Characterization of the Trypanosoma brucei cap hypermethylase Tgs1

Ruan, Jiapeng; Ullu, Elisabetta; Tschudi, Christian

doi:10.1016/j.molbiopara.2007.05.008

Cited by 19 publications

(38 citation statements)

References 22 publications

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“…TbMTAP and/or TbMTr1 may be recruited to the SLA1 snoRNA genomic locus as part of a cotranscriptional assembly pathway or join the preformed SLA1 snoRNP. T. brucei Nop10-GFP fusion protein (55) and TbNhp2-TAP tag immunolocalization (65) show accumulation in the nucleolus, consistent with H/ACA snoRNP proteins in other eukaryotic systems. The purification of the TbMTr1 on May 9, 2018 by guest http://mcb.asm.org/ complex in association with nucleolar RNP components required reexamination of TbMTr1 localization.…”

Section: Discussionsupporting

confidence: 70%

Trypanosoma bruceiSpliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex

Zamudio¹,

Mittra²,

Chattopadhyay

et al. 2009

Molecular and Cellular Biology

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Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2-O-ribose methylation of the first four nucleotides and pseudouridine () formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL 28 formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3-end maturation, and 28 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate.Mature RNA molecules contain extensive posttranscriptional nucleotide modifications of both base and ribose moieties. The isomerization of uracil to pseudouridine (5-ribosyluracil) and 2Ј-O-ribose methylation occurs by either site-specific (31, 50) or small nucleolar RNA (snoRNA)-guided enzymes (13). Conserved snoRNA-guided rRNA nucleotide modifications occur in functionally relevant regions (14), with their absence resulting in disease states due to defective catalytic RNA activity (76). Cap ribose methylations of RNA polymerase II transcripts by capspecific methyltransferases (MTases) are conserved in higher eukaryotes and implicated in the enhancement of translational efficiency (28a).Kinetoplastid protozoa, including human-pathogenic Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi, transcribe all protein-encoding genes polycistronically (28, 40). Maturation to translatable monocistronic units requires resolution of each coding region by trans splicing of a 39-nucleotide (nt) spliced leader (SL) exon and 3Ј-end polyadenylation. The SL RNA is involved in the maturation of each and every nuclear mRNA, accounting for approximately 7% of total RNA synthesis (8, 23). Rapid substrate SL consumption supports the argument for a dynamic processing mechanism. Substrate SL RNA is modified by eight methylations of the 5-nt cap structure and pseudouridylation at nt 28 ( 28 ). Along with those of the m 7 G (cap 0), the methylations of the kinetoplastid cap 4 are the most extensive, with 2Ј-O-ribose methylation of the first four nucleotides and additional base methylations on the first (m 2 6 A) and fourth (m 3 U) positions (5, 21, 49). The SL cap 4 and/or exon primary sequence and pseudouridylation have been implicated in kinetoplast...

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Section: Discussionsupporting

confidence: 70%

Trypanosoma bruceiSpliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex

Zamudio¹,

Mittra²,

Chattopadhyay

et al. 2009

Molecular and Cellular Biology

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“…Comparisons of Tgs1 family homologies indicate that hypermethylases from lower eukaryotes are mainly composed of the conserved MTase domain, whereas proteins from higher eukaryotes have long N-terminal domains with no obvious similarities to other known proteins (32). Concerning the former class, it has been shown that Tgs1 proteins from Saccharomyces cerevisiae and Trypanosoma brucei are essential for m 3 G cap formation of both snRNAs and snoRNAs (13,33), indicating that the absence of N-terminal regions does not restrict the hypermethylation capabilities of the enzymes. Biochemical studies also showed that S. pombe Tgs1 is a bona fide guanine-specific methyltransferase and that the chemical steps of m 3 G synthesis do not require input from RNA or protein cofactors in vitro (14).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Short Isoform of Human Tgs1 Hypermethylase Associating with Small Nucleolar Ribonucleoprotein Core Proteins and Produced by Limited Proteolytic Processing

Girard

Verheggen

Neel

et al. 2008

Journal of Biological Chemistry

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Tgs1 is the hypermethylase responsible for m 3 G cap formation of U small nuclear RNAs (U snRNAs) and small nucleolar RNAs (snoRNAs). In vertebrates, hypermethylation of snRNAs occurs in the cytoplasm, whereas this process takes place in the nucleus for snoRNAs. Accordingly, the hypermethylase is found in both compartments with a diffuse localization in the cytoplasm and a concentration in Cajal bodies in the nucleoplasm. In this study, we report that the Tgs1 hypermethylase exists as two species, a full-length cytoplasmic isoform and a shorter nuclear isoform of 65-70 kDa. The short isoform exhibits methyltransferase activity and associates with components of box C/D and H/ACA snoRNPs, pointing to a role of this isoform in hypermethylation of snoRNAs. We also show that production of the short Tgs1 isoform is inhibited by MG132, suggesting that it results from proteasomal limited processing of the full-length Tgs1 protein. Together, our results suggest that proteasome maturation constitutes a mechanism regulating Tgs1 function by generating Tgs1 species with different substrate specificities, subcellular localizations, and functions.

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“…Induction of expression and purification of the N-terminal His-tagged Tgs were carried out as described (25). Protein purity and concentration were evaluated by SDS–PAGE and densitometry using BSA standards and employing ImageJ software.…”

Section: Methodsmentioning

confidence: 99%

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

Simões-Barbosa

Louly

Franco

et al. 2008

Nucleic Acids Research

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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.

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Characterization of the Trypanosoma brucei cap hypermethylase Tgs1

Cited by 19 publications

References 22 publications

Trypanosoma bruceiSpliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex

Trypanosoma bruceiSpliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex

Characterization of a Short Isoform of Human Tgs1 Hypermethylase Associating with Small Nucleolar Ribonucleoprotein Core Proteins and Produced by Limited Proteolytic Processing

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

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