MEIOSIS-SPECIFIC SPLICING IN FISSION YEAST

描述(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，在后生动物中，其控制的生物过程的复杂性构成了许多挑战。这项拟议研究的总体目标是使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，并详细研究了两个编码周期蛋白的前mRNAs。这些研究表明，rem1和crs1的剪接受到一种新的抑制机制的限制，该机制要求非内含子元件位于编码区之外，距离目标内含子相当远。虽然这一共同的(非常不寻常的)调控策略表明了crs1和rem1之间的相似之处，但它们的剪接在时间和机制上是不同的：阻止营养细胞中crs1剪接的主要元件的同一性和最近发现的反式作用因子的性质表明了多聚腺苷化机制的作用，而主要的rem1元件的位置以及异源表达对剪接的影响表明了转录机制的作用。将追求三个具体目标。为了验证在剪接水平上调控的遗传电路有助于减数分裂分化的假设，将从剪接动力学、共享的调控机制和层级组织方面鉴定更多的新的减数分裂剪接转录本。为了进一步从机制上深入了解rem1和crs1剪接的调控，将通过嵌合结构和突变的分析，找出在营养细胞中抑制剪接和在减数分裂细胞中负责正调控的顺式作用元件。这些信息将被用于设计开放式和候选基因策略，以确定反式作用因子，其中可能有新的角色，在执行不同基因表达步骤的机器之间进行沟通。因此，这项研究为探索mRNA生产工厂在本地生物学背景下的功能提供了一个前所未有的机会。所获得的见解最终将与人类健康和发育障碍有关，因为异常的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.