Gene name - U2 small nuclear riboprotein auxiliary factor 50
Cytological map position - 14B17--18
Function - RNA binding
Keywords - nuclear mRNA splicing, spliceosome assembly protein
Symbol - U2af50
FlyBase ID: FBgn0005411
Genetic map position - X
Classification - RNA-binding region RNP-1
Cellular location - nuclear and cytoplasmic
|Recent literature||Abramczuk, M. K., Burkard, T. R., Rolland, V., Steinmann, V., Duchek, P., Jiang, Y., Wissel, S., Reichert, H. and Knoblich, J. A. (2017). The splicing co-factor Barricade/Tat-SF1, is required for cell cycle and lineage progression in Drosophila neural stem cells. Development [Epub ahead of print]. PubMed ID: 28935704
Stem cells need to balance self-renewal and differentiation for correct tissue development and homeostasis. Defects in this balance can lead to developmental defects or tumor formation. In recent years, mRNA splicing has emerged as one important mechanism regulating cell fate decisions. This study addresses the role of the evolutionary conserved splicing co-factor Barricade (Barc)/Tat-SF1/CUS2 in Drosophila neural stem cell (neuroblast) lineage formation. Barc is required for the generation of neurons during Drosophila brain development by ensuring correct neural progenitor proliferation and differentiation. Barc associates with components of the U2 small nuclear ribonucleic proteins (snRNP), and its depletion causes alternative splicing in form of intron retention in a subset of genes. Using bioinformatics analysis and a cell culture based splicing assay, Barc-dependent introns were found to share three major traits: they are short, GC rich and have weak 3' splice sites. These results show that Barc, together with the U2snRNP, plays an important role in regulating neural stem cell lineage progression during brain development and facilitates correct splicing of a subset of introns.
The removal of non-coding introns and the joining of coding exons via splicing, an essential step in the eukaryotic pre-mRNA processing pathway, requires accurate splice site selection by the spliceosome. Initial recognition of the 5' exon/intron boundary is achieved via base pairing with the U1 snRNA component of the U1 snRNP (see U1A snRNP also known as sans fille), while U2AF (U2 snRNP Auxiliary Factor) is the first factor bound to the 3' splice site (Ruskin, 1988). Biochemical complementation assays have demonstrated that U2AF is required for the subsequent ATP-dependent association of U2 snRNP with pre-mRNA branchpoints (Ruskin, 1988; Valcarcel, 1996). Purified U2AF is a heterodimer composed of large and small subunits in humans (Zamore, 1989), Drosophila melanogaster (Kanaar, 1993; Rudner, 1996), Caenorhabditis elegans (Zorio, 1997; Zorio, 1999a) and Schizosaccharomyces pombe. Functional conservation of this splicing factor is evidenced by (1) the restoration of splicing activity to U2AF-depleted HeLa nuclear splicing extracts via addition of Drosophila U2AF large subunit (dmU2AFLG; Zamore, 1991) and (2) the ability of human U2AF35 (hsU2AFSM: see U2 small nuclear riboprotein auxiliary factor 35) to restore growth to a S. pombe strain lacking the small subunit (Webb, 2004a; Webb 2004b and references therein).
The structural domains of U2AF are also conserved except in Saccharomyces cerevisiae, where the large subunit is highly divergent and the small subunit is absent entirely. The small subunit of U2AF consists of two zinc-binding domains (ZBDs) surrounding a central pseudo-RNA recognition motif (ØRRM; Rudner, 1998b), also known as a PUMP (PUF60/U2AF/MUD2 Protein-protein interaction) domain (Page-McCaw, 1999) or a UHM (U2AF Homology Motif; Kielkopf, 2004). These are both highly conserved between S. pombe and humans, followed by a C-terminal domain that consists of RS or RS/glycine repeats in metazoan orthologues that are not present in the fission yeast protein. The three conserved domains including both ZBDs and the ØRRM of S. pombe U2AF small subunit (spU2AFSM) contribute to RNA binding and are essential for function in vivo, while the more divergent C-terminal domain is dispensable (Webb, 2004a). A comparable domain ablation analysis in vivo has not been carried out for the U2AF large subunit, which consists of an N-terminal RS domain, a linker region, two classical RRMs (RNA Recognition Motifs) and a ØRRM (Zamore, 1992). Such an analysis would complement extensive biochemical data demonstrating that this subunit is the major contributor to RNA binding (Rudner, 1998a; Wu, 1999; Kielkopf, 2001) and interacts with multiple protein partners implicated in splicing (Webb 2004b and references therein).
RNA binding assays (Zamore, 1992; Rudner, 1998a) and in vitro selection studies (Singh, 1995; Wu, 1999; Banerjee, 2004) demonstrated that the large subunit of U2AF (U2 small nuclear riboprotein auxiliary factor 50 or U2af50 in Drosophila) interacts with the 3' polypyrimidine tract, while the small subunit (U2 small nuclear riboprotein auxiliary factor 38 or U2af38 in Drosophila) functions in recognition of the 3' AG dinucleotide (Wu, 1999; Zorio, 1999b; Merendino, 1999). The bipartite nature of the RNA target sequences for the two subunits, in combination with biochemical complementation data demonstrating that addition of the human large subunit (hsU2AFLG) alone to U2AF-depleted HeLa nuclear extracts can rescue splicing of substrates that contain long polypyrimidine tracts (Wu, 1999; Guth, 2001), led to a widely accepted model for 3' splice site recognition by U2AF. The central tenet of this model is that the binding energy contributed by the small subunit/AG interaction is essential only for introns with less extensive polypyrimidine tracts (reviewed in Moore, 2000), consistent with earlier splicing assays of mutant human pre-mRNAs in vitro, which indicated that the requirement for a 3' AG to proceed through the first step of splicing (AG-dependence) could be eliminated by expanding the polypyrimidine tract (Reed, 1989). In S. pombe, mutating the terminal AG dinucleotide prevents the first transesterification reaction for all three introns examined (Romfo, 1997), as well as for a subset of mammalian premRNAs (Reed, 1989; Wu, 1999). However, doubling the length of the polypyrimidine tract in two different fission yeast introns did not render the AG dinucleotide dispensable in vivo (Romfo, 1997), providing the first hint that both subunits of U2AF may be important for initial recognition of a broad spectrum of introns in this organism (Webb 2004b and references therein).
In vivo analysis demonstrates that all five domains of spU2AFLG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in S. pombe. Since this is not predicted by the model for metazoan 3' splice site recognition, introns for which the spU2AFLG and spU2AFSM make distinct contributions were sought by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3' pyrimidine tract. These and other studies performed in fission yeast support a model for 3' splice site recognition in which the two subunits of U2AF functionally collaborate in vivo (Webb, 2004b).
The dynamics of the splicing process and the complexities of U2AF interactions are reviewed by Kielkopf (2004). To perform its role in RNA splicing, two central canonical RRM domains of U2AF65, the large U2AF subunit, recognize the polypyrimidine tract (Py-tract) in the pre-mRNA. Binding of U2AF65 to the Py-tract is strengthened by cooperative protein-protein interactions with SF1 at the upstream BPS (Berglund, 1998; Rain, 1998) and with U2AF35 (the small U2AF subunit), which contacts the downstream 3' splice site consensus (Merendino, 1999; Wu, 1999; Zorio, 1999a). The C-terminal U2AF homology motif (UHM) domain of U2AF65 (the third RRM motif that is specialized for protein-protein interaction) interacts with the N-terminal domain of SF1 (U2AF65-UHM/SF1-ligand; Rain, 1998). At the opposite end of the large U2AF subunit, the N-terminal domain of U2AF65 provides a ligand that interacts with the central UHM domain of U2AF35 (U2AF35-UHM/U2AF65-ligand; Zhang, 1992; Rudner, 1998b). Subsequently, entry of the U2 snRNP displaces SF1 by interacting with the pre-mRNA branch point sequence (BPS) via the U2 snRNA, and with the U2AF65 C-terminal domain via the SF3b subunit, SAP155 (Gozani, 1998; Habara, 1998). Once the U2 snRNP has contacted the pre-mRNA, U2AF is dissociated by conformational rear-rangements of the spliceosome components. In summary, key protein-protein interactions are mediated by the U2AF65-UHM, which interacts with SF1 and subsequently SAP155, and by the U2AF35-UHM, which interacts with the U2AF65 N terminus (Kielkopf, 2004 and references therein).
To gain an understanding of the mechanisms underlying splice site selection and the control of alternative splicing, genome-wide approaches have been undertaken to pursue a genetic and biochemical investigation of the Drosophila large U2AF subunit (dU2AF50). Several groups have reported the identification of temperature-sensitive mutations in the yeast S. pombe large U2AF subunit homolog that reside in conserved amino acids common to all known U2AF large subunits. The S. pombe mutations were transferred to Drosophila and new temperature-sensitive dU2AF50 transgenic strains were created. In vitro, these mutant recombinant U2AF heterodimers show a dramatic temperature-dependent reduction in RNA polypyrimidine tract binding, without exhibiting any defect on in vitro splicing of model pre-mRNAs. Genome-wide expression profiles of the mutant flies identify genes that are specifically differentially expressed at the restrictive temperature. These results have allowed the identification of U2AF-sensitive target mRNAs and specific RT-PCR analysis has confirmed that splicing is impaired in the mutant flies. Most interestingly, a high proportion of intronless genes were downregulated in the mutant flies when grown at the restrictive temperature. High-density microarrays and dU2AF50 knockdown in cultured cells were used to survey the nucleo-cytoplasmic distribution of all expressed genes. This analysis revealed that more that 28% of mRNAs accumulated in the nucleus upon dU2AF50 knockdown, regardless of their intron number. A genome-wide approach analyzing RNAs bound in nuclear RNPs as well as a bioinformatic analysis confirmed that dU2AF50 associates with intronless RNAs. These results reveal a previously unknown function for dU2AF50 in the nuclear export of intronless mRNAs (Blanchette, 2004).
Two Drosophila dU2AF50 temperature-sensitive alleles were charcterized in this study; they directly impair U2AF RNA binding to an intron polypyrimidine tract. In addition, the mutations reduced the splicing efficiency of some target genes in vivo. Surprisingly, although U2AF is a known splicing factor, a large number of intronless genes were found to be downregulated in the mutant flies at the restrictive temperature. By looking at the nucleo-cytoplasmic distribution of all expressed genes, it was found that reducing dU2AF50 expression had a dramatic and widespread effect on the nucleo-cytoplasmic mRNA localization irrespective of the intron number of the affected genes. Finally, some intronless RNAs were found to be associated with dU2AF50 in nuclear RNP complexes. Thus, in addition to the well-known role of U2AF in defining 3′ splice sites in pre-mRNAs, the results reveal an unexpected function for dU2AF50 in nuclear export of intronless mRNAs (Blanchette, 2004).
As previously reported, the large U2AF subunit is highly conserved from S. pombe to humans (Kanaar, 1993; Potashkin, 1993; Zamore, 1992; Zorio, 1997) and has been shown to be highly refractory to mutations (Romfo, 1999). Two mutations identified in S. pombe generate new temperature-sensitive dU2AF50 alleles in Drosophila. Both mutations, D204N and S284Y, lie on each side of the second RNA binding domain (RRM2) without being part of it. The structures of several RRM-RNA complexes have recently been solved and shown to conform to a canonical ß1alpha1ß2ß3alpha2ß4 fold. In the structure of the human RRM2, asparatic acid 204 is located four amino acids upstream of the first ß sheet (ß1) of RRM2, which is predicted to be part of the RNA interaction platform. Although the D204 residue was not in the structure of RRM2, it is conceivable that asparatic acid 204 is involved in a salt bridge and in absence of this putative salt bridge, the RNA-RRM2 interaction might be less stable leading to the observed temperature-sensitive reduction in RNA binding. The dU2AF50 serine 284 residue is conserved in mouse and human U2AF65 and is substituted by a cysteine in S. pombe and C. elegans. Interestingly, in the U1A:RNA structure, the C-terminal region next to ß4 contacts the RNA, and, similarly, the same region in U2AF65 also appears to contact RNA (Ito, 1999). Thus, changing a serine for a bulky aromatic tyrosine may cause steric effects that might result in reduced RNA affinity (Blanchette, 2004).
Surprisingly, although the temperature-sensitive mutations dramatically affect dU2AF50 RNA binding affinity, in vitro splicing of model substrates was not affected. This probably reflects the highly cooperative nature of spliceosome assembly. Reduction in U2AF RNA binding affinity might be compensated for by interaction with other spliceosomal factors. For instance, interaction of the small U2AF subunit with the 3′ splice site together with interaction of the large U2AF subunit with SF1/BBP, which binds to the branchpoint sequence, could be involved in stabilizing U2AF binding to the polypyrimidine tract (Berglund, 1997; Blanchette, 2004).
The most striking observation made in this study is that a very high proportion of intronless genes are downregulated in the dU2AF50 mutant flies grown at the restrictive temperature. Although this observation could be interpreted as an indirect effect, the fact that this enrichment for intronless RNAs in the dU2AF50 mutants is very different from the average genomic intron distribution suggests a direct role for dU2AF50 in the expression of intronless genes. Moreover, the observation that RNAi knockdown of dU2AF50 expression results in the nuclear accumulation of a large number of intronless mRNAs, that dU2AF50 is found to be associated with intronless mRNAs in purified nuclear RNP complexes, and that the vast majority of intronless genes possess putative U2AF binding sites support a direct role for dU2AF50 in the nuclear export of intronless mRNAs. In mammals, U2AF has been shown to directly interact with the protein factor UAP56, a putative DEAD box RNA helicase essential for splicing (Fleckner, 1997), and the essential transport receptor TAP/NXF1 (Zolotukhin, 2002). It has been proposed that UAP56 is recruited to the spliceosome through an interaction with RRM1 of U2AF65 (Fleckner, 1997). In Drosophila and yeast, UAP56 is an essential export factor that functions to bridge the mRNA to the export machinery (Gatfield, 2001; Herold, 2003; Jensen, 2001). Interestingly, UAP56 was shown to be required not only for export of spliced mRNAs but also for export of intronless mRNAs (Gatfield, 2001; Jensen, 2001; Strasser, 2001; Strasser, 2002). One attractive possibility is that U2AF, as with intron-containing genes, is involved in the recruitment of UAP56, or other members of the RNA export machinery, for instance, TAP/NXF1, to intronless mRNAs prior to their nuclear export (Blanchette, 2004).
Recently, a subset of the SR family of splicing factors has been shown to be involved in the export of a class of intronless mRNAs (Huang, 2003; Huang, 2001). The SR proteins 9G8, SRp20, and SF2/ASF are proteins that shuttle between the nucleus and cytoplasm and serve as adaptors between the intronless histone mRNA and the export factor TAP. Interestingly, mammalian U2AF has been shown to continuously shuttle between the nucleus and cytoplasm (Gama-Carvalho, 2001; Zolotukhin, 2002), and although there is no known function for U2AF in the cytoplasm, these results are suggestive of a general and direct role of U2AF in the export of intronless, as well as intron-containing mRNAs (Blanchette, 2004).
The genes that are upregulated upon growth of the mutant dU2AF50 strain at the restrictive temperature generally contain multiple introns. In addition, their pre-mRNAs are generally longer than the average genomic pre-mRNA length. Intriguingly, no splicing defect was found for any of several individual upregulated genes tested. One possible explanation for this observation could reside in the recent report that the splicing machinery can stimulate the transcription apparatus (Y. W. Fong, 2001). One might envision that mRNA maturation in the mutant flies might be slower on long pre-mRNAs or on pre-mRNAs containing multiple introns because of reduced dU2AF50 RNA binding affinity. This could increase the time during which partially spliced mRNA-containing snRNPs or partially assembled spliceosomes would colocalize on nascent transcripts with the transcriptional machinery. Those spliceosomal components could feed back on the transcription machinery, releasing potentially paused RNA polymerase II complexes. This would lead to an overall increase in transcriptional rate on some genes that are more prone to RNA polymerase II pausing. Although it is not known, it is speculated that genes upregulated in the mutant flies might be part of such a class (Blanchette, 2004).
Over the past few years it has become evident that what were originally thought to be distinct steps in the gene expression pathway are tightly coupled through an extensive network of interactions between the transcriptional RNA processing and RNA export machineries. However, most of this knowledge comes from intron-containing genes, and this, in part, accounts for the relatively poor understanding of the mechanisms controlling expression of intronless genes. The observation that the splicing factor dU2AF50 can influence nuclear export of intronless genes suggests that common mechanisms and RNA-protein interactions are probably shared between these two classes of genes (Blanchette, 2004 and references therein).
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).
The large subunit of the human pre-messenger RNA splicing factor U2 small nuclear ribonucleoprotein auxiliary factor (hU2AF65) is required for spliceosome assembly in vitro. A complementary DNA clone encoding the large subunit of Drosophila U2AF (dU2AF50) has been isolated. Recombinant dU2AF50 protein complements mammalian splicing extracts depleted of U2AF activity. Germline transformation of Drosophila with the dU2AF50 complementary DNA rescues a lethal mutation, establishing that the dU2AF50 gene is essential for viability. R/S domains have been found in numerous metazoan splicing factors, but their function is unknown. The mutation in Drosophila U2AF will allow in vivo analysis of a conserved R/S domain-containing general splicing factor (Kanaar, 1993).
The essential eukaryotic pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is required to specify the 3' splice at an early step in spliceosome assembly. U2AF binds site-specifically to the intron polypyrimidine tract and recruits U2 small nuclear ribonucleoprotein to the branch site. Human U2AF (hU2AF) is a heterodimer composed of a large (hU2AF65) and small (hU2AF35) subunit. Although these proteins associate in a tight complex, the biochemical requirement for U2AF activity can be satisfied solely by the large subunit. The requirement for the small subunit in splicing has remained enigmatic. No biochemical activity has been found for hU2AF35 and it has been implicated in splicing only indirectly by its interaction with known splicing factors. In the absence of a biochemical assay, a genetic approach was undertaken to investigate the function of the small subunit in the fruit fly Drosophila melanogaster. A cDNA clone encoding the small subunit of Drosophila U2AF (dU2AF38) has been isolated and sequenced. The dU2AF38 protein is highly homologous to hU2AF35 containing a conserved central arginine- and serine-rich (RS) domain. A recessive P-element insertion mutation affecting dU2AF38 causes a reduction in viability and fertility and morphological bristle defects. Consistent with a general role in splicing, a null allele of dU2AF38 is fully penetrant recessive lethal, like null alleles of the Drosophila U2AF large subunit (Rudner, 1996).
The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies grown at the restrictive temperature. Surprisingly, almost 40% of the downregulated genes lack introns. Microarray analyses revealed that nuclear export of a large number of intronless mRNAs is impaired in Drosophila-cultured cells RNAi knocked down for dU2AF(50). Immunopurification of nuclear RNP complexes showed that dU2AF50 associates with intronless mRNAs. These results reveal an unexpected role for the splicing factor dU2AF50 in the nuclear export of intronless mRNAs (Blanchette, 2004).
A fully penetrant recessive lethal deletion of the gene coding for dU2AF50 has been characterized and can be rescued by dU2AF50 cDNA transgenes under the control of the dU2AF50 genomic promoter. S. pombe temperature-sensitive alleles in highly conserved residues of the large U2AF subunit have been identified and documented in vivo (Potashkin, 1993 and Romfo, 1999). The yeast mutations were individually introduced into the cDNA coding for dU2AF50 (Rudner, 1998b; Rudner, 1998c), and transgenic flies were successfully recovered for all four mutations tested. The transgenes were then assayed for their ability to genetically complement a deletion of dU2AF50 (XR15). Females carrying the XR15 deletion over a balancer chromosome were mated with the different transgenic males. A functional rescue allele was scored by the presence of non-Binsinscy males (males without the balancer chromosome) in the progeny resulting from flies carrying the XR15 deletion allele and rescued by the transgene. Of the four mutations tested only two, D204N and S284Y, successfully rescued the dU2AF50 XR15 deletion (Blanchette, 2004).
The two mutant dU2AF50 alleles were then tested for a temperature-sensitive phenotype by repeating the crosses at 25°C and 29°C and by counting the ratio of rescued males to the nonbalanced females that eclosed at the two temperatures. The wild-type dU2AF50 transgene efficiently rescued the dU2AF50 deletion with a similar rescue percentage at both 25°C and 29°C. By contrast, flies carrying the mutant transgenes efficiently rescued the dU2AF50 deletion at 25°C, but a marked reduction in rescued male viability was observed upon growth of the two mutant transgenic strains at 29°C. This strong temperature-sensitive phenotype was observed for each mutation in two independent transgenic strains. The temperature-dependent phenotype was not due to differences in either dU2AF50 expression or stability in the mutant transgenic strains since similar levels of dU2AF50 protein were observed in rescued males from the wild-type and from the two mutant transgenic strains at all temperatures tested. Homozygous males and females carrying the dU2AF50 XR15 deletion rescued by the S284Y transgene were successfully engineered. This homozygous mutant strain displayed the same temperature-sensitive phenotype when grown at the restrictive temperature (30°C), the homozygous females laid eggs with abnormal eggshell morphology, and the mutant embryos failed to developed into larvae. Taken together, these results confirm that the S. pombe temperature-sensitive mutations are transferable to the Drosophila dU2AF50 gene to generate new temperature-sensitive alleles (Blanchette, 2004).
dU2AF50 binds to the intron polypyrimidine tract upstream of the 3′ splice site and U2AF is required for 3′ splice site recognition of all characterized pre-mRNAs. The identified temperature-sensitive mutations D204N and S284Y flank the second RRM and thus might affect dU2AF50/dU2AF38 RNA binding. Using a bicistronic system to simultaneously express both dU2AF subunits in bacteria, soluble wild-type and mutant recombinant dU2AF50/dU2AF38 heterodimers (hereafter referred to as U2AF) were efficiently produced and tested for their ability to bind RNA (Rudner, 1998a). All three recombinant protein preparations were similar in composition, with dU2AF50 being expressed as a single species and dU2AF38 being slightly proteolysed, as observed previously (Rudner, 1998a). Wild-type dU2AF efficiently forms specific RNA-protein complexes with an RNA oligonucleotide carrying the polypyrimidine tract and AG of the 3′ splice site of the first intron of the adenovirus major late pre-mRNA (MINX) when incubated at different temperatures, ranging from 20°C to 35°C. However, both the D204N and S284Y mutants are defective in forming specific RNA-protein complexes at all temperatures tested. Interestingly, both dU2AF mutants formed apparent protein-RNA aggregates at the top of the gel similar to what was seen with the wild-type protein at 4°C. Those large complexes are attributed to nonspecific RNA-protein complexes which are displaced upon specific interaction between the RNA and dU2AF. At 20°C, small amounts of specific RNA-protein complexes can be seen with the D204N and S284Y U2AF mutants. By titrating the amount of protein, specific complexes can be formed at 20°C with the D204N and S284Y dU2AF, although less efficiently than with the wild-type dU2AF (apparent KD of 0.6 microM, 2.3 microM, and 7.8 microM for the wild-type, D204N and S284Y dU2AF, respectively). Incubating the binding reaction at 35°C does not affect wild-type dU2AF (apparent KD of 1.0 microM), but completely abrogates binding of both the D204N and S284Y mutant dU2AFs (apparent KD > 50 microM). These results demonstrate that the D204N and S284Y temperature-sensitive dU2AF mutations affect RNA binding in a temperature-dependent manner (Blanchette, 2004).
Although the RNA-affinity of the mutant protein is greatly reduced, in vitro splicing using a U2AF depletion-reconstitution system failed to show any defect of the mutant dU2AF recombinant proteins. Different pre-mRNAs at all temperatures tested were spliced as efficiently in the presence of the mutant proteins as they were spliced in the presence of the wild-type dU2AF. The use of efficiently spliced in vitro models might have impaired the capacity to observe splicing defect with the mutant dU2AF50 proteins. However, in vivo, more sensitive dU2AF50 targets might be less efficiently spliced, retained in the nucleus because they still contain an intron, and then degraded in the mutant dU2AF50 flies at the restrictive temperature. Individual gene expression using high-density microarrays was quantified from RNA extracted from both wild-type and mutant homozygous adult grown at the permissive and restrictive temperatures. Temperature-dependent variations in expression of individual genes were obtained by pairwise comparison of the triplicate experimental samples (nine comparisons). Significant variations in gene expression were determined using the Affymetrix statistical algorithm, and a positive score was attributed to genes that were significantly affected in at least seven out of nine pairwise comparisons (highly stringent). The analysis revealed that, at the restrictive temperature, 88 and 53 genes were downregulated in the wild-type and the mutant flies, respectively and that 35 genes were specifically downregulated only in the mutant flies. By contrast, 143 and 426 genes were significantly upregulated in the wild-type and mutant flies grown at the restrictive temperature, out of which 374 were specifically upregulated in the S284Y mutant flies. The differential expression of some genes were confirmed by semiquantitative RT-PCR (Blanchette, 2004).
While the upregulated genes from the mutant flies do not display any striking functional clustering, the downregulated genes cluster into two major categories. Eight genes (23% of the downregulated genes) are known or predicted to be trypsin-like proteases, while seven genes (20% of the downregulated genes) are known to be involved in oogenesis. The observation that several genes involved in oogenesis were downregulated fits well with the observed abnormal eggshell phenotype in the mutant strains (Blanchette, 2004).
Since U2AF is known to be a splicing factor, the intron characteristics were examined in the up- and downregulated genes in the S284Y mutant flies shifted to the restrictive temperature. In the latest release of the Drosophila genome annotation (v. 3.1), the average number of introns per gene ranges from 0 introns (17.9% of all genes) to 49 introns (CG17150) and the distribution of the average number of introns per gene is centered around 1 intron per gene (21%). Interestingly, the upregulated genes from the S284Y mutant flies grown at the restrictive temperature show a consistent overrepresentation for genes containing multiple introns with a distribution centered around three introns per gene. By contrast, the identified downregulated genes show predominantly zero or one intron per gene (37% and 45.7%, respectively). The differences in the intron number distributions are specific for the differentially expressed genes in the mutant flies since no significant differences were observed for the genes that were up- and down-regulated in the wild-type flies grown at 30°C. The observation that the distribution of introns for the specifically affected genes in the S284Y dU2AF50 mutant flies is significantly different from the average genomic distribution suggests that the dU2AF50 mutation might have a direct effect on those genes. In Drosophila, the modal intron length is 75 nt and does not vary significantly for the genes up- and down-regulated in the dU2AF50 mutant S284Y flies grown at the restrictive temperature. These results indicate that a mutation which reduces the RNA binding affinity of dU2AF50 can directly affect the expression of both intronless, as well as, intron-containing genes (Blanchette, 2004).
Whether splicing was affected was tested for some of the genes downregulated in the S284Y dU2AF50 mutant flies grown at the restrictive temperature. Using oligonucleotide primer pairs flanking the single intron of two genes (CG4783 and Ag5r2, which are the fourth and second most downregulated genes in the S284Y flies, respectively), RT-PCR was performed on RNA extracted from wild-type and mutant flies grown at both the permissive and restrictive temperatures. Splicing of CG4783 is efficient and similar in wild-type flies grown at both 25°C and 30°C temperatures. However, in the S284Y mutant flies at 25°C, the splicing efficiency of CG4783 is reduced and, moreover, is even more reduced in the S284Y flies grown at 30°C. Additionally, splicing of Ag5r2 is efficient and similar in both wild-type and mutant flies grown at the permissive temperature (25°C). However, when the flies are shifted to the restrictive temperature (30°C), the splicing efficiency of Ag5r2 is reduced in the wild-type and the reduction in splicing is even more evident in the S284Y mutant flies. This confirms that the S284Y dU2AF50 mutation affects, in a temperature-sensitive fashion, the splicing efficiency of some target genes in vivo (Blanchette, 2004).
RT-PCR assays were also performed on two genes that were upregulated in the dU2AF50 mutant flies shifted to the restrictive temperature (30°C). Oligonucleotide primer pairs flanking their multiple introns, two and six introns in CG7036 and transportin, respectively, were used to analyze splicing defects. The splicing efficiency of both pre-mRNAs was similar in both wild-type and mutant flies grown at either the permissive or restrictive temperatures. Taken together, these results suggest that the temperature-sensitive S284Y mutation in dU2AF50 reduces splicing of some of the downregulated pre-mRNAs in vivo in the mutant flies grown at the restrictive temperature but does not appear to affect the splicing of the upregulated genes that contain multiple introns (Blanchette, 2004).
It was reasoned that the reduction in the level of some intronless mRNA in the dU2AF50 mutant flies might result from nuclear retention and degradation as has been observed when RNA export factor expression is knocked down (Herold, 2003). The expression of dU2AF50 was efficiently knocked down by RNAi in cultured Drosophila SL2 cells to less than 20% of the endogenous level, and nuclear and cytoplasmic RNA was isolated from control and dU2AF50 knocked-down cells. High-density oligonucleotide microarrays were used to measure the RNA level of 13,738 genes in both the nuclear and cytoplasmic fractions isolated from control and dU2AF50 knocked-down cells. Four thousand, seven hundred, and thirty-six genes show consistent expression in L2 cells, and for each expressed gene, the nucleo-cytoplasmic ratio from the control and dU2AF50- knocked-down sample was used to calculated a nuclear retention index describing the effect of the dU2AF50 RNAi. Using an arbitrary retention index greater than 0.1, more than 28% of the analyzed genes (1334 genes) were predicted to be retained in the nucleus in the dU2AF50 knocked-down cells with 12% of the retained mRNAs (166 genes) being intronless. The microarray analysis was validated by RT-PCR with eight individual genes showing variable retention indices. The RpL32 pre-mRNA contains two introns while the other genes are intronless. The nucleo-cytoplasmic distribution predicted by the microarray and measured by RT-PCR nicely correlates for RpL32, as well as the intronless genes CG15784, CyCB3, Gip, Mpp6, and slp1, and confirms their nuclear retention in the dU2AF50 knocked-down cells. In addition, the number of genes predicted to be retained in the nucleus by the microarray analyses is likely to be an underestimate of the real number due to the normalization process. The normalization is done using the calculated population average, and this normalization process is based on the ad hoc assumption that only a small proportion of genes are significantly different between the compared samples. As an example, the mRNAs coding for CG30342 and noi, which have slightly negative retention index (−0.39 and −0.07 respectively) and thus were not predicted to be retained in the nucleus, nonetheless showed nuclear retention when assayed by RT-PCR. These results indicate that the essential pre-mRNA splicing factor dU2AF50 also plays a significant and unexpected role in the nuclear export of a large number of intronless mRNAs (Blanchette, 2004).
The previous results predict that dU2AF50 should associate with intronless mRNAs, either directly or indirectly, as part of a multicomponent RNP complex in order to promote their nuclear export. In order to test this prediction, a specific immunopurification of nuclear RNP complexes from a 0-12 hr Drosophila embryonic RNP preparation was performed using affinity-purified anti-dU2AF50 antibody (Rudner, 1998c). Genome-wide identification of the associated RNAs was performed using spotted Drosophila cDNA microarrays containing approximately 6000 different EST PCR fragments. Five independent experiments were performed, and RNAs consistently present in at least three assays were used for further analyses. In the top 200 RNAs, the dU2AF50-associated genes show an overrepresentation for pre-mRNAs with multiple introns, with a distribution centered around two introns per gene. Surprisingly, but consistent with the hypothesis, four of the first 200 dU2AF50-bound RNAs were intronless, confirming that dU2AF50 can be found stably associated with intronless RNAs. Semiquantitative RT-PCR was used to confirm that those RNAs were immunoaffinity purified together with dU2AF50. Moreover, three of them (CG30342, Slp1, and noi) are expressed in L2 cells and show nuclear retention when the expression of dU2AF50 is knocked down (Blanchette, 2004).
A statistical model of all known U2AF binding sites (3′ splice sites) was generated and used to search for binding sites in intronless genes. This approach found that more than one third of all intronless mRNAs contain at least one site that matches this U2AF model as well as the average 3′ splice site. However, no enrichment for strong U2AF binding sites was observed in the set of intronless genes as a whole. The four intronless mRNAs found by microarray analysis of affinity-selected dU2AF50-containing RNP complexes all contain putative U2AF binding sites that match the model as well as the average splice site (Blanchette, 2004).
The binding assay supports the notion that dU2AF50 can associate with intronless RNAs, and the bioinformatic analyses are consistent with this observation. Together this suggests that dU2AF50 participates in the export of a large number of intronless genes. While only a small fraction of the top mRNAs found in stable U2AF-containing nuclear RNP particles were intronless, the vast majority of intronless mRNAs are predicted to contain putative U2AF binding sites. Thus, even transient association of U2AF with these transcripts for the recruitment of RNA export factors could account for the effects of dU2AF50 mutations or depletion of the nucleo-cytoplasmic transport of intronless mRNAs (Blanchette, 2004).
Several fission yeast temperature-sensitive mutants defective in pre-mRNA processing (prp- mutants) at the nonpermissive temperature have been identified. The prp2+ gene has been cloned by its ability to complement the temperature-sensitive growth defect of a prp2- mutant. The gene also corrects the pre-mRNA splicing defect of prp2- mutants and encodes a 59-kilodalton polypeptide (PRP2). A molecular characterization indicates that PRP2 is a previously uncharacterized yeast splicing factor with extensive similarity to the mammalian splicing factor U2AF65. Thus, this study provides evidence that a U2AF homolog participates in RNA processing in vivo (Potashkin, 1993).
The modular structure of splicing factor SF1 is conserved from yeast to man and SF1 acts at early stages of spliceosome assembly in both organisms. The hnRNP K homology (KH) domain of human (h) SF1 is the major determinant for RNA binding and is essential for the activity of hSF1 in spliceosome assembly, supporting the view that binding of SF1 to RNA is essential for its function. Sequences N-terminal to the KH domain mediate the interaction between hSF1 and U2AF65, which binds to the polypyrimidine tract upstream of the 3' splice site. Moreover, yeast SF1 interacts with Mud2p, the presumptive U2AF65 homologue in yeast, and the interaction domain is conserved in yeast SF1. The C-terminal degenerate RRMs in U2AF65 and Mud2p mediate the association with hSF1 and yeast SF1, respectively. Analysis of chimeric constructs of hSF1 and yeast SF indicates that the KH domain may serve a similar function in both systems, whereas sequences C-terminal to the KH domain are not exchangeable. Thus, these results argue for hSF1 and yeast SF1, as well as U2AF65 and Mud2p, being functional homologues (Rain, 1998).
U2AF is an essential splicing factor required for recognition of the polypyrimidine tract and subsequent U2 snRNP assembly at the branch point. Because Caenorhabditis elegans introns lack both polypyrimidine tract and branch point consensus sequences but have a very highly conserved UUUUCAG/R consensus at their 3' splice sites, it was hypothesized that U2AF might serve to recognize this sequence and thus promote intron recognition in C. elegans. The gene for the large subunit of U2AF, uaf-1, has been cloned. Three classes of cDNA were identified. In the most abundant class the open reading frame is similar to that for the U2AF65 from mammals and flies. The remaining two classes result from an alternative splicing event in which an exon containing an in-frame stop codon is inserted near the beginning of the second RNA recognition motif. However, this alternative mRNA is apparently not translated. Interestingly, the inserted exon contains 10 matches to the 3' splice site consensus. To determine whether this feature is conserved, uaf-1 from the related nematode Caenorhabditis briggsae was examined. It is composed of six exons, including an alternatively spliced third exon interrupting the gene at the same location as in C. elegans. uaf-1 is contained in an operon with the rab-18 gene in both species. Although the alternative exons from the two species are not highly conserved and would not encode related polypeptides, the C. briggsae alternative exon has 18 matches to the 3' splice site consensus. It is hypothesized that the array of 3' splice site-like sequences in the pre-mRNA and alternatively spliced exon may have a regulatory role. The alternatively spliced RNA accumulates at high levels following starvation, suggesting that this RNA may represent an adaption for reducing U2AF65 levels when pre-mRNA levels are low (Zorio, 1997).
Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (polyY) tract, and AG at the splice boundary comprise the mammalian 3' splice site. Although the AG is crucial for the recognition of introns with relatively short polyY tracts, which are termed 'AG-dependent introns', the molecule responsible for AG recognition has never been identified. A key player in 3' splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the polyY tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabiditis elegans introns have a highly conserved U4CAG/ R at their 3' splice sites instead of branch-point and polyY consensus sequences. Nevertheless, C. elegans has U2AF. Both U2AF subunits crosslink to the 3' splice site. These results suggest that the U2AF65-U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG (Zorio, 1999).
In most species the 3' splice site is recognized initially by an interaction between the two-subunit splicing factor U2AF with the polypyrimidine (polyY) tract that results in recruitment of the U2 snRNP to the branch-point consensus just upstream. In contrast, in Caenorhabditis elegans, both the polyY tract and the branch-point consensus sequences are missing, apparently replaced by the highly conserved U4CAG/R 3' splice site consensus. Nevertheless C. elegans U2AF65 is very similar to its mammalian and fly counterparts and may recognize the 3' splice site consensus. The C. elegans U2AF35 gene, uaf-2, has been cloned. It lacks an identifiable RS domain, which, in flies, has been shown to play a role in RNA binding, but it contains an extended glycine-rich stretch at its C-terminus. uaf-2 is in an operon with cyp-13, a gene that encodes a cyclophilin with an RRM domain at its N-terminus. It has been demonstrated by RNA interference that both U2AF genes, uaf-1 (which encodes U2AF65) and uaf-2, are required for viability, whereas cyp-13 is apparently not (Zorio, 2000).
Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. A previously uncharacterized activity, U2AF, has been identified that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, U2AF is shown to bind to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing (Ruskin, 1988).
U2 auxiliary factor (U2AF) is a non-snRNP protein required for the binding of U2 snRNP to the pre-mRNA branch site. Purified U2AF comprises two polypeptides of 65 and 35 kd. Biochemical complementation and immunological assays were performed to characterize U2AF in greater detail. An extract lacking only U2AF activity was used to show that U2AF is an essential splicing factor. All U2AF activity in vitro is shown to reside in the 65 kd U2AF polypeptide. Based upon both immunological and functional criteria, U2AF is shown to be evolutionarily conserved. Most significantly, a Drosophila melanogaster nuclear extract contains proteins that are antigenically related to both human U2AF polypeptides and can substitute for human U2AF in vitro. Finally, it is shown that U2AF has an unexpected intranuclear distribution. Although diffusely present throughout the nucleoplasm, U2AF is also concentrated in a small number (between one and five) of nuclear 'centers'. This localization differs strikingly from that reported for snRNP antigens and splicing factors. These data suggest that these centers represent novel aspects of nuclear organization (Zamore, 1991).
The large subunit of the human U2 small nuclear ribonucleoprotein particle auxiliary factor (hU2AF65) is an essential RNA-splicing factor required for the recognition of the polypyrimidine tract immediately upstream of the 3' splice site. In the present study, the solution structures of two hU2AF65 fragments, corresponding to the first and second RNA-binding domains (RBD1 and RBD2, respectively), were determined by nuclear magnetic resonance spectroscopy. The tertiary structure of RBD2 is similar to that of typical RNA-binding domains with the beta1-alpha1-beta2-beta3-alpha2-beta4 topology. In contrast, the hU2AF65 RBD1 structure has unique features: (1) the alpha1 helix is elongated by one turn toward the C-terminus; (2) the loop between alpha1 and beta2 (the alpha1/beta2 loop) is much longer and has a defined conformation; (3) the beta2 strand, (188)AVQIN(192), was not predicted by sequence alignments, and (4) the beta2/beta3 loop is much shorter. Chemical shift perturbation experiments showed that the U2AF-binding RNA fragments interact with the four beta-strands of RBD2 whereas, in contrast, they interact with beta1, beta3 and beta4, but not with beta2 or the alpha1/beta2 loop, of RBD1. The characteristic alpha1-beta2 structure of the hU2AF65 RBD1 may interact with other proteins, such as UAP56 (Ito, 1999).
U2AF is an essential splicing factor that recognizes the 3' splice site and recruits the U2 snRNP to the branch point. The X-ray structure of the human core U2AF heterodimer, consisting of the U2AF35 central domain and a proline-rich region of U2AF65, has been determined at 2.2 Å resolution. The structure reveals a novel protein-protein recognition strategy, in which an atypical RNA recognition motif (RRM) of U2AF35 and the U2AF65 polyproline segment interact via reciprocal 'tongue-in-groove' tryptophan residues. Complementary biochemical experiments demonstrate that the core U2AF heterodimer binds RNA, and that the interacting tryptophan side chains are essential for U2AF dimerization. Atypical RRMs in other splicing factors may serve as protein-protein interaction motifs elsewhere during spliceosome assembly (Kielopf, 2001).
Requirements for intron recognition during pre-mRNA splicing in plants differ from those in vertebrates and yeast. Plant introns contain neither conserved branch points nor distinct 3' splice site-proximal polypyrimidine tracts characteristic of the yeast and vertebrate introns, respectively. However, they are strongly enriched in U residues throughout the intron, property essential for splicing. To understand the roles of different sequence elements in splicing, proteins involved in intron recognition in plants are being characterized. Nicotiana plumbaginifolia, a dicotyledonous plant, contains two genes encoding different homologs of the large 50-65-kDa subunit of the polypyrimidine tract binding factor U2AF, characterized previously in animals and Schizosaccharomyces pombe. Both plant U2AF65 isoforms, referred to as NpU2AF65a and NpU2AF65b, support splicing of an adenovirus pre-mRNA in HeLa cell nuclear extracts depleted of the endogenous U2AF factor. Both proteins interact with RNA fragments containing plant introns and show affinity for poly(U) and, to a lesser extent, poly(C) and poly(G). The branch point or the 3' splice site regions do not contribute significantly to intron recognition by NpU2AF65. The existence of multiple isoforms of U2AF may be quite general in plants because two genes expressing U2AF65 have been identified in Arabidopsis, and different isoforms of the U2AF small subunit are expressed in rice (Domon, 1998).
The mammalian splicing factor U2AF65 binds to the polypyrimidine tract adjacent to the 3' splice site and promotes assembly of U2 small nuclear ribonucleoprotein on the upstream branch point, an interaction that involves base pairing with U2 small nuclear RNA (snRNA). U2AF65 contains an RNA binding domain, required for interaction with the polypyrimidine tract, and an arginine-serine-rich (RS) region, required for U2 snRNP recruitment and splicing. Binding of U2AF65 to the polypyrimidine tract directs the RS domain to contact the branch point and promotes U2 snRNA-branch point base pairing even in the absence of other splicing factors. Analysis of RS domain mutants indicates that the ability of U2AF65 to contact the branch point, to promote the U2 snRNA-branch point interaction, and to support splicing are related activities, requiring only a few basic amino acids. Thus, the U2AF65 RS domain plays a direct role in modulating spliceosomal RNA-RNA interactions (Valcarcel, 1996).
Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3' splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. The U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155's downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, a direct correlation is shown between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, these data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS (Gozani, 1998).
The splicing factor U2AF65 binds to pyrimidine-rich sequences at 3' splice sites to recruit U2 snRNP to pre-mRNAs. U2AF65 can also promote the recruitment of U1 snRNP to weak 5' splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF65 is critical for U1 recruitment, and the role of its RNA-RNA annealing activity in this novel function of U2AF65 is discussed (Forch, 2003).
A new pyrimidine-tract binding factor, PUF, has been identified that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions (Page-McCaw, 1999).
During the early events of pre-mRNA splicing, intronic cis-acting sequences are recognized and interact through a network of RNA-RNA, RNA-protein, and protein-protein contacts. A branchpoint sequence binding protein (BBP) has been identified in yeast. The mammalian ortholog (mBBP/SF1) also binds specifically to branchpoint sequences and interacts with the well studied mammalian splicing factor U2AF65, which binds to the adjacent polypyrimidine (PY) tract. The mBBP/SF1-U2AF65 interaction promotes cooperative binding to a branchpoint sequence-polypyrimidine tract-containing RNA, and it is suggested that this cooperative RNA binding contributes to initial recognition of the branchpoint sequence (BPS) during pre-mRNA splicing. The essential nature of the third RBD of U2AF65 has been demonstrated for the interaction between the two proteins, both in the presence and absence of RNA (Berglund, 1998).
Two sequences important for pre-mRNA splicing precede the 3' end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tract. Initial recognition of these signals involves cooperative binding of the splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF65 to the Py tract. Both factors are required for recruitment of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components. In contrast, extensive depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spliceosome assembly or splicing significantly. Since BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system. Extensive depletion of SF1/mBBP from nuclear extracts of HeLa cells results in only modest reduction of their activity in spliceosome assembly and splicing. Some of these effects reflect differences in the kinetics of U2 snRNP binding. Although U2AF65 binding is reduced in the depleted extracts, the defects caused by SF1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF65, arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF65 binding (Guth, 2000).
Mammalian splicing factor 1 (SF1; also mammalian branch point binding protein [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3' splice sites and it participates in the assembly of early spliceosomal complexes. SF1/mBBP utilizes a 'maxi-K homology' (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investigate how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2AF and possibly other proteins, a transcriptional reporter system was constructed utilizing human immunodeficiency virus type 1 Tat fusion proteins and the RNA-binding specificity of the complex was examined using KH domain and RNA-binding site mutants. SF1/mBBP and U2AF cooperatively assemble in a reporter system at RNA sites composed of the BPS, PPT, and AG dinucleotide found at 3' splice sites, with endogenous proteins assembled along with the Tat fusions. The activities of the Tat fusion proteins on different BPS variants correlate well with the known splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the maxi-KH domain was identified by mutagenesis and appears similar to that used by 'simple' KH domains, involving residues from two putative helices, a highly conserved loop, and parts of a sheet. Using a homology model constructed from the cocrystal structure of a Nova KH domain-RNA complex, a plausible arrangement for SF1/mBBP-U2AF complexes assembled at 3' splice sites is proposed (Peled-Zehavi, 2001).
The essential splicing factors SF1 and U2AF play an important role in the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. The structure of the C-terminal RRM (RRM3) of human U2AF65 complexed to an N-terminal peptide of SF1 reveals an extended negatively charged helix A and an additional helix C. Helix C shields the potential RNA binding surface. SF1 binds to the opposite, helical face of RRM3. It inserts a conserved tryptophan into a hydrophobic pocket between helices A and B in a way that strikingly resembles part of the molecular interface in the U2AF heterodimer. This molecular recognition establishes a paradigm for protein binding by a subfamily of noncanonical RRMs (Selenko, 2003).
Splicing of mRNA precursors (pre-mRNAs) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use for the interaction between U2 snRNP and the branchpoint is unclear, and, in particular, none of the known mammalian factors required for this step have the sequence characteristics of proteins that hydrolyze ATP. Entry of U2 snRNP into the spliceosome is initiated by interaction of the essential splicing factor U2AF65 with the pre-mRNA polypyrimidine tract. A new region of U2AF65 required for function has been identified, and this information is used to clone a human 56-kD U2AF65 associated protein (UAP56). UAP56 is an essential splicing factor, which is recruited to the pre-mRNA dependent on U2AF65, and is required for the U2 snRNP-branchpoint interaction. The sequence of UAP56 indicates it is a member of the DEAD box family of RNA-dependent ATPases, which mediate ATP hydrolysis during several steps of yeast pre-mRNA splicing. These results reveal a new function of U2AF65: to position a DEAD box protein required for U2 snRNP binding at the pre-mRNA branchpoint region (Fleckner, 1997).
The large subunit of the mammalian U2AF heterodimer (U2AF65) is essential for splicing in vitro. To expand the understanding of how this protein functions in vivo, a null allele was created of the gene encoding the Schizosaccharomyces pombe ortholog, U2AF59, and it was employed in a variety of genetic complementation assays. (1) Analysis of an extensive series of double amino acid substitutions indicates that this splicing factor is surprisingly refractory to mutations. (2) Despite extensive structural conservation, metazoan large subunit orthologs cannot substitute in vivo for fission yeast U2AF59. (3) Because the activity of U2AF65 in vitro involves binding to the 3' polypyrimidine tract, the splicing of introns containing or lacking this feature was examined in a U2AF59 mutant described here as well as a previously isolated temperature-sensitive mutant. The data indicate that all four introns tested, including two that lack extensive runs of pyrimidines between the branchpoint and 3' splice site, show splicing defects upon shifting to the nonpermissive condition. In all cases, splicing is blocked prior to the first transesterification reaction in the mutants, consistent with the role inferred for human U2AF65 based on in vitro experiments (Romfo, 1999).
Recognition of a functional 3' splice site in pre-mRNA splicing requires a heterodimer of the proteins U2AF65/U2AF35. U2AF65 binds to RNA at the polypyrimidine tract, whereas U2AF35 is thought to interact through its arginine/serine-rich (RS) domain with other RS-domain-containing factors bound at the 5' splice site, assembled in splicing enhancer complexes, or associated with the U4/U6.U5 small nuclear ribonucleoprotein complex. It is unclear, however, how such network interactions can all be established through the small RS domain in U2AF. The function is described of a U2AF35-related protein (Urp), which is the human homologue of a mouse imprinted gene. Nuclear extracts depleted of Urp are defective in splicing, but activity can be restored by addition of recombinant Urp. U2AF35 could not replace Urp in complementation, indicating that their functions do not overlap. Co-immunodepletion showed that Urp is associated with the U2AF65/U2AF35 heterodimer. Binding studies revealed that Urp specifically interacts with U2AF65 through a U2AF35-homologous region and with SR proteins (a large family of RS-domain-containing proteins) through its RS domain. Therefore, Urp and U2AF35 may independently position RS-domain-containing factors within spliceosomes (Tronchere, 1997).
In metazoans, spliceosome assembly is initiated through recognition of the 5' splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF. U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35. U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Site-specific crosslinking was used to show that very early during spliceosome assembly U2AF35 directly contacts the 3' splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3'-splice-site consensus, AG/G. For introns with weak polypyrimidine tracts, the U2AF35-3'-splice-site interaction is critical for U2AF binding and splicing. These results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3' splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns (Wu, 1999).
U2AF promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF65 and U2AF35. U2AF65 binds to the polypyrimidine (Py) tract upstream from the 3' splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF35 facilitates U2AF65 binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF35 remain controversial. Recombinant U2AF65 is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF65, U2AF35, and the interaction between them are required for splicing of an immunoglobulin mu pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF35 occurs without changes in U2AF65 cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF35 and a novel function for this factor in pre-mRNA splicing (Guth, 1999).
The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. U2AF65 binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF35) contacts the conserved AG dinucleotide at the 3' end of the intron. The interaction between U2AF35 and the 3' splice site AG can stabilize U2AF65 binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF35 has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF65 binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF65. In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF35 dependence of IgM have been investigated. The results indicate that (1) the IgM substrate is an AG-dependent pre-mRNA, (2) U2AF35 dependence correlates with AG dependence, and (3) the identity of the first nucleotide of exon 2 is important for U2AF35 function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF35 activity and because a truncation mutant of U2AF35 consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in complementation assays. While some of the effects of U2AF35 can be explained in terms of enhanced U2AF65 binding, other activities of U2AF35 do not correlate with increased cross-linking of U2AF65 to the Py tract. Collectively, the results argue that interaction of U2AF35 with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF65 binding, thus revealing a dual function for U2AF35 in pre-mRNA splicing (Guth, 2001).
The small subunit of U2AF, which functions in 3' splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, the small subunit of Schizosaccharomyces pombe U2AF (U2AFSM) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AFSM depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues (Webb, 2004a).
U2AF35 is encoded by a conserved gene designated U2AF1. Evidence is provided for the existence of alternative vertebrate transcripts encoding different U2AF35 isoforms. Three mRNA isoforms (termed U2AF35a-c) are produced by alternative splicing of the human U2AF1 gene. U2AF35c contains a premature stop codon that targets the resulting mRNA to nonsense-mediated mRNA decay. U2AF35b differs from the previously described U2AF35a isoform in 7 amino acids located at the atypical RNA Recognition Motif involved in dimerization with U2AF65. Biochemical experiments indicate that isoform U2AF35b, which has been highly conserved from fish to man, maintains the ability to interact with U2AF65, stimulates U2AF65 binding to a pre-mRNA, and promotes U2AF splicing activity in vitro. Real time, quantitative PCR analysis indicates that U2AF35a is the most abundant isoform expressed in murine tissues, although the ratio between U2AF35a and U2AF35b varies from 10-fold in the brain to 20-fold in skeletal muscle. It is proposed that post-transcriptional regulation of U2AF1 gene expression may provide a mechanism by which the relative cellular concentration and availability of U2AF35 protein isoforms are modulated, thus contributing to the finely tuned control of splicing events in different tissues (Pacheco, 2004).
Polypyrimidine tract-binding protein (PTB: see Drosophila Hephaestus) binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3'-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5' splice site sequence to form a pseudoexon. The binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB is antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminates enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation (Lou, 1999).
The excision of introns with weak polypyrimidine tracts at their 3' splice sites can be enhanced by sequence elements in the downstream exon or by a downstream 5' splice site. The enhancers inside the exon do not conform to a strict consensus, but they are generally rich in purines. Members of the family of SR proteins recognize these elements. Not only does SF2/ASF activate many different polypurine enhancers, but also at least one other SR protein, most likely SC35, is active as well. The degree of splicing activation varies with the polypurine enhancers and the SR proteins. Further, it is shown that the similar activation by downstream 5' splice sites requires U1 snRNP, which is not the case with purine-rich enhancers. These results are consistent with a model showing that U1 snRNP binds to the 5' splice site and SR proteins to exonic sequences upstream of the 5' splice site. Both interact with U2AF at the 3' splice site. This represents a molecular explanation for the exon recognition which is important for splice site selection in mammals (Achsel, 1996).
U2AF is a heterodimeric splicing factor composed of 65-kDa (U2AF65) and 35-kDa (U2AF35) subunits. The large subunit of U2AF recognizes the intronic polypyrimidine tract, a sequence located adjacent to the 3' splice site that serves as an important signal for both constitutive and regulated pre-mRNA splicing. The small subunit U2AF35 interacts with the 3' splice site dinucleotide AG and is essential for regulated splicing. Like several other proteins involved in constitutive and regulated splicing, both U2AF65 and U2AF35 contain an arginine/serine-rich (RS) domain. The role of RS domains in the subcellular localization of U2AF is reported. Both U2AF65 and U2AF35 are shown to shuttle continuously between the nucleus and the cytoplasm by a mechanism that involves carrier receptors and is independent from binding to mRNA. The RS domain on either U2AF65 or U2AF35 acts as a nuclear localization signal and is sufficient to target a heterologous protein to the nuclear speckles. Furthermore, the results suggest that the presence of an RS domain in either U2AF subunit is sufficient to trigger the nucleocytoplasmic import of the heterodimeric complex. Shuttling of U2AF between nucleus and cytoplasm possibly represents a means to control the availability of this factor to initiate spliceosome assembly and therefore contribute to regulate splicing (Gama-Carvalho, 2003).
TAP/NXF1 is a conserved mRNA export receptor serving as a link between messenger ribonucleoproteins (mRNPs) and the nuclear pore complex. The mechanism by which TAP recognizes its export substrate is unclear. TAP is added to spliced mRNP in human cells. A distinct region of TAP was identified that targets it to mRNP. Using yeast two-hybrid screens and in vitro binding studies, it was found that this region coincides with a direct binding site for U2AF35, the small subunit of the splicing factor U2AF. This interaction is evolutionarily conserved across metazoa, indicating its significance. In human cells the exogenously expressed large U2AF subunit, U2AF65, accumulates in spliced mRNP, leading to the recruitment of U2AF35 and TAP. Similar to TAP, U2AF65 stimulates directly the nuclear export and expression of an mRNA that is otherwise retained in the nucleus. Together with the finding that U2AF is continuously exported from the nucleus, these data suggest that U2AF participates in nuclear export, by facilitating TAP's addition to its mRNA substrates (Zolotukhin, 2002).
The general splicing factor U2AF65 recognizes the polypyrimidine tract (Py tract) that precedes 3' splice sites and has three RNA recognition motifs (RRMs). The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein-protein contacts with splicing factors mBBP/SF1 and SAP155. Unexpectedly, the human RRM3 domain was found to be dispensable for U2AF65 activity in vitro. However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155: (1) deletion of RRM3 from the human protein has no effect on Py-tract binding; (2) RRM123 and RRM12 select similar sequences from a random pool of RNA; (3) deletion of RRM3 has no effect on the splicing activity of U2AF65 in vitro. However, deletion of the RRM3 domain of S. pombe U2AF(59) abolishes U2AF function in vivo. In addition, certain amino acid substitutions on the four-stranded beta-sheet surface of RRM3 compromise U2AF function in vivo without affecting binding to mBBP/SF1 or SAP155 in vitro. It is proposed that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns (Banerjee, 2004).
Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. A U2AF-depleted HeLa nuclear extract has a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly (Kent, 2005).
Pre-mRNA splicing is frequently coupled to transcription by RNA polymerase II (RNAPII). This coupling requires the C-terminal domain of the RNAPII largest subunit (CTD), although the underlying mechanism is poorly understood. Using a biochemical complementation assay, an activity was identified that stimulates CTD-dependent splicing in vitro. This activity was purified from HeLa cells and found to consist of a complex of two well-known splicing factors: U2AF65 and the Prp19 complex (PRP19C). Evidence is provided that both U2AF65 and PRP19C are required for CTD-dependent splicing activation, that U2AF65 and PRP19C interact both in vitro and in vivo, and that this interaction is required for activation of splicing. Providing the link to the CTD, it was shown that U2AF65 binds directly to the phosphorylated CTD, and that this interaction results in increased recruitment of U2AF65 and PRP19C to the pre-mRNA. These results not only provide a mechanism by which the CTD enhances splicing, but also describe unexpected interactions important for splicing and its coupling to transcription (David, 2011).
A number of interactions linking pre-mRNA processing and the CTD have been documented. For capping, the functional connection with the CTD is straightforward; the guanylytransferase and methyltransferase enzymes necessary for capping both bind to the S5- phosphorylated CTD, which allosterically activates guanylytransferase activity (McCracken, 1997; Yue, 1997; Ho, 1999). Connections between the polyadenylation machinery and the CTD have also been demonstrated. Human CstF50 was shown to interact physically with both the phosphorylated and unphosphorylated CTD, an interaction that appears important for efficient cleavage/polyadenylation in vivo (N. Fong, 2001). The yeast CFI subunit Pcf11 also interacts with the S2-phosphorylated CTD, the functional importance of which was suggested by a genetic interaction between a Pcf11 allele and an RBP1 CTD truncation allele (Licatalosi, 2002). Also, a CTD phosphatase, Ssu72, was shown recently to be important for transcription-coupled 3' processing in vitro (Xiang, 2010; David, 2011 and references therein).
The machinery that carries out pre-mRNA splicing is considerably more complex than those responsible for capping and polyadenlyation. The spliceosome, the protein-RNA assembly that catalyzes intron removal, contains at least 150 proteins and undergoes dynamic changes in conformation and protein composition during the series of events that begin with splice site recognition and end after the execution of the two catalytic steps. In vitro, spliceosome assembly proceeds through the formation of a series of stable intermediate complexes, which are biochemically separable and amenable to proteomic analysis (Wahl, 2009). Among the earliest steps in spliceosome assembly is recognition of the 5' and 3' splice sites by the U1 snRNP and U2AF, respectively. U2AF is a dimer comprised of U2AF65 and U2AF35. U2AF65 binds to polypyrimidine-rich sequences found near the 3' end of most introns and promotes stable U2 snRNP association with the pre-mRNA, an activity that requires its N-terminal arginine-serine-rich (RS) domain. U2AF35 contacts a well-conserved AG dinucleotide at the 3' end of the intron and can interact with exon-bound SR proteins; both interactions can stabilize U2AF binding to suboptimal polypyrimidine tracts. Later steps in spliceosome assembly involve the activity of numerous additional factors, including the U4/U6.U5 tri-snRNP and the Prp19 complex, or PRP19C (Wahl, 2009). PRP19C was first discovered in yeast, where it was shown to be an essential splicing factor that does not tightly associate with snRNPs (Hogg, 2010). PRP19C, which consists of four polypeptides that form a salt-stable core (CDC5L, PRLG1, Prp19, and SPF27) and three more loosely associated polypeptides (HSP73, CTNNBL1, and AD002) (Grote, 2010), is found at the core of catalytically activated spliceosomes and plays a critical but poorly understood role in activation of the spliceosome. Because PRP19C does not contain any proteins known to bind RNA, it is likely that PRP19C recruitment to the spliceosome occurs through protein-protein interactions with RNA-bound factors, although no such interaction has yet been described (David, 2011 and references therein).
Most of what is known about the process of spliceosome assembly has come from the use of in vitro systems that are uncoupled from transcription, leaving the role of the transcriptional machinery in the process relatively poorly understood. However, a few physical interactions between splicing factors and the CTD have been documented. The yeast U1 snRNP component Prp40 was shown to bind to the phosphorylated CTD through multiple WW domains. In humans, splicing factors that have been shown to bind directly to the CTD include CA150, PSF, and p54/NRB. Of these, support for a functional significance to the CTD interaction has been provided only for PSF, which can be recruited to promoters by strong transcriptional activators to promote splicing in a CTD-dependent manner in vivo (David, 2011 and references therein).
In order to study the functional connections between the CTD and pre-mRNA splicing, a fusion between the CTD and the SR protein SRSF1 (formerly ASF/SF2) was constructed. This allowed recruitment of the CTD to splicing substrates harboring SRSF1-binding sites independent of transcription. Using this fusion protein, which is called SRSF1-CTD, in in vitro splicing assays, an increase was observed in splicing kinetics in its presence when compared with SRSF1 alone. In addition, a HeLa nuclear fraction (NF20-40) was found to be capable of activating splicing of one substrate, IgMA3, in HeLa S-100 in the presence of SRSF1-CTD but not SRSF1, suggesting that NF20-40 contains a factor capable of functionally interacting with the CTD. The factor responsible for this activity was purified and characterized, and it was found to consists of a complex containing both U2AF65 and PRP19C. U2AF65 and PRP19C interact directly in vitro and in an RNA-independent manner in vivo. Additionally, U2AF65 binds directly to the phosphorylated CTD, increasing U2AF association with the pre-mRNA and recruitment of PRP19C. U2AF65 thus bridges the transcriptional machinery and later stages of spliceosomal assembly through novel interactions with the RNAPII CTD and PRP19C (David, 2011).
A model is presented for activation of CTD-dependent splicing by a U2AF-PRP19C complex. At promoters, RNAPII is present in preinitiation complexes, but the CTD is unphosphorylated and unable to recruit splicing factors. Transcription initiation results in CTD phosphorylation by multiple kinases, resulting in the association of splicing factors (including SR proteins), U1 snRNP through unknown interactions, and the U2AF-PRP19C complex via a direct interaction with U2AF65. RNAPII-associated U1 and SR proteins recognize a transcribed exon, resulting in its tethering to the RNAPII elongation complex through multiple interactions. Transcription of the 3' splice site results in a transition from protein-protein interactions between U2AF65 and the p-CTD to protein-RNA interactions, resulting in efficient recognition of the 3' splice site. This facilitates rapid transition to a mature spliceosomal complex promoted by U2AF65-associated PRP19C (David, 2011).
Search PubMed for articles about Drosophila
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date revised: 12 January 2018
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