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Trichomonas vaginalis; snoRNA; capping; trimethylguanosine synthase; pseudouridine
Abstract:
The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential
guanine-N2 methylations of m7G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal
snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m2,7G
in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m7G, m2,7G, and m2,2,7G caps.
Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m2,7G and
m2,2,7G caps. Expression of TvTgs in yeast tgs1D cells leads to the formation of m2,7G and m2,2,7G caps and complementation of
the lethality of a tgs1D mud2D strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing
RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified
16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA
snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA.
