Unknown mechanisms exist to ensure that exons are not skipped during biogenesis of mRNA. Studies have connected transcription elongation with regulated alternative exon inclusion. To determine whether the relative rates of transcription elongation and spliceosome assembly might play a general role in enforcing constitutive exon inclusion, we measured exon skipping for a natural two-intron gene in which the internal exon is constitutively included in the mRNA. Mutations in this gene that subtly reduce recognition of the intron 1 branchpoint cause exon skipping, indicating that rapid recognition of the first intron is important for enforcing exon inclusion. To test the role of transcription elongation, we treated cells to increase or decrease the rate of transcription elongation. Consistent with the "first come, first served" model, we found that exon skipping in vivo is inhibited when transcription is slowed by RNAP II mutants or when cells are treated with inhibitors of elongation. Expression of the elongation factor TFIIS stimulates exon skipping, and this effect is eliminated when lac repressor is targeted to DNA encoding the second intron. A mutation in U2 snRNA promotes exon skipping, presumably because a delay in recognition of the first intron allows elongating RNA polymerase to transcribe the downstream intron. This indicates that the relative rates of elongation and splicing are tuned so that the fidelity of exon inclusion is enhanced. These findings support a general role for kinetic coordination of transcription elongation and splicing during the transcription-dependent control of splicing.

A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.In eukaryotes, the removal of introns from nuclear transcripts requires two transesterification reactions carried out by spliceosomes. Five small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U5, U4, and U6, and many extrinsic protein factors act in concert to build a spliceosome and execute the splicing reactions (38,39,49,50,53). The spliceosome is clearly the most dynamic of the RNP enzymes. Major changes in the secondary structure of U4, U6, and U2 snRNAs during the spliceosome cycle are inferred from genetic and crosslinking studies (reviewed in reference 63). The snRNAs arrive at the assembling spliceosome in a form unlike that necessary for the catalytic activity of splicing and must be rearranged before splicing can proceed (7,45). A less studied corollary of this finding is that these rearrangements must be undone during spliceosome disassembly, and snRNA structure must be regenerated in an appropriate form for another round of spliceosome assembly and splicing (56). Although individual proteins are clearly linked to changes in the composition and organization of splicing complexes at distinct points in the splicing pathway (for a review, see reference 63), it has been difficult to assign responsibility for a specific RNA rearrangement event to any single protein.The first ATP-dependent step during in vitro spliceosome assembly is the stable binding of the U2 snRNP to the branch point region of the intron, an event normally dependent on formation of ATP-independent complexes between the premRNA and other proteins, as well as the U1 snRNP (51). These ATP-independent complexes (called the E complex in mammalian studies and commitment complexes in yeast studies) contain pre-mRNA that has been recognized both at the 5Ј splice site by the U1 snRNP and at the branch point by the homologous mammalian (SF1) or yeast (BBP) branch pointinteracting proteins (2,13,29,38,48). Formation of this complex is expected to specify an exon joining event because the 5Ј splice site and branch point are selected,...

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Skipping of internal exons during removal of introns from pre-mRNA must be avoided for proper expression of most eukaryotic genes. Despite significant understanding of the mechanics of intron removal, mechanisms that ensure inclusion of internal exons in multi-intron pre-mRNAs remain mysterious. Using a natural two-intron yeast gene, we have identified distinct RNA-RNA complementarities within each intron that prevent exon skipping and ensure inclusion of internal exons. We show that these complementarities are positioned to act as intron identity elements, bringing together only the appropriate 5 splice sites and branchpoints. Destroying either intron self-complementarity allows exon skipping to occur, and restoring the complementarity using compensatory mutations rescues exon inclusion, indicating that the elements act through formation of RNA secondary structure. Introducing new pairing potential between regions near the 5 splice site of intron 1 and the branchpoint of intron 2 dramatically enhances exon skipping. Similar elements identified in single intron yeast genes contribute to splicing efficiency. Our results illustrate how intron secondary structure serves to coordinate splice site pairing and enforce exon inclusion. We suggest that similar elements in vertebrate genes could assist in the splicing of very large introns and in the evolution of alternative splicing.A key event in the decoding of genetic information in eukaryotes is the removal of intervening sequences or introns by nuclear pre-mRNA splicing (reviewed in refs. 1 and 2). The process of splicing requires the concerted activities of a large number of trans-acting RNA and protein factors that recognize and assemble onto splicing signals located near the branchpoint and splice sites in the pre-mRNA (1, 2). An early step in spliceosome assembly is the formation of a splicing complex within which the reactive sites of the intron destined to be removed have largely been determined and the limits of the intron defined (3-5). Distinguishing the correct splice sites from the many similar pre-mRNA sequences is a problem (1, 2), compounded by the fact that vertebrate genes frequently contain long introns (Ͼ5 kb) and short internal exons (Ͻ300 bp, ref. 6). In mammals, failure to identify splice sites often results in exon skipping, causing aberrant gene expression (7) as well as a number of genetic diseases in humans (8).Simply finding correct splice sites is insufficient to determine correct splicing. Experiments using chimeric introns indicate that nearly any 5Ј splice site can be joined to nearly any 3Ј splice site (1, 2, 9). Because most splice sites are compatible, the splicing machinery must pair them in a fashion that prevents joining of compatible splice sites from different introns. The relative sizes of adjacent exons and introns appear to influence the mechanism by which this is determined (10). This interpretation is based on the general observation that 5Ј splice site mutations in a large intron following a small exon causes skipp...

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