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) mini-exon 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')m6(2)AmpAmpCmpm3Ump. 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.
Department of Microbiology and Immunology, Stanford School of Medicine, California 94305.
J D
JD
Bangs
P F
PF
Crain
T
T
Hashizume
J A
JA
McCloskey
J C
JC
Boothroyd
D000818:Animals; D002852:Chromatography, Ion Exchange; D016831:Crithidia fasciculata / Q000235:genetics*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D011061:Poly A / Q000378:metabolism; D012315:RNA Caps / Q000737:chemistry* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases; D012263:Ribonucleosides / Q000032:analysis* / Q000737:chemistry; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D012315:RNA Caps; D012333:RNA, Messenger; D012263:Ribonucleosides; D011061:Poly A; D012260:Ribonucleases