Howe K J, Ares M
Center for the Molecular Biology of RNA, Biology Department, University of California, Santa Cruz, CA 95064, USA.
Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12467-72. doi: 10.1073/pnas.94.23.12467.
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.
为了大多数真核基因的正确表达，在从前体mRNA中去除内含子时必须避免内部外显子的跳跃。尽管对内含子去除机制有了深入了解，但确保多内含子前体mRNA中内部外显子包含的机制仍然神秘。利用一个天然的双内含子酵母基因，我们在每个内含子中鉴定出了独特的RNA-RNA互补性，这些互补性可防止外显子跳跃并确保内部外显子的包含。我们表明，这些互补性的定位是作为内含子识别元件，仅将合适的5'剪接位点和分支点聚集在一起。破坏任何一个内含子的自我互补性都会导致外显子跳跃的发生，而使用补偿性突变恢复互补性则可挽救外显子的包含，这表明这些元件是通过RNA二级结构的形成起作用的。在内含子1的5'剪接位点附近区域与内含子2的分支点之间引入新的配对潜力会显著增强外显子跳跃。在单内含子酵母基因中鉴定出的类似元件有助于剪接效率。我们的结果说明了内含子二级结构如何用于协调剪接位点配对并强制外显子包含。我们认为脊椎动物基因中的类似元件可能有助于非常大的内含子的剪接以及可变剪接的进化。
