Sequence determinants of splicing in yeast

mRNA splicing is a key process in eukaryotic gene expression. Most Intron-containing genes are constitutively spliced, and hence must undergo splicing to produce a functional mRNA. Therefore, regulation of splicing efficiency greatly affects gene expression regulation. Here we use a large synthetic oligo library of ~25,000 variants to explore how different intronic sequence determinants affect splicing efficiency and mRNA expression levels in S. cerevisiae. We found that the three splice sites (donor, acceptor, and branching point) differ in how deviations from the consensus sequence affect functionality. We also use intronic sequences from other yeast species with modified splicing machinery to show that intron architecture has co-evolved with the splicing machinery to adapt to the presence or absence of a specific splicing factor. Finally, we show that synthetic sequences containing two introns give rise to diverse RNA isoforms, exposing intronic features that control and facilitate alternative splicing. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome.

mRNA剪接（mRNA splicing）是真核生物基因表达中的核心过程。绝大多数含内含子的基因均为组成型剪接基因，因此必须经过剪接步骤才能产生功能性mRNA。因此，剪接效率的调控极大地影响基因表达调控。本研究利用包含约25000个变体的大型合成寡核苷酸文库（synthetic oligo library），探究酿酒酵母（Saccharomyces cerevisiae，简称S. cerevisiae）中不同内含子序列决定因素对剪接效率与mRNA表达水平的影响。研究发现，三类剪接位点（splice sites）——供体位点（donor site）、受体位点（acceptor site）与分支点（branching point）——其序列偏离共识序列（consensus sequence）时对功能的影响各不相同。此外，本研究借助改造后的剪接机器（splicing machinery）与其他酵母物种的内含子序列，证实内含子结构（intron architecture）已与剪接机器共同进化，以适配特定剪接因子（splicing factor）的存在与否。最后，我们发现包含两个内含子的合成序列可产生多种RNA异构体（RNA isoforms），揭示了能够控制并促进可变剪接（alternative splicing）的内含子特征。本研究阐明了进化过程中内含子塑造的全新机制，使细胞能够调控自身转录组（transcriptome）。