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

裂殖酵母中叉头因子对减数分裂中期RNA加工的调节

• 批准号: 1330788
• 负责人: Jo Ann Wise
• 金额: $ 47.69万
• 依托单位: Case Western Reserve University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-15 至 2020-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1330788&HistoricalAwards=false
• 关键词: Regulation; mid; meiotic; RNA; processing

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.

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的基因受到严格的调控，这些蛋白质或RNA仅在特定的细胞类型或发育的某些时期才需要。 该项目将利用裂殖酵母裂殖酵母作为一个简单的模型系统，以获得广泛适用的见解，负责保持严格的时间控制基因表达的分子事件。减数分裂是一种分化过程，通过分裂而不合成DNA，从二倍体前体细胞产生单倍体配子，有丝分裂是细胞在DNA合成后通过分裂增殖，是整个真核生物界的保守途径。在裂变酵母中，响应于不利环境条件从细胞增殖到分化的转换由改变的转录物积累的连续波介导，所述改变的转录物积累与早期事件（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。该项目的总体目标是剖析转录因子叉头家族成员拮抗性发挥功能的分子机制，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生产性RNA的合成和加工。 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调节起始，但主要研究者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的下游的关联。 与这种不寻常的分布相一致，补充数据表明，叉头因子调节新合成的mRNA，这是机械地连接到转录终止的聚腺苷尾的添加。该项目的前两个具体目标将测试工作假设，即多聚腺苷酸化和通读转录之间的决定是通过启动子处RNA加工因子的差异募集和/或转录复合物穿过多聚腺苷酸化信号时延伸和终止之间的竞争来执行的。 第三个目标将利用来自叉头调控的减数分裂基因的RNA的深度测序，然后通过生物信息学分析来揭示共享的序列基序和其协调调控可能需要的其他特征。 叉头因子在转录和RNA加工之间的界面上起作用的发现与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多聚腺苷酸化，这是一种重要但研究不足的调控机制，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指导来自不同背景和不同专业发展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的培训外，还将共同监督本科生和高中生。 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，裂变酵母的使用也将如此，裂变酵母在分子水平上类似于多细胞真核生物，但即使是刚刚开始实验室研究的学生，培养和操作也相对简单。 同过去一样，将作出特别努力，将妇女和一般在科学研究中代表性不足的群体成员纳入该项目。