TRYPANOSOMA-BRUCEI RNA EDITING - A FULL ROUND OF URIDYLATE INSERTIONAL EDITING IN-VITRO MEDIATED BY ENDONUCLEASE AND RNA LIGASE

RNA editing in kinetoplastids is the post-transcriptional insertion an d deletion of uridylate residues in mitochondrial transcripts, directe d by base pairing with guide RNAs. Models for editing propose transest erification or endonuclease plus RNA ligase reactions and may involve a guide RNA-mRNA chimeric intermediate. We have assessed the feasibili ty of the enzymatic pathway involving chimeras in vitro. Cytochrome b chimeras generated with mitochondrial extract were first found to have junctions primarily at the major endonuclease cleavage sites, support ing the role of endonuclease in chimera formation. Such cytochrome b c himeras are then specifically cleaved by extract endonuclease within t he oligo(U) tract at the editing site, and the mRNA cleavage products are then joined by RNA ligase to generate partially edited mRNAs with uridylate residues transferred to an editing site. These in vitro gene rated partially edited mRNAs mimic partially edited mRNAs generated in vivo. Specific endonuclease cleavage in the editing region of the par tially edited RNA demonstrates the potential for further in vitro edit ing. Finally, sensitivity to various ATP analogs suggests that all edi ting-like activities reported thus far utilize a mechanism involving R NA ligase.