The divergent eukaryote Trichomonas vaginalis has an m 7 G cap methyltransferase capable of a single N2 methylation

Abstract

Eukaryotic RNAs typically contain 5′ cap structures that have been primarily studied in yeast and metazoa. The only known RNA cap structure in unicellular protists is the unusual Cap4 on Trypanosoma brucei mRNAs. We have found that T. vaginalis mRNAs are protected by a 5′ cap structure, however, contrary to that typical for eukaryotes, T. vaginalis spliceosomal snRNAs lack a cap and may contain 5′ monophophates. The distinctive 2,2,7-trimethylguanosine (TMG) cap structure usually found on snRNAs and snoRNAs is produced by hypermethylation of an m7G cap catalyzed by the enzyme trimethylguanosine synthase (Tgs). Here, we biochemically characterize the single T. vaginalis Tgs (TvTgs) encoded in its genome and demonstrate that TvTgs exhibits substrate specificity and amino acid requirements typical of an RNA cap-specific, m7G-dependent N2 methyltransferase. However, recombinant TvTgs is capable of catalysing only a single round of N2 methylation forming a 2,7-dimethylguanosine cap (DMG) as observed previously for Giardia lamblia. In contrast, recombinant Entamoeba histolytica and Trypanosoma brucei Tgs are capable of catalysing the formation of a TMG cap. These data suggest the presence of RNAs with a distinctive 5′ DMG cap in Trichomonas and Giardia lineages that are absent in other protist lineages.

Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis

“…Atypically, but consistent with the apparent inessentiality of TMG caps in fungi, we recently observed that the spliceosomal snRNAs of the divergent unicellular eukaryote Trichomonas vaginalis lack a guanosine cap structure, though these snRNAs are transcribed by RNA Pol II, and T. vaginalis encodes both the enzymatic machinery for m 7 G capping and a homolog (called TvTgs) of yeast and human Tgs1 6 (Simoes- Barbosa et al 2008aBarbosa et al ,b, 2010. This scenario raises interesting questions about the existence and location of hypermethylated caps in Trichomonas as well as the function of TvTgs1.…”

“…The observation that Tgs enzymes from Trichomonas, Giardia, and mimivirus (Hausmann and Shuman 2005a;Simoes-Barbosa et al 2008a;Benarroch et al 2009) produce only DMG in vitro suggests the possibility that DMG cap structures are present in protozoal or viral RNAs and are potentially biologically relevant. We examined this hypothesis by characterizing the cap structures of endogenous RNAs.…”

“…It is notable that the enzymes from G. lamblia, T. vaginalis, and mimivirus are limited in vitro to only a single round of methyl transfer to a m 7 G cap substrate forming a 2,7-dimethylguanosine (DMG) cap product. These studies raised the prospect that Giardia, Trichomonas, and mimivirus might elaborate DMG-capped RNAs (Hausmann and Shuman 2005a;Simoes-Barbosa et al 2008a;Benarroch et al 2009). …”

Mutational analyses of trimethylguanosine synthase (Tgs1) and Mud2: Proteins implicated in pre-mRNA splicing

“…Indeed, we find here that the F804A mutant is defective in converting DMG to TMG (6% of wild-type activity). Phe804 is conserved in Schizosaccharomyces, Entamoeba, and Trypanosoma Tgs1 proteins, each of which has biochemically documented TMG synthase activity (Hausmann and Shuman 2005a;Simoes-Barbosa et al 2008), but is substituted by a lysine in Giardia Tgs2 and MimiTgs, which are DMG-only synthases (Hausmann and Shuman 2005b;Benarroch et al 2010). A simple speculation is that the hydrophobic-to-charged substitution might disrupt the putative m 2,7 G substrate docking site for the second transmethylation reaction.…”

“…Mutational studies of several Tgs enzymes identified a suite of essential amino acid side chains that were predicted to form the binding pockets for the AdoMet donor and m 7 G acceptor (Mouaikel et al 2003;Hausmann and Shuman 2005b;Hausmann et al 2007Hausmann et al , 2008Simoes-Barbosa et al 2008;Benarroch et al 2009). Most of the substrate interactions inferred from mutational studies and modeling exercises were confirmed by the crystal structure of human Tgs1 (hTgs1) in a ternary complex with m to the triphosphate moiety of m 7 GTP ( Fig.…”