Poster abstracts
Poster number 53 submitted by Gregory Harrison
Defining the role of MRB10130 in uridine insertion and deletion RNA Editing in trypanosomes
Gregory L. Harrison Jr. (Department of Microbiology and Immunology, School of Medicine, State University of New York at Buffalo), Michelle L. Ammerman (Department of Microbiology and Immunology, School of Medicine, State University of New York at Buffalo), Laurie K. Read (Department of Microbiology and Immunology, School of Medicine, State University of New York at Buffalo)
Abstract:
In Trypanosoma brucei, a majority of the mitochondrially encoded genes are encrypted and require the post-transcriptional addition and deletion of uridines to create translatable open reading frames. This process is referred to as Uridine Insertion and Deletion RNA Editing (RNA editing) and is essential in T. brucei. RNA editing is catalyzed by the RNA Editing Core Complex (RECC); however, in vitro and in vivo studies suggest additional factors are required for the processivity, specificity, and efficiency of this process. One of these factors is the Mitochondrial RNA Binding Complex (MRB1), which consists of a dynamic network of protein-protein and protein-RNA interactions. To begin to define MRB1 architecture, our lab previously conducted a comprehensive yeast two-hybrid (Y2H) screen, followed by in vivo immunoprecipitation and mass spectrometry. These studies identified an MRB1 core and at least two TbRGG2 subcomplexes. 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 interactions with several MRB1 components were enhanced in the presence of RNA. According to its predicted structure, MRB10130 is composed almost entirely of alpha-helical repeats that resemble ARM/HEAT repeats. Proteins containing these types of repeats often act as organizers and mediators of protein-protein interactions. The involvement of numerous proteins and RNA species in RNA editing presents the need for spatial and temporal regulation of these interactions. We hypothesize that MRB10130 is involved in coordinating interactions within the MRB1 complex. Here we show that repression of MRB10130 leads to a significant RNA editing defect and growth phenotype. We are currently purifying MRB10130-containing complexes by tandem affinity purification followed by glycerol gradient sedimentation to more precisely define the interactions of this protein with MRB1 subcomplexes. In addition, we are using a series of knockdown and epitope-tagged cell lines to determine the role of MRB10130 in maintaining interactions within and between MRB1 subcomplexes. These studies will provide insight into the mechanisms underlying coordination of MRB1 complex function in trypanosome RNA editing.
Keywords: RNA editing, trypanosome