Functional Analysis of Transcription in a Protist Model

本研究旨在明确趋异介体（MED）转录复合体在纤毛虫和原生生物嗜热四膜虫（Tt）中的功能和机制。在大多数真核生物中，RNA聚合酶II（RNAPII）亚基Rpb 1通过在其C-末端结构域（CTD）内进行翻译后修饰（PTM）的重复七肽重复序列协调共转录事件。MED转录共激活因子复合物是酵母和人类细胞中的CTD相互作用蛋白复合物，其在转录共调节中起作用。理解Tt中的MED功能/机制是普遍感兴趣的，因为1）Tt在其RNAPII的Rpb 1亚基中不具有典型的七肽重复序列，2）Tt在减数分裂期间经历全基因组MED依赖性ncRNA转录，其在mRNA转录不存在的情况下发生。我的长期目标是描述Tt中RNAPII依赖性mRNA和减数分裂特异性ncRNA的转录机制。我们最近在Tt中发现了一种不同的MED（Garg et al.（2019）Current Biology）1，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。在目标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，我们将进行选定MED蛋白的邻近生物素化标记实验，以鉴定MED共调节转录的蛋白质组。目的3将整合和利用这些结果来阐明Tt MED共调节mRNA转录的机制。意义：基因组的不稳定性往往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的可重复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，因此这项研究具有根本的重要性，并可能导致识别与人类癌症相关的新标记蛋白。因此，这项研究可能对加拿大的药理学和生物技术产业产生长期效益。培训内容：拟议的研究计划将培养四名研究生和多名本科生研究人员在基础分子生物学和分子遗传学方法以及强大的新技术，如下一代测序。这些方法共同构成了下一代基础研究人员以及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