During RNA editing in kinetoplastid parasites,
trans-acting guide RNAs (gRNAs) direct the insertion
and deletion of U residues at precise sites in mitochondrial
pre-mRNAs. We show here that some modifications to the
3′ terminal ribose of gRNA inhibit its ability to
direct in vitro U insertion. However, we found that gRNAs
lacking this moiety in some circumstances support in vitro
editing. Thus, the 3′ OH is not required. Inhibition
resulting from gRNA modification can be overcome by increasing
the gRNA–pre-mRNA base-pairing potential upstream
of the editing site, suggesting an importance for this
interaction to productive processing.