Mapping contacts between gRNA and mRNA in trypanosome RNA editing

All guide RNAs (gRNAs) identified to date have defined 5' anchor sequences, guiding sequences and a non-encoded 3' uridylate tail. The 5' anchor is re quired for in vitro editing and is thought to be responsible for selection and binding to the pre-edited mRNA. Little is known, however, about how the gRNAs are used to direct RNA editing. Utilizing the photo-reactive crossli nking agent, azidophenacyl (APA), attached to the 5'- or 3'-terminus of the gRNA, we have begun to map the structural relationships between the differ ent defined regions of the gRNA with the pre-edited mRNA. Analyses of cross linked conjugates produced with a 5'-terminal APA group confirm that the an chor of the gRNA is correctly positioning the interacting molecules. 3' Cro sslinks (X-linker placed at the 3'-end of a U-10 tail) have also been mappe d for three different gRNA/mRNA pairs. In all cases, analyses indicate that the U-tail can interact with a range of nucleotides located upstream of th e first edited site. It appears that the U-tail prefers purine-rich sites, close to the first few editing sites. These results suggest that the U-tail may act in concert with the anchor to melt out secondary structure in the mRNA in the immediate editing domain, possibly increasing the accessibility of the editing complex to the proper editing sites.