Functional Analysis of Transcription in a Protist Model

本文描述的研究将确定纤毛虫和原生动物嗜热四膜虫(TT)中发散介体(MED)转录复合体的功能和机制。在大多数真核生物中，RNA聚合酶II(RNAPII)亚基Rpb1通过在其C末端结构域(CTD)内经历翻译后修饰(PTM)的重复七肽重复来协调共转录事件。MED转录共激活复合体是酵母和人类细胞中一种与CTD相互作用的蛋白质复合体，在转录协同调节中发挥作用。理解MED在TT中的功能/机制是人们普遍感兴趣的，因为1)TT在其RNAPII的Rpb1亚基中不具有典型的七肽重复序列，2)TT在减数分裂过程中经历全基因组依赖的MED依赖的ncRNA转录，该转录发生在没有转录的情况下。我的长期目标是研究依赖RNAPII的mRNA和减数分裂特异的ncRNA在TT中的转录机制。我们最近在TT中发现了一种发散的MED(Garg等人)。(2019)Current Biology)1，并将其与mRNA和ncRNA转录有关。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，以完整地确定其功能。在目标2中，我们将对选定的MED蛋白进行邻近生物素化标记实验，以确定与MED共同调控转录的蛋白质集。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：基因组不稳定通常与随机的染色体重排有关。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复制DNA重排的不同寻常的性质。了解四膜虫如何重排其染色体将增加关于DNA重排的一般知识，因此这项研究具有重要的基础意义，并可能导致识别与人类癌症相关的新标记蛋白。出于这个原因，这项研究对加拿大的医药和生物技术行业具有潜在的长期好处。 培训：拟议的研究计划将培训四名研究生和多名本科生研究人员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，因此这项研究将使加拿大的医疗保健系统受益

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