Transcriptional regulation of non-coding RNAs in Tetrahymena thermophila

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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