Grant: Discovery Grants Program - Individual

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