Mechanisms of environmentally regulated alternative splicing in S.Pombe

摘要 前信使核糖核酸剪接是真核基因表达的重要组成部分。许多后生动物，包括 人类调节不同的剪接模式，以从有限的数量产生蛋白质组的扩张 基因。重要的是，相当一部分引起突变的人类疾病表现为 改变塑造基因剪接模式的序列。然而，这一点所依据的机制 复杂的信号转导途径是否受到调控，目前仍知之甚少。了解致病突变如何损害 这种能力需要更好地了解剪接体正确识别和识别 激活数十个“近源”异常剪接位点背景中的“同源”剪接位点序列，以及 在不断变化的发展和环境线索的背景下。在最简单的层面上，这将需要 理解两者：(1)转录本(或基因结构)中的顺式调控元件 调控；(2)跨调控因子赋予这一特定调控的机制基础。 为了更好地了解替代的前-mrna剪接的机制，我们选择研究 遗传易驯化的分裂酵母，裂殖酵母庞贝。在许多方面，在S.pombe中拼接看起来 类似于高等真核生物中的剪接。在近一半的S.pombe基因中发现了内含子，并且 单个基因被多个内含子打断。在S.pombe内含子中发现的剪接位点序列不 与紧密一致的序列一致，但看起来更像核苷酸中的人类内含子 在这些位置发现退化，这是剪接调控的已知标志。在我们最近的基础上 展示了S.pombe可以催化类似哺乳动物的环境调节的替代剪接， 我们目前工作的目标是了解顺式调节元件和反式作用因子 对这项规定来说是必要的。为此，我们将利用我们现有的高通量基因工具 为确定这些成绩单中需要进行监管的元素而开发。同样，我们也会 使用正向遗传方法来识别和表征这些必需的剪接因子 受监管的事件。这些方法的结合应该会对这些机制提供重要的见解 这个有机体可以通过这个途径来调节它的基因表达。鉴于高度的保护水平 在S.pombe和人类的剪接之间，这项工作可能会为剪接调控提供关键的见解 在高等真核生物中，包括它在许多人类疾病中的错误调控。

ABSTRACT Pre-mRNA splicing is an essential component of eukaryotic gene expression. Many metazoans, including humans, regulate alternative splicing patterns to generate expansions of their proteome from a limited number of genes. Importantly, a considerable fraction of human disease causing mutations manifest themselves by altering the sequences that shape the splicing patterns of genes. Nevertheless, the mechanisms by which this complex pathway is regulated remain poorly understood. Understanding how disease-causing mutations impair this ability will require improved knowledge of the mechanisms by which the spliceosome correctly identifies and activates ‘cognate’ splice site sequences in the background of scores of ‘near-cognate’ aberrant splice sites, and in the context of changing developmental and environmental cues. At the simplest level, this will require understanding both: (1) the cis-regulatory elements within a transcript (or gene structure) that destine it for regulation; and (2) the mechanistic bases by which trans-regulatory factors can impart this specific regulation. To better understand the mechanisms of alternative pre-mRNA splicing, we have chosen to examine the genetically tractable fission yeast, Schizosaccharomyces pombe. In many ways, splicing in S. pombe looks similar to splicing in higher eukaryotes. Introns have been identified in nearly half of all S. pombe genes, and single genes are interrupted by multiple introns. The splice site sequences found in S. pombe introns do not conform to tight consensus sequences but rather appear much more like human introns in the nucleotide degeneracy found at these positions, a known hallmark of splicing regulation. Building upon our recent demonstrations that S. pombe can catalyze mammalian-like environmentally-regulated alternative splicing, the goals of our current work are to understand the cis-regulatory elements and trans-acting factors that are necessary for this regulation. Toward this end, we will employ high-throughput genetic tools that we have developed to identify the elements within these transcripts that are required for their regulation. Similarly, we will use a forward genetic approach to identify and characterize the splicing factors that are necessary for these regulated events. The combination of these approaches should provide important insights into the mechanisms by which this organism can regulate its gene expression via this pathway. Given the high level of conservation between splicing in S. pombe and humans, this work is likely to provide critical insights into splicing regulation in higher eukaryotes, including its mis-regulation in many human diseases.