Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA

Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addi tion and deletion during mRNA editing in trypanosomes. Terminal uridylyl tr ansferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the as sociation of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (198) and II (35S to 40S) . Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing . Purification of TUTase from mitochondrial extracts resulted in the identi fication of two chromatographically distinct TUTase activities. Stable sing le-uridine addition to different substrate RNAs is performed by the 19S com plex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle t hat may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine -rich region of the cognate mRNA protects the uridine tail from a uridine e xonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pai ring interaction with the poly(U) tail of the gRNA. This RNA fails to prote ct the uridine tail of the gRNA from exoribonucleolytic trimming and is con sistent with a role for the purine-rich region of the mRNA in gRNA maturati on.