Cotranscriptional cap 4 formation on the Trypanosoma brucei spliced leaderRNA

mRNA cap formation in trypanosomatid protozoa is mediated through trans-spl icing 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 tran scriptional arrest in permeable T. brucei cells, we have analyzed the tempo ral progression of cap 4 formation on the 140-nucleotide-long SL RNA. m7G c apping of the SL RNA could be detected on prematurely terminated SL RNA tra nscripts of 56 nucleotides in length and longer. Subsequent modifications c haracteristic of the SL RNA cap 4 were added successively in a 5' to 3' dir ection 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 f ully modified cap 4 structure was present on transcripts arrested at positi on 117 and beyond, Taken together, our results are consistent with a cotran scriptional mechanism for generating the cap 4 structure on the SL RNA.