Insertional and deletional RNA editing in trypanosome mitochondria.

The mitochondrial mRNAs of trypanosomes are often post-transcriptionally modified by an RNA processing event, termed RNA editing, which results in the insertion or deletion of uridylate (U) residues in mRNAs. RNA editing is necessary for the formation of complete coding sequences for several essential mitochondrial proteins. The number and site of U addition and deletion is directed by small guide RNAs (gRNAs). Recent studies indicate that the mechanism of RNA editing in trypanosomes involves a series of enzymatic steps. We show that the initial step in this enzymatic cascade requires the formation of a binary RNA complex between the gRNA and its cognate pre-mRNA. Depletion of specific gRNAs inhibits cleavage of the pre-mRNA by an editing site specific endoribonuclease. Addition of synthetic gRNAs reverses this inhibition. All of the activities needed for RNA editing in vitro are present within a 19S ribonucleo-protein complex (RNP) composed of gRNAs, the editing site specific endonuclease, an RNA ligase, a terminal uridylate transferase (TUTase) and approximately 15 other unidentified proteins. We have recently identified and cloned the gene for a 45kDa protein, the RNA Editing Associated Protein-1 (REAP-1), which is a component of trypanosome editing complexes. REAP-1 co-purifies with RNA ligase and TUTase activities and is part of a > 700 kDa RNP containing gRNAs. Antibodies against REAP-1 inhibit in vitro RNA editing reactions confirming its role in RNA editing.