Splicing regulation in Drosophila sex determination.
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Alternative Splicing↗
Biomedical subjects
J Valcárcel
Publications and source records attributed to J Valcárcel.
The hnRNP A1 protein regulates HIV-1 tat splicing via a novel intron silencer element.
The generation of >30 different HIV-1 mRNAs is achieved by alternative splicing of one primary transcript. The removal of the second tat intron is regulated by a combination of a suboptimal 3' splice site and cis-acting splicing enhancers and silencers. Here we show that hnRNP A1 inhibits splicing of this intron via a novel heterogeneous nuclear ribonucleoprotein (hnRNP) A1-responsive intron splicing silencer (ISS) that can function independently of the previously characterized exon splicing silencer (ESS3). Surprisingly, depletion of hnRNP A1 from the nuclear extract (NE) enables splicing to proceed in NE that contains 100-fold reduced concentrations of U2AF and normal levels of SR proteins, conditions that do not support processing of other efficiently spliced pre-mRNAs. Reconstituting the extract with recombinant hnRNP A1 protein restores splicing inhibition at a step subsequent to U2AF binding, mainly at the time of U2 snRNP association. hnRNP A1 interacts specifically with the ISS sequence, which overlaps with one of three alternative branch point sequences, pointing to a model where the entry of U2 snRNP is physically blocked by hnRNP A1 binding.
Alternative Splicing↗
Modulation of msl-2 5' splice site recognition by Sex-lethal.
The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 (msl-2) transcripts. We have previously shown that SXL blocks the binding of U2 auxiliary factor (U2AF) to the polypyrimidine (Py)-tract associated with the 3' splice site of the regulated intron. We now report 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. We also show that 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-25' splice site and for splicing of this pre-mRNA, we propose that SXL antagonizes TIA-1 activity and thus prevents 5' splice site recognition by U1 snRNP. Taken together with previous data, we conclude that efficient retention of msl-2 intron involves inhibition of early recognition of both splice sites by SXL.
5' Untranslated Regions↗
Molecular mechanisms of gene expression regulation by the apoptosis-promoting protein TIA-1.
TIA-1 and the related protein TIAR promote DNA fragmentation in digitonin-permeabilized thymocytes. These proteins contain RNA Recognition Motifs (RRMs) and bind uridine-rich sequences. These observations suggested that TIA-1/TIAR are pro-apoptotic factors that influence some aspect of RNA metabolism. Here we review recent data implicating TIA-1 as a regulator of translation of Tumor Necrosis Factor-alpha mRNA and as regulator of alternative splicing of a variety of pre-mRNAs, including those of the Fibroblast Growth Factor Receptor 2 and the Fas receptor. We also discuss how some of these activities could be integrated in the control of programmed of cell death.
Alternative Splicing↗
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.
Binding Sites↗
Switch in 3' splice site recognition between exon definition and splicing catalysis is important for sex-lethal autoregulation.
Maintenance of female sexual identity in Drosophila melanogaster involves an autoregulatory loop in which the protein Sex-lethal (SXL) promotes skipping of exon 3 from its own pre-mRNA. We have used transient transfection of Drosophila Schneider cells to analyze the role of exon 3 splice sites in regulation. Our results indicate that exon 3 repression requires competition between the 5' splice sites of exons 2 and 3 but is independent of their relative strength. Two 3' splice site AG's precede exon 3. We report here that, while the distal site plays a critical role in defining the exon, the proximal site is preferentially used for the actual splicing reaction, arguing for a switch in 3' splice site recognition between exon definition and splicing catalysis. Remarkably, the presence of the two 3' splice sites is important for the efficient regulation by SXL, suggesting that SXL interferes with molecular events occurring between initial splice site communication across the exon and the splice site pairing that leads to intron removal.
Adenine Nucleotides↗
Kinetic role for mammalian SF1/BBP in spliceosome assembly and function after polypyrimidine tract recognition by U2AF.
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 U2AF(65) 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. As BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system. We report here that 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 U2AF(65) binding was 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 U2AF(65), arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF(65) binding.
Amino Acid Sequence↗
Alternative pre-mRNA splicing: the logic of combinatorial control.
Alternative splicing of mRNA precursors is a versatile mechanism of gene expression regulation that accounts for a considerable proportion of proteomic complexity in higher eukaryotes. Its modulation is achieved through the combinatorial interplay of positive and negative regulatory signals present in the RNA, which are recognized by complexes composed of members of the hnRNP and SR protein families.
Alternative Splicing↗
The apoptosis-promoting factor TIA-1 is a regulator of alternative pre-mRNA splicing.
We report here that the apoptosis-promoting protein TIA-1 regulates alternative pre-mRNA splicing of the Drosophila melanogaster gene male-specific-lethal 2 and of the human apoptotic gene Fas. TIA-1 associates selectively with pre-mRNAs that contain 5' splice sites followed by U-rich sequences. TIA-1 binding to the U-rich stretches facilitates 5' splice site recognition by U1 snRNP. This activity is critical for activation of the weak 5' splice site of msl-2 and for modulating the choice of splice site partner in Fas. Structural and functional similarities with the Saccharomyces cerevisiae splicing factor Nam8 suggest striking evolutionary conservation of a mechanism of pre-mRNA splicing regulation that controls biological processes as diverse as meiosis in yeast, dosage compensation in fruit flies, or programmed cell death in humans.
Alternative Splicing↗
The Drosophila fl(2)d gene, required for female-specific splicing of Sxl and tra pre-mRNAs, encodes a novel nuclear protein with a HQ-rich domain.
The Drosophila gene female-lethal(2)d [fl(2)d] interacts genetically with the master regulatory gene for sex determination, Sex-lethal. Both genes are required for the activation of female-specific patterns of alternative splicing on transformer and Sex-lethal pre-mRNAs. We have used P-element-mediated mutagenesis to identify the fl(2)d gene. The fl(2)d transcription unit generates two alternatively spliced mRNAs that can encode two protein isoforms differing at their amino terminus. The larger isoform contains a domain rich in histidine and glutamine but has no significant homology to proteins in databases. Several lines of evidence indicate that this protein is responsible for fl(2)d function. First, the P-element insertion that inactivates fl(2)d interrupts this ORF. Second, amino acid changes within this ORF have been identified in fl(2)d mutants, and the nature of the changes correlates with the severity of the mutations. Third, all of the phenotypes associated with fl(2)d mutations can be rescued by expression of this cDNA in transgenic flies. Fl(2)d protein can be detected in extracts from Drosophila cell lines, embryos, larvae, and adult animals, without apparent differences between sexes, as well as in adult ovaries. Consistent with a possible function in posttranscriptional regulation, Fl(2)d protein has nuclear localization and is enriched in nuclear extracts.
Alternative Splicing↗
Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG.
The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts. Here we report that 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. Our 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.
Adenine Nucleotides↗
Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65).
U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) 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 U2AF(35) facilitates U2AF(65) binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF(35) remain controversial. Here we show that recombinant U2AF(65) is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin micro; pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF(35) occurs without changes in U2AF(65) cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF(35) and a novel function for this factor in pre-mRNA splicing.
Animals↗
Multiple forms of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF subunits expressed in higher plants.
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, we are characterizing proteins involved in intron recognition in plants. In this work we show that 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 extend, 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.
Amino Acid Sequence↗
Functional association of nuclear protein 4.1 with pre-mRNA splicing factors.
Protein 4.1 is a multifunctional polypeptide that links transmembrane proteins with the underlying spectrin/actin cytoskeleton. Recent studies have shown that protein 4.1 is also present in the nucleus, localized in domains enriched in splicing factors. Here we further analyze the relationship between protein 4. 1 and components of the splicing machinery. Using HeLa nuclear extracts capable of supporting the splicing of pre-mRNAs in vitro, we show that anti-4.1 antibodies specifically immunoprecipitate pre-mRNA and splicing intermediates. Immunodepletion of protein 4.1 from HeLa nuclear extracts results in inhibition of their splicing activity, as assayed with two different pre-mRNA substrates. Coprecipitation of protein 4.1 from HeLa nuclear extracts with proteins involved in the processing of pre-mRNA further suggests an association between nuclear protein 4.1 and components of the splicing apparatus. The molecular cloning of a 4.1 cDNA encoding the isoform designated 4.1E has allowed us to show that this protein is targeted to the nucleus, that it associates with the splicing factor U2AF35, and that its overexpression induces the redistribution of the splicing factor SC35. Based on our combined biochemical and localization results, we propose that 4.1 proteins are part of nuclear structures to which splicing factors functionally associate, most likely for storage purposes.
Animals↗
The Drosophila splicing regulator sex-lethal directly inhibits translation of male-specific-lethal 2 mRNA.
Male-specific expression of the protein male-specific-lethal 2 (MSL-2) controls dosage compensation in Drosophila. msl-2 gene expression is inhibited in females by Sex-lethal (SXL), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5' untranslated region (UTR) of msl-2 mRNA contains putative SXL binding sites and is retained in female flies. Here we show that SXL plays a dual role in the inhibition of msl-2 expression. Cotransfection of Drosophila Schneider cells with an SXL expression vector and a reporter containing the 5' UTR of msl-2 mRNA resulted in retention of the 5' UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation was blocked by SXL, but not by the intron per se. Both splicing and translation inhibition by SXL were recapitulated in vitro and found to be dependent upon SXL binding to high-affinity sites within the intron, showing that SXL directly regulates these events. Our data reveal a coordinated mechanism for the regulation of msl-2 expression by the same regulatory factor: SXL enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm.
Animals↗
Post-transcriptional regulation: the dawn of PTB.
The 'polypyrimidine-tract-binding protein' (PTB) participates in the control of alternative processing and translation of various RNAs, and may operate as a multifunctional regulator of tissue-specific gene expression.
Alternative Splicing↗
U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction.
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. In this report we identify a new region of U2AF65 required for function, and use this information to clone a human 56-kD U2AF65 associated protein (UAP56). We show that 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. Our results reveal a new function of U2AF65: to position a DEAD box protein required for U2 snRNP binding at the pre-mRNA branchpoint region.
Adenosine Triphosphatases↗
Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal.
The protein Sex-lethal (SXL) controls pre-mRNA splicing of two genes involved in Drosophila sex determination: transformer (tra) and the Sxl gene itself. Previous in vitro results indicated that SXL antagonizes the general splicing factor U2AF65 to regulate splicing of tra. In this report, we have used transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF65 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.
Animals↗