RRM2 is bound near the 5'-end, which is consistent with all known X-ray structures of the proteins containing two RRMs. Both RRMs of Sxl and only RRM1 and RRM2 of U2AF65 together constitute the minimal Py-tract recognition domain; the RRM3 motif of U2AF65 is not cross-linked to any of the Py tracts. There are two unusual observations: (1) certain 5-IU positions are cross-linked to both RRM1 and RRM2 for all of the Py tracts tested; (2) the size of the cross-linking site for RRM2 is variable and can greatly exceed the RNA site size for other RRMs. The efficient cross-linking site of RRM1 is limited to two-four uridines at the 3'-end of the Py tract. Below, a model for Py-tract recognition is presented that explains various observations, and the biological significance of this mode of RNA recognition is discussed (Banerjee, 2003).
It is postulated that RRM1 as well as RRM2 of both proteins can bind to the Py tract in multiple registers, and RNA at the junction of RRM1 and RRM2 can form a loop of variable length, resulting in an ensemble of complexes. Although, the number of different possible RNA-protein complexes could exceed 40, assuming that each RRM contacts 4 residues in the 17-nt-long Py tract of tra, only three are dealt with in detail. The actual number of residues contacted by each RRM could be different. In complex A, both RRMs are bound to two adjacent uridine stretches, which is similar to the sharp boundary observed at the RRM junction in the X-ray structure of Sxl. In complex B, whereas the binding of RRM1 is unchanged, the binding of RRM2 is shifted by 3 nt upstream. As a consequence, residues 5-8 are looped out. In complex C, the binding of RRM1 is shifted by 1 nt upstream and of RRM2 by 2 nt, resulting in a loop of 2 nt. It should be emphasized that the size and location of the loop will vary depending on the interactions of each RRM. The complexes discussed here as well as those not shown are likely in rapid equilibrium. It is possible that various RNA-protein complexes have different binding energies. This unusual situation of multiple modes of binding likely arises because it is hard for an RRM to discriminate between adjacent uridines. Several observations led to this proposal: (1) the size of the cross-linking site is variable, which can be large for RRM2 on longer Py tracts; (2) both RRMs are cross-linked to certain residues on all of the Py tracts tested; (3) although efficient cross-linking of RRM1 is restricted to the 3'-end of the Py tract, it does not cross-link to a unique set of residues. The preference of RRM1 near the 3'-end of the Py tract could limit the number of possible complexes. (4) A lack of duplicated RRM2-RRM1 cross-linking pattern supports the possibility that in the majority of complexes a single protein molecule binds to the Py tract (Banerjee, 2003).
What does the model explain? First, it explains how certain residues can be cross-linked to both RRMs. The possibility of subpopulations of various complexes implies that a particular residue could contact either RRM1 or RRM2 in a given complex. However, the reason cross-linking of the same residue to both RRMs was observed is because the experiment reflects data from a mixture of complexes. Second, the model provides the basis for the extended site size of RRM2. Although the site size for each RRM is typically 4-7 residues for a given complex, the extended site size for RRM2 can be explained by RNA looping for certain members of the ensemble. The malleable nature of uridine-rich sequences, which are known to be largely unstructured, makes them particularly suited for adopting flexible RNA loops. Third, this model could explain previous chemical interference/protection and saturation mutagenesis data for Sxl, in which the binding site appeared larger than would have been expected for two RRMs. Fourth, in the absence of RRM3 cross-linking, the idea is favored that only two of the three RRMs of U2AF65, and both RRMs of Sxl, likely contributed to the selection of the consensus sequences. This suggestion is consistent with the interaction of RRM3 with other splicing factors such as mBBP/SF1 and SAP155. However, the possibility that RRM3, which was shown to be required for Py-tract binding, lacks appropriate amino acids for 5-IU cross-linking cannot be ruled out. Fifth, the positioning of the RRM1 of U2AF65 at the 3'-end of the Py tract would allow interaction with the small subunit (U2AF35), and thus ready recognition of the 3'-splice-site AG dinucleotide by U2AF35. Sixth, the model explains how Sxl could bind uridine tracts of variable length in the Sxl-regulated pre-mRNAs, and how U2AF65 could bind to natural Py tracts that differ widely in length. Finally, although a comparison of the cross-linking pattern and the Sxl X-ray structure indicates differences in binding, the idea is favored that the Sxl structure represents only one member of the ensemble, perhaps chosen because of the crystal contacts that favored crystallization (Banerjee, 2003).
The cross-linking pattern observed here is inconsistent with an alternative model(s) in which RRM1 and RRM2 would contact a fixed site in a single register with a sharp boundary at the junction of two RRMs. In this scenario, somehow RRM2 would contact a much larger site at the same time; this is inconsistent with the known interactions for RRMs, including the Sxl structure in which RRM2 contacts only 3 residues. Alternatively, if RRM1 and RRM2 are constrained with respect to each other upon RNA binding, the entire protein could bind at different locations. This would result in an increased site size for RRM1 and an increased number of residues cross-linked to both RRMs. The observed cross-linking pattern -- restricted cross-linking of RRM1 to the 3'-end of the Py tract and cross-linking of only 2-4 residues to both RRMs -- is incompatible with the alternative model (Banerjee, 2003).
Although recognition of a Py tract in multiple modes explains several observations, it begs the question of whether or how one member of the ensemble might convert to another. Either RRM2 could slide on the RNA with respect to RRM1 or the protein may undergo dissociation/reassociation. Also the molecular basis for the preferential cross-linking of RRM1 to the 3'-end of the Py tract is not understood; perhaps there is a signal at the 3' boundary. The exact site size for an RRM or the amount of each complex cannot be accurately determined because the observed cross-linking depends on the product of occupancy and the intrinsic cross-linking efficiency of a given binding site. It is not possible to distinguish whether RRM1 is flexible or constrained with respect to RRM2 when bound to RNA in solution. The X-ray structure shows that although two RRMs of Sxl are tethered by a flexible linker in the absence of RNA, the linker region forms a short 310-helix upon RNA binding. In addition, the RRM2 of Sxl when bound to RNA interacts with RRM1 as well as the linker region. Similar interactions are also seen for HuD. However, the energetics of these interactions for Sxl as well as the structure of the first linker region of U2AF65 when bound to RNA remain to be determined (Banerjee, 2003).
The model has important biological consequences. In general, the strength of 3'-splice sites correlates well with the length of the adjacent Py tracts, and the binding affinity for U2AF65. Two possibilities for this correlation have been envisioned. All three RRMs of U2AF65 could contact longer Py tracts, whereas only a subset of the RRMs contact shorter Py tracts. Alternatively, all three RRMs of U2AF65 could contact Py tracts, regardless of the length of the Py tract, but the number of interactions differs depending on the length of the Py tract. It was found that both RRM1 and RRM2 of U2AF65 are cross-linked to all three Py tracts, including the shortest FS Py tract of tra, and that RRM3 is not cross-linked to any of the Py tracts tested, including the longest, NSS Py tract of tra. Therefore, it is proposed that changes in the number of interactions with only RRM1 and RRM2, the number of possible complexes or both, rather than interactions with a subset of RRMs (one, two, or three RRMs), provide the most likely basis for different affinities for various-length Py tracts, and thus 3'-splice-site strength. In this scenario, longer Py tracts would provide additional registers or binding sites, thereby resulting in increased apparent binding affinity. For example, if an RNA offers a single register for binding, only one of the possible encounters with the protein will lead to productive binding; others would require continued sampling until the correct register is found. In contrast, if there are multiple correct registers, encounters with any of them will be productive, thereby increasing the chances of finding the binding site. A homopolymeric sequence like poly(U) provides a much larger set of binding sites because different registers, rather than being contiguous, extensively overlap, thereby offering a significantly large advantage in increasing the apparent binding affinity (Banerjee, 2003).
In conclusion, these studies provide insight into Py-tract recognition. These findings should be applicable to the entire family of proteins that recognize uridine-rich sequences, contain multiple RRMs, and show sequence and structural similarities with Sxl. The modified NCS cleavage protocol and the tryptophan-based domain mapping strategy described in this study provide a useful means for defining recognition of RNA, DNA, or protein sequences by any protein that has multiple recognition domains. This detailed biochemical analysis underscores the importance of independent evaluation of conclusions from structural studies (Banerjee, 2003).
The sequence of the SPF45 protein is significantly conserved, yet functional studies have identified it as a splicing factor in animal cells and as a DNA-repair protein in plants. Using a combined genetic and biochemical approach to investigate this apparent functional discrepancy, both of these studies have been unified and validated by demonstrating that the Drosophila protein is bifunctional, with independent functions in DNA repair and splicing. SPF45 associates with the U2 snRNP and mutations that remove the C-terminal end of the protein disrupt this interaction. Although animals carrying this mutation are viable, they are nevertheless compromised in their ability to regulate Sex-lethal splicing, demonstrating that Sex-lethal is an important physiological target of SPF45. Furthermore, these mutant animals exhibit phenotypes diagnostic of difficulties in recovering from exogenously induced DNA damage. The conclusion that SPF45 functions in the DNA-repair pathway is strengthened by finding both genetic and physical interactions between SPF45 and RAD201, a previously uncharacterized member of the RecA/Rad51 protein family. Together with these finding that the fly SPF45 protein increases the survival rate of mutagen-treated bacteria lacking the RecG helicase, these studies provide the tantalizing suggestion that SPF45 has an ancient and evolutionarily conserved role in DNA repair (Chaouki, 2006; full text of article).
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