GUIDE RNA-MESSENGER-RNA CHIMERAS, WHICH ARE POTENTIAL RNA EDITING INTERMEDIATES, ARE FORMED BY ENDONUCLEASE AND RNA LIGASE IN A TRYPANOSOMEMITOCHONDRIAL EXTRACT

RNA editing in kinetoplast mitochondrial transcripts involves the inse rtion and/or deletion of uridine residues and is directed by guide RNA s (gRNAs). It is thought to occur through a chimeric intermediate in w hich the 3' oligo(U) tail of the gRNA is covalently joined to the 3' p ortion of the mRNA at the site being edited. Chimeras have been propos ed to be formed by a transesterification reaction but could also be fo rmed by the known mitochondrial site-specific nuclease and RNA ligase. To distinguish between these models, we studied chimera formation in vitro directed by a trypanosome mitochondrial extract; This reaction w as found to occur in two steps. First, the mRNA is cleaved in the 3' p ortion of the editing domain, and then the 3' fragment derived from th is cleavage is ligated to the gRNA. The isolated mRNA 3' cleavage prod uct is a more efficient substrate for chimera formation than is the in tact mRNA, inconsistent with a transesterification mechanism but suppo rting a nuclease-ligase mechanism. Also, when normal mRNA cleavage is inhibited by the presence of a phosphorothioate, normal chimera format ion no longer occurs. Rather, this phosphorothioate induces both cleav age and chimera formation at a novel site within the editing domain, F inally, levels of chimera-forming activity correlate with levels of mi tochondrial RNA ligase activity when reactions are conducted under con ditions which inhibit the ligase, including the lack of ATP containing a cleavable alpha-beta bond. These data show that chimera formation i n the mitochondrial extract occurs by a nuclease-ligase mechanism rath er than by transesterification.