Alterations in the conserved SL1 trans-spliced leader of Caenorhabditis elegans demonstrate flexibility in length and sequence requirements in vivo.
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Approximately 70% of mRNAs in Caenorhabditis elegans are trans spliced to conserved 21- to 23-nucleotide leader RNAs. While the function of SL1, the major C. elegans trans-spliced leader, is unknown, SL1 RNA, which contains this leader, is essential for embryogenesis. Efforts to characterize in vivo requirements of the SL1 leader sequence have been severely constrained by the essential role of the corresponding DNA sequences in SL1 RNA transcription. We devised a heterologous expression system that circumvents this problem, making it possible to probe the length and sequence requirements of the SL1 leader without interfering with its transcription. We report that expression of SL1 from a U2 snRNA promoter rescues mutants lacking the SL1-encoding genes and that the essential embryonic function of SL1 is retained when approximately one-third of the leader sequence and/or the length of the leader is significantly altered. In contrast, although all mutant SL1 RNAs were well expressed, more severe alterations eliminate this essential embryonic function. The one non-rescuing mutant leader tested was never detected on messages, demonstrating that part of the leader sequence is essential for trans splicing in vivo. Thus, in spite of the high degree of SL1 sequence conservation, its length, primary sequence, and composition are not critical parameters of its essential embryonic function. However, particular nucleotides in the leader are essential for the in vivo function of the SL1 RNA, perhaps for its assembly into a functional snRNP or for the trans-splicing reaction. (+info)
Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA.
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Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism. (+info)
Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor.
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Splicing of certain pre-mRNA introns is dependent on an enhancer element, which is typically purine-rich. It is generally thought that enhancers increase the use of suboptimal splicing signals, and one specific proposal is that enhancers stabilize binding of U2AF65 to weak polypyrimidine (Py) tracts. Here, we test this model using an IgM pre-mRNA substrate, which contains a well-characterized enhancer. Although the enhancer was required for in vitro splicing, we found it had no effect on U2AF65 binding. Unexpectedly, replacement of the natural IgM Py tract, branchpoint, and 5' splice site with consensus splicing signals did not circumvent the enhancer requirement. These observations led us to identify a novel regulatory element within the IgM M2 exon that acts as a splicing inhibitor; removal of the inhibitor enabled splicing to occur in the absence of the enhancer. The IgM M2 splicing inhibitor is evolutionarily conserved, can inhibit the activity of an unrelated, constitutively spliced pre-mRNA, and acts by repressing splicing complex assembly. Interestingly, the inhibitor itself forms an ATP-dependent complex that contains U2 snRNP. We conclude that splicing of IgM exons M1 and M2 is directed by two juxtaposed regulatory elements-an enhancer and an inhibitor-and that a primary function of the enhancer is to counteract the inhibitor. (+info)
Transcription of the human U2 snRNA genes continues beyond the 3' box in vivo.
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The 3' box of the human class II snRNA genes is required for proper 3' processing of transcripts, but how it functions is unclear. Several lines of evidence suggest that termination of transcription occurs at the 3' box and the terminated transcript is then a substrate for processing. However, using nuclear run-on analysis of endogenous genes, we demonstrate that transcription continues for at least 250 nucleotides beyond the 3' box of the U2 genes. Although in vivo footprinting analysis of both the U1 and U2 genes detects no protein-DNA contacts directly over the 3' box, a series of G residues immediately downstream from the 3' box of the U1 gene are clearly protected from methylation by dimethylsulfate. In conjunction with the 3' box of the U1 gene, this in vivo footprinted region causes termination of transcription of transiently transfected U2 constructs, whereas a 3' box alone does not. Taken together, these results indicate that the 3' box is not an efficient transcriptional terminator but may act as a processing element that is functional in the nascent RNA. (+info)
The SRm160/300 splicing coactivator is required for exon-enhancer function.
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Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing "coactivator," SRm160/300, which contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound "activators" and the snRNP machinery of the spliceosome. (+info)
Partial purification of the yeast U2 snRNP reveals a novel yeast pre-mRNA splicing factor required for pre-spliceosome assembly.
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We have partially purified the U2 snRNP of Saccharomyces cerevisiae. Identification of some proteins consistently found in the purified fractions by nanoelectrospray mass spectrometry indicated the presence of a novel splicing factor named Rse1p. The RSE1 gene is essential and codes for a 148.2 kDa protein. We demonstrated that Rse1p associates specifically with U2 snRNA at low salt concentrations. In addition, we showed that Rse1p is a component of the pre-spliceosome. Depletion of Rse1p and analysis of a conditional mutant indicated that Rse1p was required for efficient splicing in vivo. In vitro Rse1p is required for the formation of pre-spliceosomes. Database searches revealed that Rse1p is conserved in humans and that it belongs to a large protein family that includes polyadenylation factors and DNA repair proteins. The characteristics of Rse1p suggest that its human homologue could be a subunit of the SF3 splicing factor. (+info)
Identification of both shared and distinct proteins in the major and minor spliceosomes.
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In metazoans, two distinct spliceosomes catalyzing pre-messenger RNA splicing have been identified. Here, the human U11/U12 small nuclear ribonucleoprotein (snRNP), a subunit of the minor (U12-dependent) spliceosome, was isolated. Twenty U11/U12 proteins were identified, including subsets unique to the minor spliceosome or common to both spliceosomes. Common proteins include four U2 snRNP polypeptides that constitute the essential splicing factor SF3b. A 35-kilodalton U11-associated protein homologous to the U1 snRNP 70K protein was also identified. These data provide fundamental information about proteins of the minor spliceosome and shed light on its evolutionary relationship to the major spliceosome. (+info)
Combined biochemical and electron microscopic analyses reveal the architecture of the mammalian U2 snRNP.
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The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5' half of U2 small nuclear RNA, whereas SF3a associates with the 3' portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain. (+info)