Transcriptional regulation of non-coding RNAs in Tetrahymena thermophila

纤毛虫原生动物嗜热四膜虫在其性发育过程中经历了新的大规模程序化DNA重排。 这些基因组重排的潜在机制在很大程度上是未知的，除了需要特定的染色质构型，其组装是由减数分裂的非编码RNA（ncRNA）的核范围转录加工的小RNA（sRNA）指导。 我研究的长期目标是完全理解这些大规模DNA重排的机制和功能。 由于sRNA，ncRNA和染色质相关蛋白之间的联系在真核生物学中广泛存在，我希望获得有关四膜虫中它们的机制和调控的知识将产生关于蛋白质和分子途径的新信息，这些蛋白质和分子途径对所有真核生物（包括人类）都是至关重要的。 在短期内，我们将确定转录的ncRNA从四膜虫减数分裂核的潜在机制。 由于机制和蛋白质参与转录的任何DNA序列在四膜虫是知之甚少，我们已经采取了有针对性的蛋白质组学的方法来确定一套蛋白质复合物的转录功能。 接下来，我们将确定RNA聚合酶II（RNAPII），转录引擎，在生长的四膜虫蛋白质编码基因的调节模式。 最后，我们将确定RNAPII是如何调节在四膜虫减数分裂过程中转录成ncRNA的独特DNA序列。 从长远来看，我们将利用我们获得的知识，以了解新转录的ncRNA，细胞的RNAi机制和染色质组装/重塑途径之间的联系。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 ciliate protozoan Tetrahymena thermophila undergoes novel large-scale programmed DNA rearrangements during the course of its sexual development. The underlying mechanism of these genome rearrangements is largely unknown except for the requirement of specific chromatin configurations whose assembly is directed by small RNAs (sRNAs) processed from meiotic, nucleus-wide transcription of non-coding RNAs (ncRNAs). The long-term objective of my research is to completely understand the mechanism and function of these large-scale DNA rearrangements. Because the connections between sRNAs, ncRNAs and chromatin related proteins are widespread in eukaryotic biology, I expect that acquiring knowledge concerning their mechanism and regulation in Tetrahymena will yield novel information on proteins and molecular pathways that are fundamental to all eukaryotes, including humans. In the short-term we will identify the underlying mechanism of transcription of ncRNAs from the meiotic nucleus of Tetrahymena. Because the mechanisms and proteins involved in the transcription of any DNA sequence in Tetrahymena is poorly understood, we have adopted a targeted proteomic approach to identify the set of protein complexes that function in transcription. Next we will identify the mode(s) of regulation of RNA polymerase II (RNAPII), the engine of transcription, at protein coding genes in growing Tetrahymena. Finally, we will determine how RNAPII is regulated at the unique DNA sequences that are transcribed into ncRNAs during Tetrahymena meiosis. In the longer term we will leverage our acquired knowledge in order to understand connections between newly transcribed ncRNAs, the cell's RNAi machinery, and chromatin assembly/remodeling pathways.***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