MEIOSIS-SPECIFIC SPLICING IN FISSION YEAST

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无处不在，但主要在后生动物中进行了研究，其中它控制的生物过程的复杂性带来了许多挑战。提出的研究的总体目标是利用分裂酵母中独特的遗传，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个减数剪接转录本，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，通过一种新的抑制机制，rem1和crs1的剪接被限制在减数分裂中，该机制需要位于编码区之外的非内含子元件，距离目标内含子有相当大的距离。虽然这种共同的（非常不寻常的）调控策略表明crs1和rem1之间存在相似性，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剪接的主要元件的身份和最近发现的反式作用因子的性质表明其与聚腺苷酸化机制有关，而主要rem1元件的位置以及异源表达对剪接的影响表明其与转录机制有关。将实现三个具体目标。为了验证在剪接水平上调控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剪接转录本，并对剪接动力学、共享调节机制和分层组织进行表征。为了进一步深入了解rem1和crs1剪接的调控机制，将通过嵌合结构和突变分析确定在营养细胞中抑制剪接和在减数分裂细胞中积极控制剪接的顺式作用元件。这些信息将用于开放式和候选基因策略的设计，以确定反作用因子，其中可能是在执行基因表达不同步骤的机制之间介导通信的新参与者。因此，这项研究为探索mRNA生产工厂在原生生物环境中的功能提供了前所未有的机会。所获得的见解最终将与人类健康和发育障碍有关，因为异常基因表达是许多疾病的潜在原因。

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.