Characterization of the Trypanosoma brucei cap hypermethylase Tgs1.

Ribonucleoprotein particles (RNPs) containing either U1, U2, U4/U6, and U5 snRNAs reside in the nucleoplasm and are essential for the splicing of pre-mRNA. Mammalian U-snRNAs, with the exception of U6, are synthesized by RNA polymerase (pol) II and initially contain a 5'-terminal 7-monomethylguanosine (m7G) cap. Following export to the cytoplasm, they bind the Sm core proteins at the highly conserved Sm binding site [5'-PuA(U)nGPu-3'], which is an obligatory process for the hypermethylation of the m7G cap to a trimethylguanosine (TMG) cap structure [1,2]. Subsequent nuclear import of the U-snRNPs is dependent on signals provided by both the Sm core complex and the TMG cap [3,4]. Hypermethylation of the m7G cap is also known to occur on a subset of small nucleolar RNAs (snoRNAs), including U3, which is required for the maturation of pre-rRNA [5]. However, snoRNAs do not associate with Sm proteins, but form an RNP with a specific set of proteins. Early studies in vertebrates have shown that biogenesis of snoRNPs does not involve a cytoplasmic phase, leading to the hypothesis that hypermethylation can take place in the nucleus in the absence of Sm core proteins [6,7]. The above synopsis raises the question whether TMG cap formation of snRNAs and snoRNAs is catalyzed by the same or different enzyme(s). Mouaikel et al. [8] provided the answer by a genetic characterization of the yeast trimethylguanosine synthase Tgs1p, a protein required for hypermethylation of both U-snRNAs and snoRNAs. A Tgs1 deletion was viable, but produced a cold-sensitive phenotype. This protein is conserved throughout eukaryotic evolution, with Tgs1 in lower eukaryotes consisting mainly of the catalytic domain, while in higher eukaryotes Tgs1 possesses in addition a large N-terminal domain of unknown function [8]. In yeast, Tgs1 localized to the nucleolus [8], whereas the mammalian protein was detected both in Cajal bodies and in the cytoplasm [9,10]. Subsequent biochemical studies with recombinant fission yeast Tgs1 validated the genetic experiments and showed that this protein is indeed the catalyst of guanosine hypermethylation [11]. Similar to other eukaryotes, the U1, U2 and U4 snRNAs and the U3 snoRNA in the protist Trypanosoma brucei contain a 2,2,7-trimethyl guanosine cap [12]. The 5' end of the U5 snRNA appears to lack a TMG or m7G cap structure and most likely has a 5' phosphate end [12]. One distinctive feature of T. brucei U-snRNAs is that the genes are transcribed by pol III [12]. Although T. brucei U-snRNAs do not have a highly conserved sequence motif comparable to the Sm site, the snRNPs share a common set of Sm proteins that are organized in the classical heptamer ring [13] and more recent experiments provided evidence for a variation in the Sm core of the snRNPs, specifically in the U2 snRNP [14,15]. Surprisingly, in vitro reconstitution experiments revealed that the Sm-analogous region was not essential for assembly of the U2 core RNP [16,17]. Consistent with these observations, the analysis of mutant U2 snRNAs in vivo showed that binding of the common proteins is not dependent on the region analogous to the Sm site and that binding of common proteins is not a pre-requisite for TMG cap formation [18]. Given that little is known about the maturation pathway of U-snRNAs and snoRNAs in trypanosomatids, we initiated a characterization of the Tgs1 ortholog for T. brucei. The T. brucei Tgs1 protein (TbTgs1) was initially identified by Mouaikel et al. [8] as a partial sequence of 159 amino acids. Current database searches reveal proteins of 251 amino acids in T. brucei (GenDB accession # Tb11.02.5090), of 250 amino acids in T. cruzi (GeneDB accession # Tc00.1047053510381.60) and 248 amino acids in T. vivax (GeneDB accession # Tviv852c01.q1k_0). Of note is that we did not recognize a Tgs1 homologue in any of the Leishmania databases. The trypanosome proteins are mainly composed of the conserved catalytic domain and lack a large N-terminal domain present in higher eukaryotes (not shown). Overall, T. brucei Tgs1 exhibits 35% identity and 48% similarity with its yeast counterpart and the T. brucei protein is 40% identical and 57% similar to the catalytic domain of the mammalian Tgs1. Furthermore, residues shown to be important for function of the S. cerevisiae protein by site-directed mutagenesis [8,19] are conserved in the trypanosome proteins (not shown). To begin an investigation of the function of TbTgs1, we down-regulated expression of the protein by RNA interference (RNAi) using a hairpin-type construct under the control of a tetracycline (tet)-inducible promoter. tet-induced TbTgs1-RNAi resulted in a significant decline of mRNA abundance after 24 h (data not shown). However, compared to wild-type and uninduced cells, downregulation of TbTgs1 mRNA had no noticeable effect on cell viability. In fact, maintaining the induction for over 10 days did not reveal any detectable change in growth characteristics. Attempts to generate a TbTgs1 knock-out cell line, similar to the S. cerevisiae reports [8], were not successful, suggesting that the T. brucei cap hypermethylase might be encoded by an essential gene. Next, we determined whether TbTgs1 downregulation had any effect on hypermethylation of snRNAs and snoRNAs. Cells were induced with tet for 1 and 8 days, RNA was prepared and assayed by immunoprecipitation with anti-TMG antibodies followed by primer extension analysis (Fig. 1A). To control for immunoprecipitation specificity, the 7SL RNA, which lacks a TMG cap, was also monitored by primer extension analysis. As expected, the majority of U2 snRNA and U3 snoRNA were affinity selected by anti-TMG antibodies from RNA prepared from uninduced cells, whereas the 7SL RNA was not detected in the immunoprecipitate (Fig. 1A, lane 4). A similar result was obtained with RNA isolated from 1 day-induced cells (lane 7). However, following 8 days of induction, 42% of U2 snRNA and 34% of U3 snoRNA were recovered in the supernatant (lane 9), suggesting that TbTgs1 is involved in hypermethylation of the snRNA cap. Fig. 1 RNAi-induced downregulation of Tgs1. To generate the TbTgs1 RNAi cell line, the 756 bp of the T. brucei Tgs1 coding region were assembled as two inverted repeats separated by a stuffer fragment, inserted downstream of a tetracycline- (tet-) inducible ... At present, we do not know whether TMG formation in trypanosomes follows the eukaryotic paradigm, i.e. the RNA is first capped with an m7G moiety and subsequently converted to TMG. One prediction of this pathway is that the observed reduction of the TMG cap structure on the snRNAs should result in a concomitant increase in the accumulation of m7G-capped RNAs. To test this possibility, we performed immunoprecipitations with anti-m7G antibodies (Fig. 1B). As expected, U2 snRNA and U3 snoRNA present in RNA isolated from uninduced cells were not recovered in the immunoprecipitate (Fig. 1B, lane 4). After 1 day of induction, a small amount of U3 snRNA was detectable in the pellet fraction (lane 7), and following 8 days of Tgs1 downregulation, 23% of U2 snRNA and 50% of U3 snoRNA were immunoprecipitated with anti-m7G antibodies (lane 10). These results were in agreement with Tgs1 playing a role in the conversion of m7G cap to TMG cap. To determine the cellular localization of Tgs1, we generated a fusion with the green fluorescent protein (gfp) and expressed this construct transiently in cultured procyclic trypanosomes (Fig. 2). Tgs1-gfp fluorescence was observed throughout the nucleoplasm and in addition it was found concentrated in one or two dots outside the nucleolus (panels A-C), identified as the area of the nucleus that stains poorly with Hoechst and where the nucleolar protein Nop10-gfp localizes (panel D). Nop10 is a core protein of H/ACA snoRNPs [20]. In contrast, the fluorescence of gfp itself was equally distributed in the cytoplasm and the nucleus of the cell (data not shown, but see Fig. 1B in ref. [21]). A light and mostly diffuse staining of the cytoplasm was also observed in Tgs1-gfp cells. However, it remains to be determined whether or not this cytoplasmic staining is the result of Tgs1-gfp overexpression. Fig. 2 Tgs1 predominantly localizes to the nucleus. Fluorescence analysis of Tgs1-gfp (panels A-C) and Nop10-gfp (panel D). T. brucei procyclics were transiently transfected with appropriate constructs made in the plasmid vector pXSGFPM3FUS [21] expressing Tgs1-gfp ... In conclusion, the data presented here provide a first insight in the machinery responsible for TMG cap biogenesis in trypanosomatids. Similar to studies in yeast [8], T. brucei Tgs1 appears to be responsible for hypermethylation of the cap of both snRNAs and snoRNAs.