Structure-function analysis of the essential RNA editing accessory factor, TbRGG2
Foda, Bardees Mohamed
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RNA editing is a post-transcriptional gene regulation process that involves insertion and deletion of uridine residues at specific sites in most mitochondrial mRNAs. TbRGG2 is an essential RNA editing factor implicated in both editing initiation and progression of pan-edited RNAs. TbRGG2 possesses several activities including RNA binding, RNA annealing, RNA melting, and interactions with multiple components of mitochondrial RNA binding complex 1 (MRB1). TbRGG2 contains an N-terminal glycine rich (G-rich) region with overlapping repeats of GWG and RGs. At the C-terminus, TbRGG2 has RNA recognition motif (RRM) containing domain. In the dissertation, we performed in vitro and in vivo analysis of TbRGG2 to separate the protein's functions and determine the contribution of each domain/function to the role of TbRGG2 in RNA editing. In the first part of the study, we purified recombinant proteins representing TbRGG2 variants including TbRGG2, G-rich, RRM, GWG domains, FL-GWG (full length lacking GWG repeats), and RGG2VF-AA (TbRGG2 with two RRM point mutations). We analyzed the RNA binding and annealing activities in vitro, and used an E. coli based assay to determine RNA melting activity. G-rich region displayed high RNA binding affinity and robust RNA annealing similar to TbRGG2 as well as remarkable preference for pre-edited RNA. Significantly, RRM domain exhibited RNA melting activity independent of G-rich region. Thus, in vitro analysis revealed distinct separation of TbRGG2 activities. In second part of the study, we tested these activities in vivo for their abilities to support RNA editing and growth. We generated T. brucei cell lines depleted of TbRGG2, and expressing exogenous TbRGG2 variants. Exogenous TbRGG2 restored cell growth and RNA editing. G-rich protein supported partial restoration, while the RRM domain failed to rescue the cells. RGG2VF-AA mutant efficiently complemented depletion of endogenous TbRGG2. Thus, we conclude that both G-rich and RRM domains are required for TbRGG2 functions in RNA editing, while RNA melting is apparently dispensable. Finally, we used yeast two-hybrid analysis and co-immunoprecipitation to determine the contribution of TbRGG2 domains to interaction with MRB1 components. Generally, we detected different protein interaction profiles for each domain. Based on the data presented here, we propose a model in which the substantial RNA based activities of TbRGG2 are effected primarily by the G-rich region, but a cross-talk takes place between G-rich and RRM domains whose aspects are partially dependent on protein interactions and domain-domain regulation. This interplay is suggested to facilitate the role of TbRGG2 in RNA editing.