The Role of TNRC6 in RNA Interference




Johnson, Samantha Tori

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Small RNAs can influence translation, splicing, transcriptional activation, and transcriptional repression, through the RNA interference (RNAi) pathway. TNRC6, also known as GW182, and Argonaute (AGO) are the core proteins of RNAi. TNRC6 is a scaffolding protein that associates with AGO and bridges its interactions with other proteins to control gene expression in many different processes. There are three paralogs in mammalian cells, TNRC6A, TNRC6B, and TNRC6C. These paralogs share approximately 40% amino acid sequence identity. Whether the paralogs have unique or redundant functions is unclear. Much is known about the mechanisms of cytoplasmic RNAi but the world of nuclear RNAi remains murky. We understand that RNAi factors are present and active in the nucleus, but endogenous nuclear RNAi functions are unknown. I have used a suite of gene knockout cell lines for the TNRC6 paralogs to learn more about TNRC6 paralogs and their roles in RNAi. I examined if TNRC6 paralogs were required in several functions of small duplex RNA-mediated control of gene expression, including translational silencing by miRNAs, translational silencing by siRNAs, and transcriptional activation. On a global scale, I used high-throughput RNA sequencing, mass spectrometry, and enhanced crosslinking immunoprecipitation (eCLIP) to gather definitive answers about the TNRC6 paralogs, their roles in the cell, and their relation to AGO knockout cell lines. I found that that despite less than 40% sequence identity, the TNRC6 paralogs are functionally redundant and can replace one another for core RNAi functions. Each subsequent TNRC6 paralog knockout caused more gene changes and few genes overlapped between the single TNRC6 paralog knockouts. Changes in levels of gene expression in TNRC6 knockout cell lines are well-correlated with those observed in AGO knockout cell lines, emphasizing the important regulatory function of the partnership between AGO and TNRC6 in endogenous RNAi. Further, I found TNRC6 plays a role in splicing changes initiated by small RNA binding to intronic regions. Taken together these data further define the roles of the TNRC6 paralogs as part of the RNA interference machinery. TNRC6 is a close protein partner of AGO and serves as a cooperativity coordinator for RNAi.

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Argonaute Proteins, MicroRNAs, RNA Splicing, RNA-Binding Proteins, Transcription, Genetic


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