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Results for “Splicing Factor U2AF”

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PubMed2000

Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal.

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, we used chemical interference and protection assays, saturation mutagenesis, and RNAs containing modified nucleotides. We find that these proteins recognize distinct features of the RNA. First, 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 SXL only. Second, the 2' hydroxyl groups or backbone conformation appeared important for the binding of SXL, but not U2AF65. Third, although any of the bases (cytosine >> adenine > guanine) could substitute for uracils for U2AF65 binding, only guanine partially substituted for certain uracils for SXL binding. The different dependence on individual contacts and nucleotide preference may provide a basis for the different RNA-binding specificities and thus functions of U2AF65 and SXL in 3' splice site choice.

Amino Acid Motifs↗

PubMed2002

A general role for splicing enhancers in exon definition.

Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection.

Alternative Splicing↗

PubMed1998

Conservation of functional domains involved in RNA binding and protein-protein interactions in human and Saccharomyces cerevisiae pre-mRNA splicing factor SF1.

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 (y) SF1 interacts with Mud2p, the presumptive U2AF65 homologue in yeast, and the interaction domain is conserved in ySF1. The C-terminal degenerate RRMs in U2AF65 and Mud2p mediate the association with hSF1 and ySF1, respectively. Analysis of chimeric constructs of hSF1 and ySF 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 ySF1, as well as U2AF65 and Mud2p, being functional homologues.

Amino Acid Sequence↗

PubMed1999

Transient interaction of BBP/ScSF1 and Mud2 with the splicing machinery affects the kinetics of spliceosome assembly.

Removal of introns from pre-mRNA is an essential step of gene expression. The splicing reaction is catalyzed in a large complex termed the spliceosome. Introns are recognized during the early steps of spliceosome assembly with the formation of commitment complexes. Intron recognition is mediated by the interaction of splicing factors with conserved sequences present in the pre-mRNA. BBP/SF1 participates in this recognition by interacting with the pre-mRNA branch point in both yeast and mammals. This protein, which is essential in yeast, also interacts with the U2AF65/Mud2 splicing factor. However, its precise role in splicing complex formation is still unclear. We have now analyzed the presence of BBP and Mud2 in yeast splicing complexes using supershift and coprecipitation assays. We found that BBP is present together with Mud2 in commitment complex 2 (CC2), but is not detectable in commitment complex 1 (CC1). Furthermore, genetic and biochemical depletion of BBP demonstrated that it is required for CC2 formation. In addition we observed that BBP and Mud2 are not detectable in pre-spliceosomes. These are the first commitment complex components that are shown to be released during or immediately after pre-spliceosome formation. Interestingly, depletion of BBP or disruption of MUD2 had no significant effect on pre-spliceosome formation and splicing in vitro but led to a transient accumulation of CC1. These observations support a model in which BBP and Mud2 are recycled during transition from CC2 to pre-spliceosome.

DNA-Binding Proteins↗

PubMed1999

Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition.

Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition.

Amino Acid Sequence↗

PubMed2006

The SF3b155 N-terminal domain is a scaffold important for splicing.

Recruitment of U2 snRNP to the branch point sequence of introns is a necessary step in pre-mRNA splicing. In the current model, U2AF65, bound at the polypyrimidine tract of the intron, recruits U2 snRNP to the branch point sequence by interacting with the U2 snRNP protein SF3b155. We demonstrate that the N-terminal domain of SF3b155 contains multiple U2AF65 binding sites that are distinct from the binding site for the U2 snRNP protein p14, mapped to amino acids 396-424 of SF3b155. The N-terminal domain of SF3b155 appears to adopt a primarily unfolded structure but is functional to inhibit splicing in vitro. RNA binding studies show that the N-terminal domain of SF3b155 binds RNA nonspecifically and that the sites for U2AF65 binding and RNA binding are overlapping (or the same) within SF3b155. We propose that the N-terminal domain of SF3b155 adopts a primarily unfolded structure that functions as a scaffold to facilitate SF3b155's multiple protein-protein and protein-RNA interactions. The multiple U2AF65 binding sites on SF3b155 suggest a model in which multiple U2AF65 molecules bound to the intron could enhance U2 snRNP recruitment to the branch point sequence.

Amino Acid Sequence↗

PubMed2000

Intron recognition comes of AGe.

The molecular basis for the consensus sequence at the 3' ends of introns in higher eukaryotes has now been elucidated. However, this discovery does not explain all aspects of 3' splice site selection.

Animals↗

PubMed2005

RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit.

Structural studies of RNA polymerase II have suggested two possible exit paths for the nascent RNA: groove 1, which points toward the subcomplex of subunits Rpb4 and Rpb7, and groove 2, which points toward Rpb8. These alternatives could not be distinguished previously because less than 10 nucleotides (nt) of transcript were resolved in the structures. We have approached this question by UV cross-linking nascent RNA to components of the transcription complex through uridine analogs located within the first six nucleotides of the RNA. We find that the emerging transcript cross-links to the Rpb7 subunit of RNA polymerase II in various complexes containing 26- to 32-nt transcripts. This interaction is greatly reduced in complexes with 41- or 43-nt RNAs and absent when the transcript is 125 nt. Our results are consistent with groove 1 being the exit path for nascent RNA.

Base Sequence↗

PubMed2000

The human papillomavirus type 16 negative regulatory RNA element interacts with three proteins that act at different posttranscriptional levels.

In human papillomaviruses, expression of the late genes L1 and L2, encoding the capsid proteins, is restricted to the upper layers of the infected epithelium. A 79-nt GU-rich negative regulatory element (NRE) located at the 3' untranslated region of the human papillomavirus 16 L1 gene was identified previously as key to the posttranscriptional control of late gene expression. Here, we demonstrate that in epithelial cells, the NRE can directly bind the U2 auxiliary splicing factor 65-kDa subunit, the cleavage stimulation factor 64-kDa subunit, and the Elav-like HuR protein. On induction of epithelial cell differentiation, levels of the U2 auxiliary splicing factor 65-kDa subunit decrease, levels of the cleavage stimulation factor 64-kDa subunit increase, and the levels of HuR remain unchanged, although redistribution of the HuR from the nucleus to the cytoplasm is observed. Late gene transcripts, which appear to be fully processed, are detected in undifferentiated W12 cells, but are confined in the nucleus. We propose that repression of late gene expression in basal epithelial cells may be caused by nuclear retention or cytoplasmic instability of NRE-containing late gene transcripts.

Cell Differentiation↗

PubMed1995

Enhancer-dependent interaction between 5' and 3' splice sites in trans.

Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing enhancers that act by promoting the activity of upstream splice sites. Here we show that RNA molecules containing a 3' splice site and enhancer sequence are efficiently spliced in trans to RNA molecules containing normally cis-spliced 5' splice sites or to normally trans-spliced spliced leader RNAs from lower eukaryotes. In addition, we show that this reaction is stimulated by (Ser + Arg)-rich splicing factors that are known to promote protein-protein interactions in the cis-splicing reaction. Thus, splicing enhancers facilitate the assembly of protein complexes on RNAs containing a 3' splice site, and this complex is sufficiently stable to functionally interact with 5' splice sites located on separate RNAs. This trans-splicing is mediated by interactions between (Ser + Arg)-rich splicing factors bound to the enhancer and general splicing factors bound to the 5' and 3' splice sites. These same interactions are likely to play a crucial role in alternative splicing and splice-site selection in cis.

Animals↗

PubMed1996

Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.

We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) particles that sediment at the 200S region in sucrose gradients. The InRNP particles contain all uridine-rich small nuclear ribonucleoprotein complexes required for pre-mRNA splicing, as well as protein splicing factors. In this paper we show that all of the phosphorylated, mAb 104 detectable, Ser/Arg-rich essential splicing factors (SR proteins) in the nucleoplasm are integral components of the InRNP particles, whereas only part of the essential splicing factor U2AF65 (U2 snRNP auxiliary factor) and the polypyrimidine tract binding protein (PTB) are associated with these particles. This finding suggests a limiting role for SR proteins in the assembly of the InRNP particles. We further show that the structural integrity of InRNP particles is sensitive to variations in the phosphorylation levels of the SR proteins.

Animals↗

PubMed1997

A cellular 65-kDa protein recognizes the negative regulatory element of human papillomavirus late mRNA.

Papillomavirus late gene expression is tightly linked to the differentiation state of the host cell. Levels of late mRNAs are only in part controlled by regulation of the late promoter, other posttranscriptional mechanisms exist that reduce the amount of late mRNA in undifferentiated cells. Previously we described a negative regulatory element (NRE) located upstream of the human papillomavirus type 16 late poly(A) site. We have delineated the NRE to a 79-nt region in which a G+U-rich region was the major determinant of NRE activity. UV-crosslinking assays identified a prominent nuclear protein of 65 kDa as the only factor in close contact with the NRE, and a complex of at least five proteins, including the 65-kDa protein, was enriched on NRE-RNA. Binding of the 65-kDa protein was depleted by preincubation with poly(U) Sepharose in high salt, a property characteristic of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF65 and bacterially expressed U2AF65 exhibited NRE binding. The 65-kDa protein bound to the G+U-rich NRE 3' half which shows homology to the B2P2 sequence a known U2AF65 binding site in the alpha-tropomyosin gene, and the G+U-rich element can be replaced by B2P2 in the binding assay. Treatment of cells with phorbol 12-myristate 13-acetate reduced binding of the 65-kDa protein, induced NRE binding of a cytoplasmic protein, and relieved the NRE block on reporter gene expression.

Base Sequence↗

PubMed1995

The U1 small nuclear ribonucleoprotein/5' splice site interaction affects U2AF65 binding to the downstream 3' splice site.

In the gene of the neural cell adhesion molecule, the 5' splice site of the alternate exon 18 plays an important role in establishing regulated splicing profiles. To understand how the 5' splice site of exon 18 contributes to splicing regulation, we have investigated the interaction of the U2AF65 splicing factor to pre-mRNAs that contained portions of the constitutive exon 17 or the alternate exon 18 fused to exon 19 and separated by a shortened intron. Despite sharing an identical 3' splice site, only the pre-mRNA that contained a portion of exon 17 and its associated 5' splice site displayed efficient U2AF65 cross-linking. Strikingly, a G-->U mutation at position +6 of the intron, converting the 5' splice site of exon 18 into that of exon 17, stimulated U2AF65 crosslinking. The improved cross-linking efficiency of U2AF65 to a pre-mRNA carrying the 5' splice site of exon 17 required the integrity of the 5' end of U1 but not of U2 small nuclear RNA. Our results indicate that neural cell adhesion molecule 5' splice site sequences influence U2AF65 binding through a U1 small nuclear ribonucleoprotein/U2AF interaction that occurs at the commitment stage of spliceosome assembly, before stable binding of the U2 small nuclear ribonucleoprotein. Thus, the 5' splice sites of exons 17 and 18 differentially affect U2AF65 binding to the 3' splice site of exon 19. Factors that modulate U1 small nuclear ribonucleoprotein binding to these 5' splice sites may play a critical role in regulating exon 18 skipping.

Base Sequence↗

PubMed1997

Aberrant methylation of an imprinted gene U2af1-rs1(SP2) caused by its own transgene.

Genomic imprinting refers to the parental allele-specific expression of genes. The precise mechanism underlying this phenomenon, which may involve DNA methylation, is not yet known. U2af1-rs1(SP2) is an imprinted gene expressed from the paternal allele and is methylated on the maternal allele. Here we report an artificial system in which expression and methylation of the endogenous imprinted gene U2af1-rs1 can be affected by interaction with its own transgene in the testis. We suggest that there is a mechanism in male gametogenesis by which the U2af1-rs1 gene is kept unmethylated to be expressed in the offspring in addition to a mechanism in female gametogenesis by which the U2af1-rs1 gene is methylated and is not expressed in the offspring.

Alleles↗

PubMed1998

Role of the constitutive splicing factors U2AF65 and SAP49 in suboptimal RNA splicing of novel retroviral mutants.

Retroviruses display a unique form of alternative splicing in which both spliced and unspliced RNAs accumulate in the cytoplasm. Simple retroviruses, such as avian sarcoma virus, do not encode regulatory proteins that affect splicing; this process is controlled solely through interactions between the viral RNA and the host cell splicing machinery. Previously, we described the selection and characterization of novel avian sarcoma virus mutants. These viruses were separated into two classes based upon analysis of splicing intermediates produced in infected cells and in a cell-free system. One class, which included mutants with altered polypyrimidine tract or branch point sequences, showed significant accumulation of intermediates, suggesting that splicing was regulated in step 2. The other class, which included mutants with deletions of exonic enhancer sequences, did not accumulate splicing intermediates, suggesting that splicing was regulated before step 1 of the splicing reaction. In this report, we show that a mutant blocked at step 1 fails to form a stable spliceosomal complex, whereas one blocked at step 2 shows a defect in its ability to transit through the last spliceosomal complex. Using UV cross-linking methods, we show that regulation at each step is associated with specific changes in the binding of cellular splicing factors. Regulation at step 1 is correlated with decreased cross-linking of the factor U2AF65, whereas regulation at step 2 is correlated with enhanced cross-linking of the factor SAP49. Because these mutations were isolated by selection for replication-competent viruses, we conclude that retroviral splicing may be regulated in vivo through altered binding of constitutive splicing factors.

Alternative Splicing↗

PubMed2001

Disruption of imprinted expression of U2afbp-rs/U2af1-rs1 gene in mouse parthenogenetic fetuses.

The present study shows that the U2afbp-rs gene, a paternally expressed imprinted gene, is activated and expressed in a biallelic manner from maternal alleles in parthenogenetic mouse fetuses on day 9.5 of gestation. The mean expression was detected to be 88% (31-134%) of that in control biparental fetuses, using real-time quantitative reverse transcription and polymerase chain reaction. This disrupted expression of the gene was associated with changes in the chromatin structure but not with the methylation pattern in the regulation region. The present results show that parental specific expression of imprinted genes is not always maintained in uniparental embryos. This suggests that both parental genomes are necessary to establish parental specific expression of the U2afbp-rs gene.

Animals↗

PubMed2002

Inhibition of histone deacetylases alters allelic chromatin conformation at the imprinted U2af1-rs1 locus in mouse embryonic stem cells.

Most loci that are regulated by genomic imprinting have differentially methylated regions (DMRs). Previously, we showed that the DMRs of the mouse Snrpn and U2af1-rs1 genes have paternal allele-specific patterns of acetylation on histones H3 and H4. To investigate the maintenance of acetylation at these DMRs, we performed chromatin immunoprecipitation on trichostatin-A (TSA)-treated and control cells. In embryonic stem (ES) cells and fibroblasts, brief (6-h) TSA treatment induces global hyperacetylation of H3 and H4. In ES cells only, TSA led to a selective increase in maternal acetylation at U2af1-rs1, at lysine 5 of H4 and at lysine 14 of H3. TSA treatment of ES cells did not affect DNA methylation or expression of U2af1-rs1, but was sufficient to increase DNase I sensitivity along the maternal allele to a level comparable with that of the paternal allele. In fibroblasts, TSA did not alter U2af1-rs1 acetylation, and the parental alleles retained their differential DNase I sensitivity. At Snrpn, no changes in acetylation were observed in the TSA-treated cells. Our data suggest that the mechanisms regulating histone acetylation at DMRs are locus and developmental stage-specific and are distinct from those effecting global levels of acetylation. Furthermore, it seems that the allelic U2af1-rs1 acetylation determines DNase I sensitivity/chromatin conformation.

Acetylation↗

PubMed2004

Newly Initiated RNA encounters a factor involved in splicing immediately upon emerging from within RNA polymerase II.

We employed RNA-protein cross-linking to map the path of the nascent RNA as it emerges from within RNA polymerase II. A UV-cross-linkable uridine analog was incorporated at two positions within the first five nucleotides of the transcript. Only the two largest subunits of RNA polymerase II cross-linked to the transcript in complexes containing 17-24-nucleotide (nt) RNAs. Extension of the RNA to 26 or 28 nt revealed an additional strong cross-link to the splicing factor U2AF65. In U17 complexes, in which the RNA is still contained within the polymerase, U2AF65 is tightly bound. In contrast, U2AF65 is more loosely bound in C28 transcription complexes, in which about 10 nt of transcript have emerged from the RNA polymerase. Cross-linking of U2AF65 to RNA in a C28 complex was eliminated by the addition of an excess of an RNA oligonucleotide containing the consensus U2AF65 binding site, but U2AF65 was not displaced by a nonconsensus RNA. These findings indicate that U2AF65 shifts from protein-protein to protein-RNA interactions as the RNA emerges from the polymerase. During transcription of one particular template at low UTP concentration, RNA polymerase II pauses just after synthesizing a transcript segment that is a U2AF65 binding site. Dwell time of the polymerase at this pause site was significantly and specifically reduced by the addition of recombinant U2AF65 to the transcription reaction. Therefore, the association of U2AF65 with RNA polymerase II may function not only to deliver U2AF65 to the nascent transcript but also to modulate efficient transcript elongation.

Cell Nucleus↗