Trypanosome RNA editing: Simple guide RNA features enhance U deletion 100-fold

Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPa se (A6) pre-mRNA and gRNA. Here we examine which gRNA features are importan t for this U deletion. We find that, foremost, this editing depends critica lly on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA nee ds to direct unexpectedly efficient in vitro U deletion, using either the p urified editing complex or whole extract. In fact, our optimized gRNA const ructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro Il deletion, in which previous artificial gR NAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bri dge, 3' OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.