TRYPANOSOME-U-DELETIONAL RNA EDITING INVOLVES GUIDE RNA-DIRECTED ENDONUCLEASE CLEAVAGE, TERMINAL U-EXONUCLEASE, AND RNA LIGASE ACTIVITIES

We have studied the mechanism of accurate in vitro RNA editing of Tryp anosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs t hat specify deletion of 2, 3, or 4 U residues at editing site 1 and mi tochondrial extract. This extract not only catalyzes deletion of the s pecified number of U residues but also exhibits a novel eudonuclease a ctivity that cleaves the input pre-mRNA in a gRNA-directed manner, pre cisely at the phosphodiester bond predicted in a simple enzymatic mode l of RNA editing, This cleavage site is inconsistent with a chimera-ba sed editing mechanism, The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase, RNA ligase can then join the mRNA halves through their n ewly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can t hen undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model, All of these en zymatic activities cofractionate with the U-deletion activity and may reside in a single complex, The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pr e-mRNA at the bond immediately 5' of the region base paired to the gRN A, (ii) U deletion from or U addition to the 3' OH of the upstream mRN A half, (iii) ligation of the mRNA halves, and (iv) formation of addit ional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.