Mechanisms of environmentally regulated alternative splicing in S.Pombe

粟酒裂殖酵母中环境调控的选择性剪接机制

• 批准号: 9753758
• 负责人: JEFFREY A PLEISS
• 金额: $ 32.17万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753758
• 关键词: Alternative Splicing; Architecture; Biological; Biology; Cells; Collection; Complex; Consensus Sequence; Cues; Development; Disease; Distant; Elements; Environment; Essential Genes; Etiology; Eukaryota; Event; Exons; Family; Fission Yeast; Frequencies; Gene Expression; Gene Structure; Genes; Genetic; Genetic Screening; Genome; Genomics; Goals; Human; Impairment; Interruption; Introns; Knowledge; Laboratories; Malignant Neoplasms; Messenger RNA; Molecular; Molecular Biology; Monitor; Mutation; Nuclear; Nucleotides; Organism; Orthologous Gene; Pathway interactions; Pattern; Positioning Attribute; Proteins; Proteome; RNA Splicing; Regulation; Regulatory Element; Role; Saccharomyces cerevisiae; Saccharomycetales; Set protein; Shapes; Site; Spliceosome Assembly Pathway; Spliceosomes; Structural Genes; Structure; System; Technology; Testing; Trans-Activators; Transcript; Variant; Work; Yeasts; base; disease-causing mutation; exon skipping; genetic approach; genetic regulatory protein; human disease; improved; insight; mRNA Precursor; member; mutant; novel strategies; response; tool

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.

摘要 前体mRNA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，调节可变剪接模式，从有限数量的蛋白质组中产生扩增， 基因。重要的是，相当一部分人类致病突变通过以下方式表现出来： 改变基因剪接模式的序列。然而，这一机制 复杂途径的调控仍然知之甚少。了解致病突变如何损害 这种能力将需要更好地了解剪接体正确识别和 在大量“近同源”异常剪接位点的背景下激活“同源”剪接位点序列，和 in the context上下文of changing变化developmental发展and environmental环境cues线索.在最简单的层面上，这将需要 理解两个：（1）转录本（或基因结构）内的顺式调节元件， 调节;（2）反式调节因子可以赋予这种特定调节的机制基础。 为了更好地理解选择性前体mRNA剪接的机制，我们选择了检查 遗传上易处理的裂殖酵母，粟酒裂殖酵母。在许多方面，S.蓬贝外观 类似于高等真核生物中的剪接。在所有S.粟酒基因，和 单个基因被多个内含子中断。在S.粟酒属内含子不 符合紧密的共有序列，但在核苷酸中看起来更像人类内含子， 在这些位置发现简并，这是剪接调节的已知标志。基于我们最近 证明S.粟酒裂殖酵母可以催化类酵母的环境调节的可变剪接， 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， 这是必要的。为此，我们将采用我们现有的高通量遗传工具， 制定这些规则是为了确定这些记录中对其进行监管所需的要素。同样，我们将 使用正向遗传方法来识别和表征这些所必需的剪接因子， 规范的事件。这些方法的结合应提供重要的洞察机制 这种生物体可以通过这种途径调节其基因表达。鉴于保护程度很高 在S. pombe和人类，这项工作可能会提供关键的见解剪接调控 在高等真核生物中，包括它在许多人类疾病中的错误调节。