RNA editing in Trypanosoma brucei: characterization of gRNA U-tail interactions with partially edited mRNA substrates

Guide RNAs (gRNAs), key components of the RNA editing reaction in Trypanoso ma brucei, direct the insertion and deletion of uridylate (U) residues, Ana lyses of gRNAs reveal three functional elements, The 5'-end of the gRNA con tains the anchor, which is responsible for selection and binding to the pre -edited mRNA. The second element (the guiding region) provides the informat ion required for editing, At the 3'-end of the gRNA is a non-encoded U-tail , whose function remains unclear, However, the cleavage-ligation model for editing proposes that the U-tail binds to purine-rich regions upstream of e diting sites, thereby strengthening the interaction and holding onto the 5' cleavage product, Our previous studies demonstrated that the U-tail intera cts with upstream sequences and may play roles in both stabilization and te thering, These studies also indicated that the U-tail interactions involved mRNA regions that were to be subsequently edited. This raised the question of what happens to the mRNA-U-tail interaction as editing proceeds in the 3'-->5' direction. We examined gCYb-558 and its U-tail interaction with 5'C YbUT and two partially edited 5'CYb substrates. Our results indicate that t he 3'-end of the U-tail interacts with the same sequence in all three mRNAs . Predicted secondary structures using crosslinking data suggest that a sim ilar structure is maintained as editing proceeds, These results indicate th at the role of the U-tail may also involve maintenance of important seconda ry structure motifs.