Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA

Abstract

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...

Conservation and regulation of alternative splicing by dynamic inter- and intra-intron base pairings in Lepidoptera 14-3-3z pre-mRNAs

“…34 Indeed, some examples have indicated that RNA secondary structures are involved in splicing regulation. 7,8,19,21,[35][36][37][38] In yeast, new discoveries using high-throughput sequencing have lent strong support to the general hypothesis that the information encoded in large mRNAs is regulated and organized by RNA structure. 39 Therefore, RNA secondary structure strongly contributes to alternative splicing regulation in higher organisms.…”

Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae

“…1A, scheme i; Materials and Methods). Levels of Dyn2-Cup1 mRNA measured by reverse transcription (RT) by using a primer specific to CUP1 (Howe and Ares 1997) show that the internal exon is efficiently included in the mRNA (E2+; Fig. 1B, lane 1).…”

“…There are at least eight two-intron genes in S. cerevisiae (Blandin et al 2000;Davis et al 2000); thus, there are at least eight internal exons for which inclusion in the mature mRNA must be enforced. We previously found that exon inclusion in the two-intron YL8A (RPL7A) pre-mRNA in yeast is enforced in cis by intron selfcomplementarities that orchestrate correct pairing between each set of splice sites (Howe and Ares 1997). This mechanism is inadequate for genes such as DYN2 (Dick et al 1996), which clearly lack intron self-complementarities.…”

High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5??? splice site selection in support of a common looping out and repression mechanism

“…The hnRNP A1 protein has also been implicated in the control of 39 splice site use (Bai et al+, 1999;Caputi et al+, 1999;Del Gatto-Konczak et al+, 1999;Bilodeau et al+, 2001;Tange et al+, 2001;Zhu et al+, 2001)+ In several of these cases, high-affinity A1 binding sites located in the exon near the target 39 splice site have been identified+ In the HIV-1 tat exon 3, recent studies have reported that a high-affinity A1 binding site can antagonize the interaction of SC35 with a nearby enhancer element (Zhu et al+, 2001), and that A1 binding sites in the intron near the 39 splice junction can compromise U2 snRNP binding to the branch site (Tange et al+, 2001)+ Thus, hnRNP A1 may employ different strategies to control splicing decisions depending on the position of high-affinity binding sites and the proximity of other cis-acting elements and trans-acting factors+ Such versatility has also been observed for the RNA binding protein ASF/SF2, which mediates exon enhancer activity in many systems but can also function as a repressor in some pre-mRNAs+ HnRNP A1 could therefore directly interfere with the binding of specific splicing factors in some situations, whereas in others, interactions between distantly bound hnRNP A1 molecules could change the conformation of premRNAs to help carry out switches in splice site selection+ Finally, it is worth noting that the frequency of putative A1 binding sites is high in introns, particularly near splice junctions (Blanchette & Chabot, 1999)+ Thus, in addition to a role in splice site selection, an interaction between bound A1 proteins (and possibly other A1-related hnRNP proteins) may help loop out introns to facilitate commitment between distant splice sites in a manner that is similar to short RNA duplexes in yeast introns (Newman, 1987;Libri et al+, 1995;Charpentier & Rosbash, 1996;Howe & Ares, 1997)+ We are currently exploring the role of high-affinity A1 binding sites in the splicing of large introns+ Because a splice site positioned in a loop becomes repressed, the A1/A1 interaction may also contribute toward preventing the utilization of splice site-like sequences that are invariably present in long introns+…”