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

智力优势：细胞进化了复杂的机制，以确保编码蛋白质或RNA的基因受到严格调控，这些基因只在特定类型的细胞中或在发育过程中的某些时候才需要。这个项目将利用裂解酵母作为一个简单的模型系统，以获得对负责维持对基因表达的严格时间控制的分子事件的广泛适用的见解。减数分裂是从二倍体前体细胞通过分裂产生单倍体配子的分化过程，不需要DNA合成；有丝分裂是细胞在DNA合成后通过分裂增殖的过程，它们是真核生物王国中保守的途径。在分裂酵母中，细胞对不利环境条件的反应从增殖到分化的转换是由一系列转录积累变化所介导的，这些变化与早期事件(DNA复制和重组)、中期事件(细胞分裂)和晚期事件(孢子形成)相关。该项目的主要目标是剖析转录因子叉头家族成员发挥拮抗作用的分子机制，以防止有丝分裂生长细胞中减数分裂基因的不适当表达，并在减数分裂过程中协同促进生产性RNA的合成和处理。尽管一些叉头家族成员作为常规的启动子限制性转录因子来调节启动，但首席研究员实验室最近的工作揭示了这些因子沿着整个基因体甚至在减数分裂细胞编码区下游的关联。与这种不寻常的分布相一致的是，补充数据表明，叉头因子调节着新合成的mRNAs中多聚腺苷尾巴的添加，这与转录终止有机械上的联系。该项目的前两个特定目标将检验工作假说，即在多腺化和通读转录之间的决定是通过在启动子上的RNA处理因子的差异招募和/或当转录复合体穿过聚腺苷信号时在延伸和终止之间的竞争来实现的。第三个目标将利用对叉头调控的减数分裂基因的RNA进行深度测序，然后进行生物信息学分析，以揭示共同的序列基序和其他可能需要的协调调控特征。叉头因子在转录和RNA加工之间的界面作用的发现与基因表达领域的最新发展作为一个整体共振，并提供了一个独特的机会来研究聚腺苷酸化，一个重要的但研究不足的调节机制，在生物学相关的背景下。更广泛的影响：这个项目将建立在首席研究员指导来自不同背景和专业发展不同阶段的科学家的良好记录的基础上。博士后研究员将发挥关键作用，除了接受尖端实验策略培训外，还将共同指导本科生和高中生。CWRU RNA分子生物学中心的大学氛围将有助于确保实现更广泛的影响目标，分裂酵母的使用也将如此，它在分子水平上类似于多细胞真核生物，但即使对于刚刚开始板凳研究的学生来说，培养和操作也相对简单。同以往一样，将作出特别努力，将妇女和一般在科学研究中任职人数偏低的群体成员纳入该项目。

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.