Poster abstracts
Poster number 47 submitted by Gregory Harrison
Defining the role of MRB10130 in uridine insertion and deletion RNA editing in trypanosoma brucei
Gregory L. Harrison Jr. (Department of Microbiology and Immunology, SUNY Buffalo School of Medicine), Michelle Ammerman (Department of Microbiology and Immunology, SUNY Buffalo School of Medicine), Laurie K. Read (Department of Microbiology and Immunology, SUNY Buffalo School of Medicine)
Abstract:
In Trypanosoma brucei, the majority of the mitochondrially-encoded RNAs require the post-transcriptional addition and deletion of uridines to create translatable open reading frames through an essential process termed Uridine Insertion/Deletion RNA Editing. The enzymes that catalyze RNA editing are contained in the RNA Editing Core Complex. Recent studies have also identified the Mitochondrial RNA Binding Complex (MRB1), comprising a dynamic network of protein-protein and protein-RNA interactions, as an essential component of the editing machinery. Combining a comprehensive yeast two-hybrid (Y2H) screen and in vivo immunoprecipitation and mass spectrometry, we identified an MRB1 core and at least two TbRGG2 subcomplexes in MRB1. Interestingly, one of the proteins in the Y2H screen, MRB10130, interacted with a large number of MRB1 components within both the MRB1 core and TbRGG2 subcomplexes. MRB10130 is predicted to be composed almost entirely of alpha-helical repeats that resemble ARM/HEAT repeats, and such proteins often act as organizers of protein-protein interactions. Thus, we hypothesize that MRB10130 is involved in coordinating interactions within the MRB1 complex. Here, we show that MRB10130 is essential for both growth and RNA editing in T. brucei. We performed glycerol gradient sedimentation analysis of affinity purified MRB10130-PTP and compared that to a similar analysis of whole cell extracts. We show that MRB10130 co-purifies MRB1 core and TbRGG2 subcomplexes, but our data suggest that MRB10130 is more loosely associated with MRB1 than are other proteins, consistent with an organizing function. Furthermore, we use a cell line harboring inducible MRB10130 RNAi and an epitope-tagged MRB1 core component to show an RNA-enhanced MRB10130 involvement in maintaining interactions between the MRB1 core and TbRGG2 subcomplexes. We also show that MRB10130 is able to weakly bind RNA in vitro. We propose a model in which MRB10130 and RNA work synergistically to stabilize interactions between the MRB1 core and TbRGG2 subcomplexes.
Keywords: Trypanosoma brucei, RNA editing