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研究，其中它控制的生物过程的复杂性带来了许多挑战。拟议研究的总体目标是使用裂变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个减数分裂剪接的转录本，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的剪接被一种新的抑制机制限制在减数分裂中，这种机制需要位于编码区之外的非内含子元件，与靶内含子有相当大的距离。虽然这份共享（和非常不寻常的）调控策略表明crs1和rem1之间的相似性，它们的剪接在时间上和机制上是不同的：在营养细胞中阻止CRS 1剪接的主要元件的特性和最近发现的反式作用因子的性质表明多腺苷酸化机制的作用，而主要REM1元件的位置以及异源表达对剪接的影响提示了转录机制的作用。将追求三个具体目标。为了检验在剪接水平调节的遗传电路有助于减数分裂分化的假设，将鉴定另外的新的减数分裂剪接的转录物，并在剪接动力学、共享的调节机制和分级组织方面进行表征。为了进一步深入了解rem1和crs1剪接的调控机制，将通过分析嵌合构建体和诱变来确定负责营养细胞中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，其中可能是介导基因表达不同步骤的机制之间通信的新参与者。因此，这项研究提供了一个前所未有的机会来探索mRNA生产工厂在天然生物环境中的功能。所获得的见解最终将涉及人类健康和发育障碍，因为异常基因表达是许多疾病的根本原因。

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.