Trans splicing in trypanosomes requires methylation of the 5' end of the spliced leader RNA.

Abstract

Trypanosoma brucei spliced leader (SL) RNA contains an unusual cap 4 structure consisting of 7-methylguanosine linked to four modified nucleosides. During RNA maturation, trans splicing transfers the first 39 nucleotides of the SL RNA including the cap structure to the 5' end of all mRNAs. Here we show that exposure of permeable trypanosome cells to S-adenosyl-L-homocysteine inhibits methylation of the nucleosides adjacent to 7-methylguanosine of newly synthesized SL RNA and prevents utilization of the SL RNA in trans splicing. However, trans splicing of the SL RNA preexisting in the cells is not inhibited by S-adenosyl-L-homocysteine as shown by the observation that newly synthesized alpha-tubulin RNA is trans spliced at the same level as in control cells. Therefore, it appears that the newly synthesized SL RNA is the only known component of the trans-splicing machinery that is impaired in its function by inhibition of methylation. Undermethylation does not alter either the stability of the SL RNA or the electrophoretic mobility and chromatographic behavior of the core SL ribonucleoprotein particle. Taken together, our data suggest that the cap 4 structure of the SL RNA plays an essential role in the trans-splicing process.