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).
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