Analysis of mutant phenotypes and splicing defects demonstrates functional collaboration between the large and small subunits of the essential splicing factor U2AF in vivo

The heterodimeric splicing factor U2AF plays an important role in 3' splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3' splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating 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 Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3' splice site recognition, we sought introns for which the spU2AFLG and spU2AFSM make distinct contributions 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.     

A conditional role of U2AF in splicing of introns with unconventional polypyrimidine tracts

Recognition of polypyrimidine (Py) tracts typically present between the branch point and the 3' splice site by the large subunit of the essential splicing factor U2AF is a key early step in pre-mRNA splicing. Diverse intronic sequence arrangements exist, however, including 3' splice sites lacking recognizable Py tracts, which raises the question of how general the requirement for U2AF is for various intron architectures. Our analysis of fission yeast introns in vivo has unexpectedly revealed that whereas introns lacking Py tracts altogether remain dependent on both subunits of U2AF, introns with long Py tracts, unconventionally positioned upstream of branch points, are unaffected by U2AF inactivation. Nevertheless, mutation of these Py tracts causes strong dependence on the large subunit U2AF59. We also find that Py tract diversity influences the requirement for the conserved C-terminal domain of U2AF59 (RNA recognition motif 3), which has been implicated in protein-protein interactions with other splicing factors. Together, these results suggest that in addition to Py tract binding by U2AF, supplementary mechanisms of U2AF recruitment and 3' splice site identification exist to accommodate diverse intron architectures, which have gone unappreciated in biochemical studies of model pre-mRNAs.     

The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF 65) is essential in vivo but dispensable for activity in vitro

The general splicing factor U2AF(65) 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, we find that the human RRM3 domain is dispensable for U2AF(65) 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. First, deletion of RRM3 from the human protein has no effect on Py-tract binding. Second, RRM123 and RRM12 select similar sequences from a random pool of RNA. Third, deletion of RRM3 has no effect on the splicing activity of U2AF(65) 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. We propose that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns. We discuss the implications of these observations on previous models of U2AF function.     

The small subunit of the splicing factor U2AF is conserved in fission yeast.

The human splicing factor U2 auxiliary factor (hsU2AF) is comprised of two interacting subunits of 65 and 35 kDa. Previously we identified the Schizosaccharomyces pombe homolog, spU2AF59, of the human large subunit. We have screened a fission yeast cDNA library in search of proteins that interact with spU2AF59 using the yeast two-hybrid system and have identified a homolog of the hsU2AF35 subunit. The S. pombe U2AF small subunit is a single copy gene that encodes a protein which shares 55% amino acid identity and 17% similarity with the human small subunit. Unlike the human protein, the yeast protein lacks an arginine/serine-rich region. The predicted molecular mass of the spU2AF small subunit is 23 kDa. The region of spU2AF59 that interacts with spU2AF23 is similar to the region in which the human small and large subunits interact.     

Molecular characterization and phylogeny of U2AF35 homologs in plants

U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is an essential splicing factor with critical roles in recognition of the 3'-splice site. In animals, the U2AF small subunit (U2AF35) can bind to the 3'-AG intron border and promote U2 small nuclear RNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit. Two copies of U2AF35-encoding genes were identified in Arabidopsis (Arabidopsis thaliana; atU2AF35a and atU2AF35b). Both are expressed in all tissues inspected, with atU2AF35a expressed at a higher level than atU2AF35b in most tissues. Differences in the expression patterns of atU2AF35a and atU2AF35b in roots were revealed by a promoter::beta-glucuronidase assay, with atU2AF35b expressed strongly in whole young roots and root tips and atU2AF35a limited to root vascular regions. Altered expression levels of atU2AF35a or atU2AF35b cause pleiotropic phenotypes (including flowering time, leaf morphology, and flower and silique shape). Novel slicing isoforms were generated from FCA pre-mRNA by splicing of noncanonical introns in plants with altered expression levels of atU2AF35. U2AF35 homologs were also identified from maize (Zea mays) and other plants with large-scale expressed sequence tag projects. A C-terminal motif (named SERE) is highly conserved in all seed plant protein homologs, suggesting it may have an important function specific to higher plants.     

茄子SmU2AF65基因的可变剪接

摘要: U2AF(U2 snRNP auxiliary factor)是参与前体mRNA剪接的重要辅助因子, 在进化上具有较高保守性。文章根据茄子BAC 77N19(GenBank登录号: EF517791)的基因组序列信息和烟草NpU2AF⁶⁵a和NpU2AF⁶⁵b基因的全长cDNA序列, 设计特异引物, 经cDNA末端快速扩增法(RACE)获得了1 986 bp的茄子同源基因(SmU2AF⁶⁵)全长cDNA, GenBank登录号为EU543263。序列分析表明该序列包含1 665 bp的可阅读框, 编码554个氨基酸, 在氨基酸序列的C末端有3个保守的RNA识别结构域RRM。RT-PCR分析表明, SmU2AF⁶⁵基因在不同组织中均有表达,但是该基因通过可变剪接至少能够产生两个转录本, 在根中产生与其他组织中不同的剪切子     

Genome-wide analysis reveals an unexpected function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs

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 dU2AF50. 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.     

Alternative modes of binding by U2AF65 at the polypyrimidine tract

During initial recognition of an intron in pre-mRNA, the 3' end of the intron is bound by essential splicing factors. Notably, the consensus RNA sequences bound by these proteins are highly degenerate in humans. This raises the question of 3' splicing factor function in introns lacking canonical binding sites. Investigating the introns of the model organism Neurospora crassa revealed a different organization at the 3' end of the intron compared to most eukaryotic organisms. The predicted branch point sequences of Neurospora introns are much closer to the 3' splice site compared to those in human introns. In addition, Neurospora introns lack the canonical polypyrimidine tract found at the end of introns in most eukaryotic organisms. The large subunit of the U2 snRNP associated factor (U2AF65), which is essential for splicing of human introns and specifically recognizes the polypyrimidine tract, is also present in Neurospora. We show that Neurospora U2AF65 binds RNA with low affinity and specificity, apparently evolving with its disappearing binding site. The arginine/serine rich domain at the N-terminus of Neurospora U2AF65 regulates its RNA binding. We find that this regulated binding can be recapitulated in human U2AF65 which has been mutated to decrease both affinity and overall charge. Finally, we show that the addition of the small U2AF subunit (U2AF35) to U2AF65 with weakened RNA binding affinity significantly enhances the affinity of the resulting U2AF heterodimer.     

Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65

The U2 auxiliary factor large subunit (U2AF65) is an essential pre-mRNA splicing factor for the initial stages of spliceosome assembly. Tandem RNA recognition motifs (RRM)s of U2AF65 recognize polypyrimidine tract signals adjacent to 3' splice sites. Despite the central importance of U2AF65 for splice site recognition, the relative arrangement of the U2AF65 RRMs and the energetic forces driving polypyrimidine tract recognition remain unknown. Here, the solution conformation of the U2AF65 RNA binding domain determined using small angle x-ray scattering reveals a bilobal shape without apparent interdomain contacts. The proximity of the N and C termini within the inter-RRM configuration is sufficient to explain the action of U2AF65 on spliceosome components located both 5' and 3' to its binding site. Isothermal titration calorimetry further demonstrates that an unusually large enthalpy-entropy compensation underlies U2AF65 recognition of an optimal polyuridine tract. Qualitative similarities were observed between the pairwise distance distribution functions of the U2AF65 RNA binding domain and those either previously observed for N-terminal RRMs of Py tract-binding protein that lack interdomain contacts or calculated from the high resolution coordinates of a U2AF65 deletion variant bound to RNA. To further test this model, the shapes and RNA interactions of the wild-type U2AF65 RNA binding domain were compared with those of U2AF65 variants containing either Py tract-binding protein linker sequences or a deletion within the inter-RRM linker. Results of these studies suggest inter-RRM conformational plasticity as a possible means for U2AF65 to universally identify diverse pre-mRNA splice sites.     

Molecular genetic analysis of U2AF59 in Schizosaccharomyces pombe: differential sensitivity of introns to mutational inactivation.

The large subunit of the mammalian U2AF heterodimer (U2AF65) is essential for splicing in vitro. To expand our understanding of how this protein functions in vivo, we have created a null allele of the gene encoding the Schizosaccharomyces pombe ortholog, U2AF59, and employed it in a variety of genetic complementation assays. First, analysis of an extensive series of double amino acid substitutions indicates that this splicing factor is surprisingly refractory to mutations. Second, despite extensive structural conservation, we find that metazoan large subunit orthologs cannot substitute in vivo for fission yeast U2AF59. Third, because the activity of U2AF65 in vitro involves binding to the 3'' polypyrimidine tract, we examined the splicing of introns containing or lacking this feature in a U2AF59 mutant described here as well as a previously isolated temperature-sensitive mutant (Potashkin et al., 1993, Science 262:573-575). Our 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.     

RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) 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 recently 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 previously 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. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF³⁸) was complexed with the large-subunit (dU2AF⁵⁰) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF⁵⁰. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF⁶⁵. 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 (dU2AF⁵⁰ΔRS) severely impaired its binding activity. However, if the dU2AF³⁸ RS domain was supplied in a complex with dU2AF⁵⁰ΔRS, high-affinity binding was 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.     

A description of the Mei2-like protein family; structure,phylogenetic distribution and biological context

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a "mei2-dot" in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.     

Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed.     

U2 small nuclear RNA is remarkably conserved between Schizosaccharomyces pombe and mammals.

We report the molecular cloning and sequencing of the most abundant trimethylguanosine-capped small nuclear RNA from the fission yeast Schizosaccharomyces pombe, a highly conserved homolog of mammalian U2 small nuclear RNA. This RNA is 186 nucleotides in length, just 2 nucleotides shorter than its human counterpart; this is in contrast to Saccharomyces cerevisiae U2, which is 1,175 nucleotides long. Moreover, the secondary structure of Schizosaccharomyces pombe U2 is virtually identical to that of mammalian U2, including the 3' half of the RNA, which shows limited primary sequence identity. Northern (RNA) blot analysis revealed that the size of this RNA is conserved not only in fission yeasts but in many organisms, including other ascomycetes.     

Dual function for U2AF(35) in AG-dependent pre-mRNA splicing

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) 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 U2AF(65) 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 U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) 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 U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.