Mature mRNAs of Trypanosoma brucei possess a 5' cap acquired by discontinuous RNA synthesis

Abstract

Mature mRNAs of Trypanosoma brucei have a common 5' terminal sequence of 35 nucleotides. This is acquired by an unknown mechanism from the 5' end of a separately transcribed precursor RNA of about 140 nt called the mini-exon-derived RNA or medRNA. We have investigated the nature of the 5' ends of mature mRNAs and of the medRNA by chemical decapping and enzymic recapping. We infer that a 5' cap is present on both of these RNAs and conclude that the mini-exon-derived RNA donates its 5' cap along with the mini-exon sequence to the pre-mRNA. Using nuclear run-on experiments we show that medRNA synthesis is much more sensitive to alpha-amanitin than 5S RNA synthesis and only slightly less sensitive than tubulin gene transcription. This result, together with the presence of a cap at the 5' end of the medRNA indicates that the mini-exon is transcribed by an RNA polymerase II type enzyme. Our experiments also confirm the existence of a second minor medRNA of about 125 nt and show the presence of other small capped RNAs possibly analogous to the small nuclear RNAs of other organisms.