Transcript Specificity in Yeast Pre-mRNA Splicing Revealed by Mutations in Core Spliceosomal Components. Saccharomyces cerevisiae

Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs), a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well. Keywords: time course, splicing mutant, splicing-specific microarray Overall design: Splicing-specific microarrays were used to assay the phenotypes of 23 different conditional mutations in splicing and mRNA processing factors. The data includes time courses of shifts to the non-permissive temperature for each factor as well as dye-flipped technical replicates of each time point.

绝大多数真核生物基因的正确表达，均需从前体信使RNA（pre-messenger RNA，pre-mRNA）中移除内含子，这一过程由剪接体（spliceosome）催化完成。在高等真核生物中，存在一类被称为SR蛋白（SR proteins）的庞大辅助因子家族，它们可通过结合前述前体信使RNA中的独特序列，提升包含弱剪接位点的转录本的剪接效率。酿酒酵母（Saccharomyces cerevisiae）缺乏这类因子的功能性同源物，因此此前学界并不明确剪接体能否有效区分不同转录本。为解答这一问题，我们采用基于微阵列（microarray）的实验方法，对剪接体复合物中18种高度保守的核心组分的突变效应展开了检测。动力学谱显示，不同前体信使RNA底物的剪接缺陷存在显著差异。尤为值得注意的是，在若干剪接体突变的影响下，核糖体蛋白基因转录本的整体行为与其他含内含子的转录本截然不同。然而，部分编码核糖体蛋白基因旁系同源物（paralogs）的转录本对突变的响应却呈现出截然不同的行为，这表明剪接体能够轻松区分高度相似的前体信使RNA。由此可见，剪接体对不同底物的区分能力，或许为酿酒酵母实现转录本依赖性的基因表达调控提供了重要途径。鉴于核心剪接体组分在真核生物中高度保守，我们认为本研究结果将显著深化学界对高等真核生物中调控性剪接调控机制的理解。
关键词：时间进程（time course）、剪接突变体（splicing mutant）、剪接特异性微阵列（splicing-specific microarray）
整体设计：采用剪接特异性微阵列检测23种不同剪接与mRNA加工因子的条件性突变表型。数据集包含各因子转移至非许可温度后的时间进程数据，以及每个时间点的染料翻转技术重复样本。