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性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，其组装由小rna （sRNAs）指导，这些小rna是由非编码rna （ncRNAs）的减数分裂和全核转录加工而成。我研究的长期目标是完全了解这些大规模DNA重排的机制和功能。由于sRNAs， ncRNAs和染色质相关蛋白之间的联系在真核生物中广泛存在，我希望获得关于它们在四膜虫中的机制和调控的知识，将产生关于包括人类在内的所有真核生物基础的蛋白质和分子途径的新信息。在短期内，我们将确定来自四膜虫减数分裂核的ncrna转录的潜在机制。由于对四膜虫中任何DNA序列转录的机制和蛋白质知之甚少，我们采用了靶向蛋白质组学方法来鉴定一组在转录中起作用的蛋白质复合物。接下来，我们将确定RNA聚合酶II （RNAPII）的调节模式，转录引擎，在生长的四膜虫蛋白质编码基因。最后，我们将确定RNAPII是如何在四膜虫减数分裂过程中转录成ncrna的独特DNA序列中被调节的。从长远来看，我们将利用我们获得的知识来了解新转录的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 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