Mechanisms of environmentally responsive splicing in S.Pombe

粟酒裂殖酵母环境响应性剪接机制

• 批准号: 8306895
• 负责人: JEFFREY A PLEISS
• 金额: $ 28.09万
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8306895
• 关键词: Affect; Alternative Splicing; Architecture; Beryllium; ChIP-seq; Chromatin Structure; Complement; Data; Elements; Environment; Eukaryota; Event; Exons; Fission Yeast; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Materials; Genetic Screening; Genome; Goals; Heat-Shock Response; Human; Human Genome; Introns; Knowledge; Laboratories; Libraries; Mammalian Cell; Measures; Messenger RNA; Methods; Modeling; Molecular; Mutagenesis; Mutation; Organism; Pathway interactions; Production; Protein Isoforms; Proteins; RNA; RNA Degradation; RNA Splicing; RNA, Messenger, Splicing; Regulation; Regulatory Element; Role; Saccharomyces cerevisiae; Saccharomycetales; Signal Pathway; Signal Transduction; Spliceosomes; System; Testing; Trans-Activators; Transcript; Transcriptional Regulation; Work; abstracting; clinically significant; design; exon skipping; genome-wide; human disease; insight; novel; positional cloning; promoter; research study; response; tool

DESCRIPTION (provided by applicant): Abstract All organisms require robust methods for regulating expression of their genetic material. For the past five decades, transcriptional control has been the dominant paradigm by which regulation of gene expression has been described. More recently, however, it has become clear that regulation of pre-mRNA splicing can be an equally important regulatory mechanism. Whereas initial estimates suggested that only a small fraction of human genes would be alternatively spliced, recent evidence demonstrates that as many as 95% of all human genes are subject to this level of regulation. Nevertheless, the mechanisms by which this regulation is manifested have not been widely elucidated. To better understand how pre-mRNA splicing functions as a regulatory control point, 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 as many as 16 introns. The evolutionary retention of these introns suggested to us that splicing regulation might be a prevalent mechanism for gene regulation in S. pombe, making it a compelling system in which to undertake these experiments. Indeed, results of our initial experiments have borne out this hypothesis. Using a novel set of genome-wide tools that we developed, we have now identified distinct subsets of transcripts whose splicing is regulated in response to changing cellular environment. Strikingly, many of these regulated events mimic the paradigms that define alternative splicing in mammalian cells. The goals of our current work are to understand the trans-acting factors and cis-regulatory elements that are necessary for this regulation. Toward this end, we will employ a novel, high-throughput reverse genetic screen that we recently developed to identify the full complement of cellular proteins that are necessary for these regulated events. Similarly, we will use a forward genetic approach to identify all of the elements within these transcripts that are required for their regulation. The combination of these approaches should provide tremendous 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 important insights into splicing regulation in higher eukaryotes. Moreover, because a significant number of human diseases are associated with mis-regulation of splicing, our understanding of the underlying parameters of this pathway should be of immediate clinical significance.

描述（由申请人提供）： 摘要所有生物体都需要强有力的方法来调节其遗传物质的表达。在过去的五十年里，转录调控一直是描述基因表达调控的主导范式。然而，最近已经清楚的是，前体mRNA剪接的调节可以是同样重要的调节机制。尽管最初的估计表明只有一小部分人类基因会进行选择性拼接，但最近的证据表明，多达95%的人类基因都受到这种水平的调节。然而，这种调节的表现机制尚未得到广泛阐明。为了更好地理解前mRNA剪接如何作为调控点发挥作用，我们选择了研究遗传上易于处理的裂殖酵母，粟酒裂殖酵母。在许多方面，S.粟酒裂殖看起来类似于高等真核生物中的剪接。在所有S. pombe基因，单个基因被多达16个内含子中断。这些内含子的进化保留提示剪接调控可能是S. pombe，使其成为进行这些实验的引人注目的系统。事实上，我们最初的实验结果已经证实了这一假设。使用我们开发的一套新的全基因组工具，我们现在已经确定了转录本的不同子集，这些转录本的剪接响应于不断变化的细胞环境而受到调节。引人注目的是，许多这些受调控的事件模仿了哺乳动物细胞中定义选择性剪接的范例。我们目前工作的目标是了解这种调节所必需的反式作用因子和顺式调节元件。为此，我们将采用一种新的，高通量的反向遗传筛选，我们最近开发的，以确定这些调节事件所必需的细胞蛋白质的完整补充。同样，我们将使用正向遗传方法来识别这些转录本中对其进行调控所需的所有元素。这些方法的结合应该提供了巨大的洞察力，这种生物体可以通过这种途径调节其基因表达的机制。鉴于S. pombe和人类，这项工作很可能提供重要的见解剪接调节高等真核生物。此外，由于大量的人类疾病与剪接的错误调节有关，因此我们对该途径的基本参数的理解应该具有直接的临床意义。