Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite

Multi-Substrate Specificity and the Evolutionary Basis for Interdependence in tRNA Editing and Methylation Enzymes

Among tRNA modification enzymes there is a correlation between specificity for multiple tRNA substrates and heteromultimerization. In general, enzymes that modify a conserved residue in different tRNA sequences adopt a heterodimeric structure. Presumably, such changes in the oligomeric state of enzymes, to gain multi-substrate recognition, are driven by the need to accommodate and catalyze a particular reaction in different substrates while maintaining high specificity. This review focuses on two classes of enzymes where the case for multimerization as a way to diversify molecular recognition can be made. We will highlight several new themes with tRNA methyltransferases and will also discuss recent findings with tRNA editing deaminases. These topics will be discussed in the context of several mechanisms by which heterodimerization may have been achieved during evolution and how these mechanisms might impact modifications in different systems

The divergent eukaryote Trichomonas vaginalis has an m7G cap methyltransferase capable of a single N2 methylation

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