Functional Analysis of Transcription in a Protist Model

本文所述的研究将确定在纤毛虫和原生动物嗜热四膜虫（Tt）中发散介质（MED）转录复合物的功能和机制。在大多数真核生物中，RNA聚合酶II （RNAPII）亚基Rpb1通过在其c端结构域（CTD）内进行翻译后修饰（PTM）的七核苷酸重复迭代来协调共转录事件。MED转录共激活因子复合物是酵母和人类细胞中ctd相互作用的蛋白复合物，在转录共调节中起作用。了解MED在Tt中的功能/机制具有普遍的意义，因为1)Tt在其RNAPII的Rpb1亚基中不具有规范的七肽重复，2)Tt在减数分裂期间发生在缺乏mRNA转录的情况下，会进行全基因组MED依赖的ncRNA转录。我的长期目标是表征rnapii依赖性mRNA和减数分裂特异性ncRNA在Tt中的转录机制。我们最近在Tt (Garg et al. (2019) Current Biology)1中发现了一种不同的MED，并将其与mRNA和ncRNA转录联系起来。在接下来的五年里，我的短期目标是了解Tt MED在mRNA转录中的功能和机制。在Aim 1中，我们将评估MED对全基因组mRNA转录的贡献，以完全确定其功能。在目标2中，我们将对选定的MED蛋白进行近距离生物素化标记实验，以确定MED共同调节转录的一组蛋白。目的3将整合和利用这些结果来阐明Tt MED共同调节mRNA转录的机制。意义：基因组不稳定常与随机染色体重排有关。四膜虫是一种实验模型，具有在全基因组范围内进行发育调节可重复DNA重排的不寻常特性。了解四膜虫如何重排其染色体将增加有关DNA重排的一般知识，因此这项研究具有重要的基础意义，并可能导致鉴定与人类癌症相关的新标记蛋白。因此，这项研究对加拿大的药理学和生物技术产业具有潜在的长期效益。培训：拟开展的研究项目将培养4名研究生和多名本科生，涉及基础分子生物学和分子遗传学方法，以及下一代测序等强大的新技术。总的来说，这些方法构成了下一代基础研究人员和越来越多的临床和诊断实验室研究人员所需的专业知识，因此这项研究将有利于加拿大的医疗保健系统

The research described herein will identify function and mechanism of a divergent Mediator (MED) transcription complex in the ciliate and protist Tetrahymena thermophila (Tt). In most eukaryotes, RNA Polymerase II (RNAPII) subunit Rpb1 coordinates co-transcriptional events via iterated heptad repeats subject to post-translational modification (PTM) within its C-terminal domain (CTD). The MED transcriptional co-activator complex is a CTD-interacting protein complex in yeast and human cells that functions in transcriptional co-regulation. Understanding MED function/mechanism in Tt is of general interest because 1) Tt does not possess canonical heptad repeats in its Rpb1 subunit of RNAPII and 2) Tt undergoes genome-wide MED-dependent ncRNA transcription during meiosis that occurs in the absence of mRNA transcription. My long-term objective is to characterize the mechanism of transcription of RNAPII-dependent mRNA and meiotic-specific ncRNA in Tt. We have recently identified a divergent MED in Tt (Garg et al. (2019) Current Biology)1 and implicated it in mRNA and ncRNA transcription. Over the next five years my short-term objectives are to understand the function and mechanism of Tt MED in transcription of mRNA. In Aim 1, we will assess contribution of MED to genome-wide mRNA transcription to completely identify its function. In Aim 2, we will perform proximity biotinylation labeling experiments of selected MED proteins to identify the set of proteins with which MED co-regulates transcription. Aim 3 will integrate and leverage these results to elucidate the mechanism by which Tt MED co-regulates mRNA transcription. Significance: Genome instability is often associated with random chromosome rearrangements. Tetrahymena is an experimental model that has the unusual property of undergoing developmentally regulated reproducible DNA rearrangements on a genome-wide scale. Understanding how Tetrahymena rearranges its chromosomes will increase general knowledge concerning DNA rearrangements and therefore this research is of fundamental importance and could lead to the identification of new marker proteins relevant to human cancer. For this reason, this research is potentially of long-term benefit to Canada's pharmacological and biotechnology industries. Training: The proposed research program will train four graduate students and multiple undergraduate researchers in fundamental molecular biology and molecular genetic methods as well as powerful new technologies such as Next Generation Sequencing. Collectively these methods compose the expertise required by the next generation of basic researchers and, increasingly, researchers in clinical and diagnostic laboratories and therefore this research will benefit to Canada's healthcare system