Abstract
Uridine insertion/deletion RNA editing in kinetoplastid mitochondria corrects encoded frameshifts in mRNAs. The genetic information for editing resides in small guide RNAs (gRNAs), which form anchor duplexes just downstream of an editing site and mediate editing within a single editing "block." Many mRNAs require multiple gRNAs; the observed overall 3' to 5' polarity of editing is determined by the formation of upstream mRNA anchors by downstream editing. Hel61, a mitochondrial DEAD-box protein, was previously shown to be involved in RNA editing, but the functional role was not clear. Here we report that down-regulation of Hel61 [renamed REH1 (RNA editing helicase 1)] expression in Trypanosoma brucei selectively affects editing mediated by two or more overlapping gRNAs but has no effect on editing within a single block. Down-regulation produces an increased abundance of the gRNA/edited mRNA duplex for the first editing block of the A6 mRNA. Recombinant REH1 has an ATP-dependent double strand RNA unwinding activity in vitro with a model gRNA-mRNA duplex. These data indicate that REH1 is involved in gRNA displacement either directly by unwinding the gRNA/edited mRNA duplex or indirectly, to allow the 5' adjacent upstream gRNA to form an anchor duplex with the edited mRNA to initiate another block of editing. Purified tagged REH1 is associated with the RNA editing core complex by RNA linkers and a colocalization of REH1, REL1, and two kinetoplast ribosomal proteins with the kinetoplast DNA was observed by immunofluorescence, suggesting that editing, transcription, and translation may be functionally linked.
Highlights
Uridine insertion/deletion RNA editing in kinetoplastid mitochondria corrects encoded frameshifts in mRNAs
An 80% decrease in the abundance of REH1 mRNA by day 3 was demonstrated by RT-PCR and real-time RT-PCR (Fig. 1 B and D) and REH1 protein was not detectable by Western blot analysis (Fig. 1B)
Repression of REH1 expression showed no effect on the stability or length of the guide RNAs (gRNAs) 3′ oligo U tails (Fig. 1C), as was reported for the trypanosome REH2 mitochondrial RNA helicase [14, 16]
Summary
Uridine insertion/deletion RNA editing in kinetoplastid mitochondria corrects encoded frameshifts in mRNAs. The genetic information for editing resides in small guide RNAs (gRNAs), which form anchor duplexes just downstream of an editing site and mediate editing within a single editing “block.” Many mRNAs require multiple gRNAs; the observed overall 3′ to 5′ polarity of editing is determined by the formation of upstream mRNA anchors by downstream editing. The editing of most mRNAs is mediated by multiple overlapping gRNAs. Editing utilizing the adjacent upstream gRNA cannot proceed until the first block is completely edited because the gRNA can only form an anchor duplex with edited sequence to initiate the second editing block. An RNA helicase has been suggested to be involved in this process by displacing the initial gRNA, thereby allowing the adjacent upstream gRNA to form an anchor duplex with the edited sequence [19]. Gene disruption had no effect on either an experimentally observed mito-
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