The TbMTr1 Spliced Leader RNA Cap 1 2 ′-O-Ribose Methyltransferase from Trypanosoma brucei Acts with Substrate Specificity

Mittra, Bidyottam; Zamudio, Jesse R.; Bujnicki, Janusz M.; Stępiński, Janusz; Darżynkiewicz, Edward; Campbell, David A.; Sturm, Nancy R.

doi:10.1074/jbc.m707367200

Cited by 21 publications

(24 citation statements)

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“…The methyltransferase activity of hMTr1 was observed whether the mRNA was capped with a GpppG or m 7 GpppG moiety and is consistent with similar experiments from previous studies using partially purified cap1 activity from HeLa extracts (23). TbMTr1, the T. brucei homolog of hMTr1, also has no preference for terminal guanosine N7 methylation of the cap structures (18).…”

Section: Discussionmentioning

confidence: 99%

Characterization of hMTr1, a Human Cap1 2′-O-Ribose Methyltransferase*

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Stępiński

Darżynkiewicz

et al. 2010

Journal of Biological Chemistry

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Cellular eukaryotic mRNAs are capped at their 5 ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo.Eukaryotic cytoplasmic mRNAs are capped at their 5Ј end with a 7-methylguanosine (m 7 GpppN, where N is any nucleotide) (1). This cap is important for mRNA stability and for efficient translation initiation (for reviews, see Refs. 2, 3). The capping of mRNAs is a co-transcriptional process that requires RNA triphosphatase, RNA guanylyltransferase, and RNA methyltransferase activities (4, 5). In higher eukaryotes, the RNA triphosphatase and RNA guanylyltransferase reactions are carried out by a single bifunctional protein, whereas the methyltransferase reaction depends on a separate enzyme. The 5Ј cap structure in yeast has no additional modification and is referred to as cap0 (6). However, in higher eukaryotes, the cap structure contains 2Ј-O-ribose methylations at either the first nucleotide (named cap1) or the first and second nucleotide (cap2) (7, 8) (Fig. 1A). When the first nucleotide of the transcript is an adenosine, the base can also be methylated at the N6 position (9). Viruses that replicate in the cytoplasm, such as vesicular stomatitis virus (10), coronavirus (11), and West Nile virus (12), often encode their own capping enzymes as well as a cap 2Ј-O-ribose methyltransferase activity.The kinetoplastids such as Trypanosoma brucei are a special case. Their mRNAs are capped with a spliced leader sequence containing a total of seven methylations, including 2Ј-O-ribose methylations on the first four nucleotides (cap4) (13-15). The T. brucei 2Ј-O-ribose methyltransferases responsible for methylation of the first three positions have been identified and characterized (16 -20). Knock out of the enzymes required for methylating nucleotides 2 and 3 was shown to decrease translation efficiency by 50% (21). In higher eukaryotes, cap ribose methylation was reported to improve translation during Xenopus oocyte maturation (22).Although the existence of cap1 and cap2 have been known for Ͼ30 years, their role in mRNA biogenesis remains obscure, and the enzymes involved in their formation have not yet been identified in mammals. Cap1 and cap2 methyltransferase activity have been partially purif...

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Section: Discussionmentioning

confidence: 99%

Characterization of hMTr1, a Human Cap1 2′-O-Ribose Methyltransferase*

Bart

Stępiński

Darżynkiewicz

et al. 2010

Journal of Biological Chemistry

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155

142

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“…CBF5 silencing induces trans-splicing defects at the first step of splicing. Interestingly, the modification of cap 4 was perturbed in the CBF5-silenced cells (3), suggesting that SLA1 may function as a chaperone to maintain the proper secondary structure of SL RNA, essential for recognition by the methyltransferases (1,22,28,44) that mediate RNA cap 4 synthesis. SL RNA is transcribed by RNA polymerase II in a very special nuclear compartment near the nucleolus described for Trypanosoma cruzi (7).…”

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confidence: 99%

Trypanosome Spliced-Leader-Associated RNA (SLA1) Localization and Implications for Spliced-Leader RNA Biogenesis

et al. 2009

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Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position ؊12 (relative to the 5 splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function in trans splicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeable Trypanosoma brucei cells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. In T. brucei cells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.H/ACA RNA is a group of small nucleolar RNAs that direct pseudouridylation on rRNA and snRNAs. In almost all eukaryotes, these RNAs consist of two stem-loop structures connected by a single-stranded hinge and a tail region carrying the conserved H (AnAnnA) and ACA boxes. Two short motifs of the snoRNA base pair with the target site flanking the uridine to be isomerized (9, 25). In trypanosomes, these guide RNAs have a unique structure, composed of a single stem-loop, and carry an AGA instead of an ACA box (15, 16). All of the H/ACA molecules identified so far in trypanosomes can guide rRNA pseudouridylation; however, molecules that guide analogous modifications on U snRNAs (U1 to U5) have not been identified to date. In higher eukaryotes, the guide RNAs that direct such modifications are localized in special subnuclear domains, the Cajal bodies (6). In trypanosomes, all mRNAs are processed by trans splicing. During the trans-splicing reaction, a common spliced-leader (SL) sequence of 39 nucleotides (nt) is added to the pre-mRNA from a small RNA, the SL RNA. A novel snoRNA, termed SLA1 (SL-associated RNA), in Trypanosoma brucei was initially discovered by virtue of its efficient cross-linking with the bifunctional reagent aminomethyltrimethyl (AMT) to the SL RNA (39). It was originally proposed that SLA1 is the trypanosome U5 sn...

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“…Several SL-specific RNA modifications, including the responsible enzymatic activities, have been biochemically characterized: the unique cap4 modifications at the 5' end, 3'-end processing and an internal pseudouridine modification at U28, guided by the spliced-leader-associated (SLA1) RNA. [24][25][26][27][28][29][30][31] Some of these enzymes and modification reactions have been localized: The cap4 modifications are introduced cotranscriptionally in the nucleus and the SLA1-guided internal pseudouridylation of the SL RNA is also nuclear. 32,33 The multistep process of 3'-end formation involves both initial cytoplasmic and final nuclear 3' trimming.…”

Section: Resultsmentioning

confidence: 99%