Defining the editing ‘reaction’

Abstract

E xactly a decade after the initial report of RNA editing in the mitochondrion of the parasitic protozoan Trypanosoma brucei by Benne and co-workers l, the mechanism of uridylate (U) insertion and deletion has been resolved. In a recent article published in Science 2, Moffett Kable, Scott Seiwart and Stefan Heidmann, all working in Ken Stuart's laboratory, were able to establish, in vitro, an accurate U insertion RNA-editing reaction. The 'reaction' proceeds via a series of enzyme-catalyzed steps, with both the site and number of inserted Us directed by small transacting RNAs. This report follows closely on the heels of recent studies reporting the mechanism of deletion editing 3'4. These initial reports have been verified by others s. The papers from the Stuart lab have answered many of the fundamental questions concerning the general mechanism of RNA editing in trypanosomes. An inventory of the mitochondrial mRNAs of T. brucei reveals that 12 out of 17 mRNAs are processed by U insertion or deletion 6,v. The cytochrome oxidase II mRNA is only modestly changed, by the addition of four Us within the 663nucleotide transcript 1, from the sequence encoded by the mitochondrial genome. In contrast, in cytochrome oxidase III mRNA, the extent of editing is bewildering with 547 Us added and 41 Us deleted, resuiting in an mRNA that is so radically changed from the gene that greater than half of its residues are formed by RNA editing 8. In order to achieve the precision necessary to edit an mRNA correctly with this number of insertions and deletions, a template was postulated 9. A group of small, heterogeneous RNAs was identified as a candidate for directing the editing of mRNA. These guide RNAs (gRNA) are largely encoded by the minicircle DNA in the mitochondrion of trypanosomes and are complementary to short segments of edited mRNA. The gRNAs were also found in mitochondrial ribonucleoprotein complexes with pre-edited mRNAs and several enzymatic activities postulated to be involved in RNA editingl°'lL However, direct evidence of the role of gRNAs in RNA editing awaited the development of an in vitro system for accurate deletion and insertion editing. The recent studies from the Stuart lab have provided such a system. Their studies indicate that gRNAs have a dual role in editing. First, gRNAs recognize editing sites within the pre-edited mRNA and form the initial editing complex. This is mediated by the formation of a short 7-10-bp duplex between the gRNA and mRNA sequences immediately 3' to the editing site. This duplex is required for the subsequent enzymatic steps in editing. Second, the gRNA directs the number of Us added or deleted at an editing site. The results presented in the recent Science papers show that the Us are added or deleted to form a complementary sequence with the gRNA. The results of Kable et al. 2 and Seiwert et al. 3,4 also provide a great deal of insight into the mechanism of RNA editing in trypanosomes. Two general mechanisms have been proposed. The first is an enzyme cascade model 12 in which the pre-edited mRNA is cleaved sequentially by an editing-site-specific endoribonuclease 13, Us are added to or deleted from the 3' end of the premRNA 5' cleavage product by the action of a terminal-U transferase (TUTase) TM and the two halves are rejoined by the action of a mitochondrial RNA ligase 1-5,16 to form the edited mRNA. The second model is very different and proposes that