Poster abstracts

Poster number 90 submitted by Natalie Merlino

Functional characterization of the essential RNA editing complex, MRB1, reveals an ABC ATPase protein that acts as an editing specificity factor

Natalie M. Merlino (Department of Microbiology and Immunology, University at Buffalo School of Medicine), Michelle L. Ammerman (Department of Microbiology and Immunology, University at Buffalo School of Medicine), John C. Fisk (Department of Microbiology and Immunology, University at Buffalo School of Medicine), Maria Schumacher (Department of Biochemistry, Duke University), Laurie K. Read (Department of Microbiology and Immunology, University at Buffalo School of Medicine)

Abstract:
Mitochondrial mRNAs in kinetoplastid parasites require extensive remodeling by a unique process termed uridine insertion/deletion RNA editing. For the majority of transcripts, RNA editing is required to create translatable open reading frames, and thus editing is essential for proliferation of both human and insect vector stages of the kinetoplastid, Trypanosoma brucei. Small trans-acting guide RNAs (gRNAs), encoded in the minicircle component of the mitochondrial genome, direct proper U insertion or deletion. Editing is catalyzed by the multiprotein RNA editing core complex (RECC) or 20S editosome, although additional components are emerging as equally critical to the editing process. We have recently characterized a dynamic multiprotein complex termed MRB1 (mitochondrial RNA binding complex 1) as a key component of the editing machinery. We reported the presence of an MRB1 core that contains at least 6 proteins and is essential for RNA editing initiation. We have also identified subcomplexes containing the TbRGG2 RNA binding/annealing protein; TbRGG2 subcomplexes are essential for 3’ to 5’ progression of editing on pan-edited RNAs. Numerous additional proteins interact in a substoichiometric manner with the MRB1 core and TbRGG2 subcomplexes, and their functions are as yet unknown. Here, we present ongoing studies of one such protein, MRB1590. RNAi-mediated knockdown of MRB1590 in insect stage T. brucei leads to a modest growth defect and a specific inhibition of the 3’ to 5’ progression of editing of A6 mRNA that may reflect a role in efficient utilization of one or a few gRNAs. Structural analysis shows that MRB1590 is an ABC ATPase domain with similarity to proteins involved in DNA repair. In vivo pulldown studies demonstrate RNA-dependent interactions between MRB1590 and both TbRGG2 and the GAP1/2 component of the MRB1 core. Interestingly, MRB1590 interacts in an RNA-independent manner with the Zn finger protein, MRB6070, suggesting the presence of another multiprotein component of the dynamic MRB1 RNA editing complex, which may contain editing specificity factors. These studies provide another piece in the puzzle of our ongoing characterization of the essential and multifunctional RNA editing complex, MRB1.

Keywords: RNA editing