Mechanisms of environmentally responsive splicing in S.Pombe

描述(由申请人提供)： 摘要所有生物体都需要强大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，对前mRNA剪接的调控可以是一种同样重要的调控机制。虽然初步估计只有一小部分人类基因会被选择性剪接，但最近的证据表明，多达95%的人类基因受到这种水平的调控。然而，这种调控的机制还没有被广泛阐明。为了更好地了解前mRNA剪接是如何作为调控控制点发挥作用的，我们选择了研究遗传上易驯化的分裂酵母，裂殖酵母庞贝。在许多方面，在S.pombe中的剪接看起来与高等真核生物中的剪接相似。近一半的S.pombe基因中已经发现了内含子，单个基因被多达16个内含子打断。这些内含子的进化保留向我们表明，剪接调控可能是庞氏链霉菌基因调控的一种普遍机制，使其成为进行这些实验的一个引人注目的系统。事实上，我们初步实验的结果证实了这一假设。使用我们开发的一套新的全基因组工具，我们现在已经识别出其剪接受到调控的不同转录子集，以响应不断变化的细胞环境。引人注目的是，这些调控事件中的许多都模仿了定义哺乳动物细胞中选择性剪接的范例。我们目前工作的目标是了解这种调控所必需的反式作用因子和顺式调节元件。为此，我们将使用我们最近开发的一种新颖的高通量反向遗传筛查来识别这些调控事件所需的全部细胞蛋白质。同样，我们将使用正向遗传学方法来确定这些转录本中调控它们所需的所有元素。这些方法的结合应该会为这种有机体通过这一途径调节其基因表达的机制提供巨大的见解。鉴于S.pombe和人类剪接之间的高度保守，这项工作可能为高等真核生物的剪接调控提供重要的见解。此外，由于相当多的人类疾病与剪接的错误调控有关，我们对这一途径的潜在参数的理解应该具有直接的临床意义。

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.