Talk abstracts
Talk on Saturday 08:45-09:00am submitted by Katherine Sortino
RESC8 and RESC14 cooperate to mediate RESC function and dynamics during trypanosome RNA editing
Katherine Sortino (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Brianna Tylec (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Natalie M. McAdams (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Runpu Chen (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Yijun Sun (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.), Laurie K. Read (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY 14203.)
Abstract:
Uridine insertion/deletion RNA editing is unique to kinetoplastid organisms and essential for their survival and virulence. This intricate process requires the action of several multiprotein complexes and small trans-acting RNAs called guide RNAs. The RNA editing holoenzyme comprises three dynamically interacting complexes: RECC (RNA Editing Catalytic Complex), RESC (RNA Editing Substrate Binding Complex), and REH2C (RNA Editing Helicase 2 Complex). Previous studies in Trypanosoma brucei showed that RESC components, RESC14 and RESC8, are critical in maintaining normal protein-protein and protein-RNA interactions within the editing holoenzyme. To further clarify the role of these proteins and the RESC complex in RNA editing, we performed high-throughput sequencing of ATP synthase subunit 6 (A6) mRNA in RESC14, RESC8, and RESC13 RNAi cells, and compared the phenotypes of these cell lines. We measured a highly significant overlap between exacerbated pause sites arising on A6 mRNA in cells depleted of RESC14 and RESC8, suggesting an overlapping function between these proteins during editing progression. In contrast, RESC13 depleted cells exhibited different A6 mRNA pause sites, indicative of a function distinct from that of RESC14/8. To probe the biochemical basis of the RESC14/8 interdependence, we performed Native PAGE analysis and showed that RESC8 is not incorporated into large RNA-containing editing complexes in the absence of RESC14. We also found through Native PAGE analysis that RESC14 is needed for incorporation of other RESC factors into large RNA-containing complexes, but is dispensable for RECC, KREH1 and REH2C complex formation. To better understand the interaction of RESC14 and RESC8, co-immunoprecipitation assays were performed and identified an RNA inhibited interaction between RESC14 and RESC8. We synthesize our findings into a model showing how RESC14 and RESC8 cooperate to mediate necessary protein and RNA rearrangements during editing progression. Together, our findings suggest that RESC14 and RESC8 rely on each other for necessary protein and RNA rearrangements during editing progression.
Keywords: Trypanosome, RNA editing, Holoenzyme