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A broad analysis of splicing regulation in yeast using a large library of synthetic introns.

ABSTRACT: RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5' donor site, the branch site, and the 3' acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3' splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns' 3' ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism's splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism.

SUBMITTER: Schirman D

PROVIDER: S-EPMC8496845 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAlternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described.ResultsCloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates.ConclusionWe found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution.

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A broad analysis of splicing regulation in yeast using a large library of synthetic introns.

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