MEIOSIS-SPECIFIC SPLICING IN FISSION YEAST

裂殖酵母中减数分裂特异性剪接

• 批准号: 7144353
• 负责人: JO ANN WISE
• 金额: $ 43.27万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7144353
• 关键词: RNA splicing; Schizosaccharomyces pombe; biological signal transduction; chemical kinetics; chimeric proteins; cyclins; functional /structural genomics; genetic regulation; meiosis; microarray technology; polyadenylate; precursor mRNA; site directed mutagenesis; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): Regulated pre-messenger RNA processing is nearly ubiquitous among eukaryotes but has been studied mainly in metazoans, where the complexity of the biological processes it controls have posed many challenges. The overall goal of the proposed research is to use a combination of genetic, genomic and molecular tools uniquely available in the fission yeast Schizosaccharomyces pombe to fill this gap in our understanding. In recent work, thirteen meiotically spliced transcripts were discovered and two pre-mRNAs that encode cyclins investigated in detail. These studies revealed that splicing of rem1 and crs1 is restricted to meiosis by a novel inhibitory mechanism that requires non-intronic elements located outside the coding regions, a considerable distance from the target introns. While this shared (and highly unusual) regulatory strategy suggests similarities between crs1 and rem1, their splicing is temporally and mechanistically distinct: the identity of the major element that prevents crs1 splicing in vegetative cells and the nature of a recently discovered trans-acting factor suggest a role for the polyadenylation machinery, while the location of the major rem1 element together with the effects of heterologous expression on splicing suggest a role for the transcription machinery. Three specific aims will be pursued. To test the hypothesis that genetic circuits regulated at the level of splicing contribute to meiotic differentiation, additional new meiotically spliced transcripts will be identified and characterized with respect to kinetics of splicing, shared regulatory mechanisms, and hierarchical organization. To gain further mechanistic insight into the regulation of rem1 and crs1 splicing, the cis-acting elements responsible for both suppression of splicing in vegetative cells and positive control in meiotic cells will be pinpointed via analysis of chimeric constructs and mutagenesis. This information will be used in the design of both open-ended and candidate gene strategies to identify trans- acting factors, among which may be novel players that mediate communication between the machineries that carry out different steps in gene expression. Thus, this research provides an unprecedented opportunity to explore the function of the mRNA production factory in a native biological context. The insights gained will ultimately relate to human health and developmental disabilities, as aberrant gene expression is an underlying cause of numerous diseases.

描述（由申请人提供）：受调控的前信使 RNA 加工在真核生物中几乎普遍存在，但主要在后生动物中进行研究，其控制的生物过程的复杂性提出了许多挑战。拟议研究的总体目标是利用裂殖酵母裂殖酵母中独特的遗传、基因组和分子工具的组合来填补我们理解中的这一空白。在最近的工作中，发现了 13 个减数分裂转录本，并详细研究了编码细胞周期蛋白的两个前 mRNA。这些研究表明，rem1 和 crs1 的剪接通过一种新颖的抑制机制被限制在减数分裂中，该机制需要位于编码区外部、距目标内含子相当远的非内含子元件。虽然这种共享的（且非常不寻常的）调控策略表明 crs1 和 rem1 之间有相似之处，但它们的剪接在时间和机制上是不同的：在营养细胞中阻止 crs1 剪接的主要元素的身份以及最近发现的反式作用因子的性质表明聚腺苷酸化机制发挥作用，而主要 rem1 元件的位置以及异源表达对剪接的影响表明转录机制发挥作用。将追求三个具体目标。为了检验在剪接水平上调节的遗传回路有助于减数分裂分化的假设，将鉴定更多新的减数分裂转录本，并在剪接动力学、共享调节机制和分层组织方面进行表征。为了进一步深入了解 rem1 和 crs1 剪接的调节，将通过分析嵌合结构和诱变来查明负责营养细胞剪接抑制和减数分裂细胞中阳性控制的顺式作用元件。这些信息将用于设计开放式和候选基因策略，以识别反式作用因子，其中可能是介导在基因表达中执行不同步骤的机器之间的通信的新参与者。因此，这项研究为探索 mRNA 生产工厂在天然生物环境中的功能提供了前所未有的机会。获得的见解最终将与人类健康和发育障碍相关，因为异常的基因表达是许多疾病的根本原因。