Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides

Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) miniexon is placed at the 5′ end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5′-5′) triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m⁷G(5′)ppp(5′)m₂⁶AmpAmpCmpm ³Ump. The presence of N⁶,N⁶,2′-O-trimethyladenosine and 3,2′-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.