Poster abstracts
Poster number 35 submitted by Amartya Mishra
Kinetoplast RNA Editing Helicase 1 plays a distinct role in RNA editing profile of mitochondrial transcripts in Trypanosoma brucei
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 mRNAs require post-transcriptional uridine (U) insertion/deletion (U-indel) editing, which is directed by trans-acting gRNAs and mediated by a holoenzyme comprised of the catalytic RECC, the REH2C helicase complex, and non-catalytic RNA editing substrate binding complex (RESC). Another ATP-dependent RNA helicase, Kinetoplast RNA Editing Helicase 1 (KREH1), interacts transiently with RECC and RESC. Previous evidence suggested a role for KREH1 gRNA removal during editing progression. Here, we show that a KREH1 null mutant (KO) in procyclic form T. brucei exhibits a modest decrease in a subset of edited mRNAs compared to wild type (WT), in particular ATPase (A6) mRNA. High throughput sequencing followed by TREAT analysis of A6 mRNA in KREH1 KO cells revealed no evidence for KREH1 function in gRNA removal. Rather, we identified multiple sequences in the KREH1 KO with a long stretch of pre-edited sequence followed by one or two modified editing sites outside of the first gRNA. These data suggest a role of KREH1 in modulation of RNA structure. Overexpression (OE) of WT KREH1 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 while pre-edited mRNAs were unaffected. Together, these data suggest that 3’ to 5’ editing progression is impaired in KREH1 OE. To define the role of KREH1 ATP-binding, we overexpressed KREH1 mutated in the conserved ATP-binding motif. Mutant OE leads to cell death, indicating a dominant negative (DN) phenotype. This is accompanied by a dramatic decrease in almost all edited mRNAs with concomitant accumulation of pre-edited transcripts, including 2 of 3 moderately edited mRNAs. Thus, unlike WT KREH1, the DN mutant impairs initiation of editing. Overexpression of DN KREH1 alters RESC homeostasis by interfering with the interaction between its GRBC module and a subset of additional RESC proteins. Together, these data are consistent with a model in which GRBC and RECC assemble normally on mRNA, while KREH1-mediated ATP hydrolysis is necessary for subsequent assembly of a functional RESC, capable of promoting editing initiation. Thus, KREH1 is important for both initiation and progression of U-indel editing.
Keywords: Trypanosoma, RNA editing, RNA helicase