Regulation of mid-meiotic RNA processing by forkhead factors in fission yeast

智力优势：细胞已经进化出复杂的机制，以确保编码蛋白质或rna的基因只在特定细胞类型或发育的特定时期被严格调节。该项目将利用分裂酵母Schizosaccharomyces pombe作为一个简单的模型系统，以获得广泛适用的见解，了解负责维持对基因表达的严格时间控制的分子事件。减数分裂是在没有DNA合成的情况下，二倍体前体细胞通过分裂产生单倍体配子的分化过程，而有丝分裂是细胞在DNA合成后通过分裂进行增殖的过程，它们是整个真核生物界的保守途径。在裂变酵母中，为了应对不利的环境条件，从细胞增殖到分化的转变是由与早期事件（DNA复制和重组）、中期事件（细胞分裂）和后期事件（孢子形成）相关的转录物积累的顺序波介导的。本项目的总体目标是解剖叉头转录因子家族成员拮抗功能的分子机制，以防止有丝分裂生长细胞中减数分裂基因的不适当表达，并协同促进减数分裂过程中高产RNA的合成和加工。尽管一些叉头家族成员作为传统的启动子限制性转录因子调节起始，但最近在首席研究员实验室的工作揭示了这些因子在减数分裂细胞中沿整个基因体甚至编码区下游的关联。与这种不寻常的分布一致，补充数据表明叉头因子调节新合成mrna的多腺苷尾部的添加，这与转录终止有机制联系。本项目的前两个具体目标将测试工作假设，即在启动子处RNA加工因子的不同招募和/或转录复合体穿越多聚腺苷化信号时延伸和终止之间的竞争，强制决定多聚腺苷化和读透转录之间的决定。第三个目标将利用叉头调控的减数分裂基因的RNA深度测序，然后进行生物信息学分析，以揭示共享的序列基序和其他可能需要的协调调节的特征。叉头因子在转录和RNA加工之间的界面起作用的发现与整个基因表达领域的最新发展产生了共鸣，并提供了一个独特的机会来研究聚腺苷化，这是一个重要的但尚未充分研究的调控机制，在生物学相关的背景下。更广泛的影响：该项目将建立在首席研究员对来自不同背景和不同专业发展阶段的科学家的良好指导记录的基础上。博士后除了接受尖端实验策略的培训外，还将共同指导本科生和高中生，发挥关键作用。CWRU RNA分子生物学中心的学院氛围将有助于确保实现更广泛的影响目标，就像使用裂变酵母一样，它在分子水平上类似于多细胞真核生物，但即使是刚开始实验室研究的学生，培养和操作也相对简单。与过去一样，将特别努力将妇女和在科学研究中普遍代表性不足的群体的成员纳入该项目。

INTELLECTUAL MERIT:Cells have evolved elaborate mechanisms to ensure that genes encoding proteins or RNAs that are required only in specific cell types or at certain times in development are tightly regulated. This project will utilize the fission yeast Schizosaccharomyces pombe as a simple model system to gain broadly applicable insights into the molecular events responsible for maintaining strict temporal control over gene expression. Meiosis, a differentiation process that produces haploid gametes from diploid precursor cells through division without DNA synthesis, and mitosis, through which cells proliferate through division after DNA synthesis, are conserved pathways throughout the eukaryotic kingdom. In fission yeast, the switch from cellular proliferation to differentiation in response to adverse environmental conditions is mediated by sequential waves of altered transcript accumulation that correlate with early events (DNA replication and recombination), middle events (cell division) and late events (spore formation). The overarching goal of this project is to dissect the molecular mechanisms through which members of the forkhead family of transcription factors function antagonistically to prevent inappropriate expression of meiotic genes in mitotically growing cells and collaboratively to promote productive RNA synthesis and processing during meiosis. Although some forkhead family members act as conventional promoter-restricted transcription factors that regulate initiation, recent work in the principal investigator's laboratory has revealed association of these factors along the entire gene body and even downstream from the coding region in meiotic cells. Consistent with this unusual distribution, complementary data indicate that forkhead factors regulate the addition of a polyadenosine tail to newly synthesized mRNAs, which is mechanistically linked to transcription termination. The first two specific aims of this project will test the working hypotheses that the decision between polyadenylation and read-through transcription is enforced by differential recruitment of RNA processing factors at the promoter and/or competition between elongation and termination as the transcription complex traverses the polyadenylation signals. The third aim will utilize deep sequencing of RNA from forkhead-regulated meiotic genes, followed by bioinformatic analysis to reveal shared sequence motifs and other features that may be required for their coordinate regulation. The discovery that forkhead factors function at the interface between transcription and RNA processing resonates with recent developments in the gene expression field as a whole, and provides a unique opportunity to investigate polyadenylation, a vital but understudied regulatory mechanism, in a biologically relevant context. BROADER IMPACTS: This project will build upon the principal investigator's strong track record of mentoring scientists from diverse backgrounds and at various stages of professional development. The postdoctoral fellow will play a pivotal role by co-supervising undergraduate and high school students in addition to receiving training in cutting-edge experimental strategies. The collegial atmosphere in the Center for RNA Molecular Biology at CWRU will help to ensure that broader impact goals are achieved, as will the use of fission yeast, which resembles multicellular eukaryotes at the molecular level, yet is relatively simple to culture and manipulate even by students just beginning bench research. As in the past, a particular effort will be made to include women and members of groups generally under-represented in scientific research in the project.