Poster abstracts
Poster number 94 submitted by Amartya Mishra
Modulation of RNA Editing Helicase 1 level regulates RNA editing profile of mitochondrial transcripts in trypanosomes
Amartya Mishra (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA), Ashutosh P. Dubey (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA.), Brianna L. Tylec (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA.), Laurie K. Read (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA. )
Abstract:
In trypanosomes, most mitochondrial encoded mRNA sequences require post-transcriptional insertion and deletion of uridine residues. This U-indel RNA editing is directed by trans-acting gRNAs and mediated by a holoenzyme comprised of the catalytic RNA editing core complexes (RECC) and non-catalytic RNA editing substrate binding complex (RESC). RNA Editing Helicase 1 (REH1), an ATP-dependent mitochondrial RNA helicase, interacts transiently with RECC and RESC. Previous evidence indicates a role for REH1 in RNA editing of at least some mRNAs, but its mechanism of action is unclear. Here, we confirm by co-IP interactions between REH1 and several subunits of both RECC and RESC. We show that depletion of REH1 causes modest decreases in the levels of a subset of edited mRNAs. In contrast, overexpression of wild type (WT) REH1 from an ectopic locus causes a significant growth defect and leads to a 75-90% decrease in fully edited versions of most pan-edited mRNAs in the absence of pre-edited mRNA increases. Minimally edited mRNAs are largely unaffected. Together, these data suggest that 3’ to 5’ editing progression is impaired in REH1 overexpressors. To define the role of REH1 ATP-binding, we overexpressed REH1 mutated in the conserved ATP-binding motif. Mutant overexpression leads to cell death, indicating a dominant negative (DN) phenotype. This is accompanied a dramatic decrease in almost all edited mRNAs with concomitant accumulation of pre-edited transcripts, including 2 of 3 minimally edited mRNAs. Thus, unlike WT REH1, the DN mutant impairs initiation of editing such that pre-edited mRNAs accumulate. WT and DN REH1 bind RESC and RECC to a similar extent, suggesting that DN REH1 binding to the editing holoenzyme impairs its productive interaction with mRNA or gRNA, thereby interfering with editing initiation. We have now generated REH1 knockout cells and are analyzing these and REH1 WT and DN overexpressors by high throughput sequencing to define at the single nucleotide level the step(s) at which editing is blocked. This genomic approach, together with additional protein-protein and RNA-protein interaction studies, will unveil the mechanism of REH1 action in U-indel editing at the molecular level.
Keywords: Trypanosoma, RNA editing, RNA helicase