Cotranscriptional cap 4 formation on the Trypanosoma brucei spliced leader RNA

mRNA cap formation in trypanosomatid protozoa is mediated through trans-splicing of the capped spliced leader (SL) sequence of the SL RNA onto the 5' end of all mRNAs. The SL RNA cap structure in Trypanosoma brucei is unique among eukaryotes and consists of 7-methylguanosine (m(7)G) followed by four methylated nucleotides (cap 4): m(7)Gpppm(2)(6)AmpAmpCmpm(3)Um. Using transcriptional arrest in permeable T. brucei cells, we have analyzed the temporal progression of cap 4 formation on the 140-nucleotide-long SL RNA. m(7)G capping of the SL RNA could be detected on prematurely terminated SL RNA transcripts of 56 nucleotides in length and longer. Subsequent modifications characteristic of the SL RNA cap 4 were added successively in a 5' to 3' direction and appeared to be independent of core ribonucleoprotein formation. Transcripts between 56 and 67 nucleotides in length were partially modified and carried methyl groups on the first two adenosine residues, whereas a fully modified cap 4 structure was present on transcripts arrested at position 117 and beyond. Taken together, our results are consistent with a cotranscriptional mechanism for generating the cap 4 structure on the SL RNA.