DISCONTINUOUSLY SYNTHESIZED MESSENGER-RNA FROM TRYPANOSOMA-BRUCEI CONTAINS THE HIGHLY METHYLATED 5' CAP STRUCTURE, M7GPPPA-STAR-A-STAR-C(2'-O)MU-STAR-A

Abstract

Trypanosoma brucei mRNA is discontinuously synthesized via the 5'' addition of a "mini-exon" sequence. The mini-exon-specific cap structure was purified from a complete RNase T2 and phosphatase digest of in vivo 32P-labeled poly(A)+RNA. The purified cap structure was sequenced by a series of partial and complete enzymatic digests by nuclease P1 and venom phosphodiesterase. This approach demonstrated that the T. brucei mini-exon cap structure consists of N7-methylguanosine linked in a conventional 5''-5'' triphosphate bond to five nucleotides, in the sequence A*A*C(2''-O)mU*A (asterisks denote modifications that were not fully characterized in this work). 2''-O-methylations and other modifications appear to be present in this novel cap structure, which could have a functional role in the metabolism of the mini-exon.