Poster abstracts
Poster number 31 submitted by Ashutosh Dubey
KREH1 helicase activity promotes the occurrence and proper positioning of mRNA editing initiation in Trypanosoma brucei
Ashutosh P Dubey (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Brianna L. Tylec (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Amartya Mishra (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Runpu Chen (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Yijun Sun (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Laurie K. Read (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA)
Abstract:
In trypanosomes, 12 of 18 mitochondrially encoded mRNAs require post-transcriptional insertion and deletion of uridine residues to create translatable mRNAs. Editing is directed by sequentially utilized trans-acting guide RNAs (gRNAs) that base pair with mRNA, and catalyzed by a multi-subunit holoenzyme. The ATP-dependent RNA helicase, Kinetoplast RNA Editing Helicase 1 (KREH1), interacts transiently with the editing holoenzyme and was previously suggested to be involved in the removal of gRNAs from mRNAs following their utilization. To further investigate the KREH1 function, we generated a KREH1 null mutant (KO) in procyclic form T. brucei. KREH1 KO exhibits a modest decrease in a subset of edited mRNAs compared to wild type (WT), in particular ATPase (A6) mRNA. High throughput sequence analysis of A6 mRNA in KREH1 KO cells revealed no evidence for KREH1 function in gRNA removal. Rather, we identified multiple sequences containing a long stretch of pre-edited sequence followed by one or two modified editing sites outside of the region whose editing is directed by the first gRNA. To define the role of KREH1 helicase activity, as opposed to simply its presence, we overexpressed WT KREH1 or KREH1 with mutations in either in the conserved ATP-binding motif (KA) or DEAD box domain (EQ). Both the KA and EQ mutants, but not the WT overexpressors, exhibit a dominant-negative (DN) phenotype marked by a dramatic decrease initiation of editing on most mRNAs with concomitant accumulation of pre-edited transcripts, and defects in the positioning of the initial A6 mRNA editing event similar to those observed in KO cells. Despite the differing effects of WT and KA KREH1 on editing initiation, co-IP and RIP analyses show that these proteins interact similarly with the editing holoenzyme, mRNA, and gRNA. Together, our data support a model in which KREH1 helicase activity modulates RNA-RNA interactions to promote the occurrence and proper positioning of editing initiation.
Keywords: Trypansoma, RNA Helicase, RNA editing