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1.  West Nile Virus 5′-Cap Structure Is Formed by Sequential Guanine N-7 and Ribose 2′-O Methylations by Nonstructural Protein 5 
Journal of Virology  2006;80(17):8362-8370.
Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5′-cap structure that is methylated at the guanine N-7 and the ribose 2′-OH positions of the first transcribed nucleotide, adenine (m7GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2′-O methylations and therefore is essential for flavivirus 5′-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m7GpppA-capped RNAs to m7GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m7GpppA- and m7GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2′-O methylation. Mutagenesis of a K61-D146-K182-E218 tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2′-O MTase activities. The entire K61-D146-K182-E218 motif is essential for 2′-O MTase activity, whereas N-7 MTase activity requires only D146. The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K61-D146-K182-E218 motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.
PMCID: PMC1563844  PMID: 16912287
2.  Trypanosoma brucei Spliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex ▿ ‡  
Molecular and Cellular Biology  2008;29(5):1202-1211.
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 (ψ) 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 ψ28 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 ψ28 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.
PMCID: PMC2643836  PMID: 19103757
3.  A Metazoan/Plant-like Capping Enzyme and Cap Modified Nucleotides in the Unicellular Eukaryote Trichomonas vaginalis 
PLoS Pathogens  2010;6(7):e1000999.
The cap structure of eukaryotic messenger RNAs is initially elaborated through three enzymatic reactions: hydrolysis of the 5′-triphosphate, transfer of guanosine through a 5′-5′ triphosphate linkage and N7-methylation of the guanine cap. Three distinctive enzymes catalyze each reaction in various microbial eukaryotes, whereas the first two enzymes are fused into a single polypeptide in metazoans and plants. In addition to the guanosine cap, adjacent nucleotides are 2′-O-ribose methylated in metazoa and plants, but not in yeast. Analyses of various cap structures have suggested a linear phylogenetic trend of complexity. These findings have led to a model in which plants and metazoa evolved a two-component capping apparatus and modification of adjacent nucleotides while many microbial eukaryotes maintained the three-component system and did not develop modification of adjacent nucleotides. Here, we have characterized a bifunctional capping enzyme in the divergent microbial eukaryote Trichomonas vaginalis using biochemical and phylogenetic analyses. This unicellular parasite was found to harbor a metazoan/plant-like capping apparatus that is represented by a two-domain polypeptide containing a C-terminus guanylyltransferase and a cysteinyl phosphatase triphosphatase, distinct from its counterpart in other microbial eukaryotes. In addition, T. vaginalis mRNAs contain a cap 1 structure represented by m7GpppAmpUp or m7GpppCmpUp; a feature typical of metazoan and plant mRNAs but absent in yeast mRNAs. Phylogenetic and biochemical analyses of the origin of the T. vaginalis capping enzyme suggests a complex evolutionary model where differential gene loss and/or acquisition occurred in the development of the RNA capping apparatus and cap modified nucleotides during eukaryote diversification.
Author Summary
The protozoan parasite Trichomonas vaginalis is the cause of the most common non-viral sexually transmitted disease worldwide. Evolutionary analyses place Trichomonas in a super group called the Excavata, which includes the kinetoplastids and is highly divergent from fungi, metazoa and plants. Despite the vast evolutionary distances that separate these different eukaryotic lineages, a simplified view of eukaryotic evolution based on the complexity of nucleotide modifications at the 5′ end of mRNAs and the distribution of different types of enzymatic apparatus that confer these modifications has been proposed. Our analyses of the T. vaginalis capping enzyme challenges this view and provides the first example of a two-component capping apparatus typically found in metazoa and plants in a protozoan. The 5′-end nucleotide structure of T. vaginalis mRNAs is also shown to contain additional modified nucleotides, similar to that observed for metazoan and plant mRNAs and unlike that found in most eukaryotic microbes and fungi. Evolutionary analyses of the T. vaginalis capping enzyme indicates that this multicellular type capping apparatus may have come into existence earlier than previously thought.
PMCID: PMC2904801  PMID: 20664792
4.  Distinct RNA Elements Confer Specificity to Flavivirus RNA Cap Methylation Events▿  
Journal of Virology  2007;81(9):4412-4421.
The 5′ end of the flavivirus plus-sense RNA genome contains a type 1 cap (m7GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5′ terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2′-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5′ stem-loop structure; in contrast, 2′-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5′ viral RNA of 20 nucleotides. The cap analogues GpppA and m7GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m7Gppp-terminated RNAs suggest that the 5′ termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.
PMCID: PMC1900168  PMID: 17301144
5.  C1D and hMtr4p associate with the human exosome subunit PM/Scl-100 and are involved in pre-rRNA processing 
Nucleic Acids Research  2007;35(8):2564-2572.
The exosome is a complex of 3′–5′ exoribonucleases and RNA-binding proteins, which is involved in processing or degradation of different classes of RNA. Previously, the characterization of purified exosome complexes from yeast and human cells suggested that C1D and KIAA0052/hMtr4p are associated with the exosome and thus might regulate its functional activities. Subcellular localization experiments demonstrated that C1D and KIAA0052/hMtr4p co-localize with exosome subunit PM/Scl-100 in the nucleoli of HEp-2 cells. Additionally, the nucleolar accumulation of C1D appeared to be dependent on PM/Scl-100. Protein–protein interaction studies showed that C1D binds to PM/Scl-100, whereas KIAA0052/hMtr4p was found to interact with MPP6, a previously identified exosome-associated protein. Moreover, we demonstrate that C1D, MPP6 and PM/Scl-100 form a stable trimeric complex in vitro. Knock-down of C1D, MPP6 and KIAA0052/hMtr4p by RNAi resulted in the accumulation of 3′-extended 5.8S rRNA precursors, showing that these proteins are required for rRNA processing. Interestingly, C1D appeared to contain RNA-binding activity with a potential preference for structured RNAs. Taken together, our results are consistent with a role for the exosome-associated proteins C1D, MPP6 and KIAA052/hMtr4p in the recruitment of the exosome to pre-rRNA to mediate the 3′ end processing of the 5.8S rRNA.
PMCID: PMC1885644  PMID: 17412707
6.  The 2′-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA in Trypanosoma brucei▿ †  
Molecular and Cellular Biology  2007;27(17):6084-6092.
mRNA cap 1 2′-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2′-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2′-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2′-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2′-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3′-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.
PMCID: PMC1952150  PMID: 17606627
7.  Enzymology of RNA cap synthesis 
The 5′ guanine-N7 methyl cap is unique to cellular and viral messenger RNA (mRNA) and is the first co-transcriptional modification of mRNA. The mRNA cap plays a pivotal role in mRNA biogenesis and stability, and is essential for efficient splicing, mRNA export, and translation. Capping occurs by a series of three enzymatic reactions that results in formation of N7-methyl guanosine linked through a 5′-5′ inverted triphosphate bridge to the first nucleotide of a nascent transcript. Capping of cellular mRNA occurs co-transcriptionally and in vivo requires that the capping apparatus be physically associated with the RNA polymerase II elongation complex. Certain capped mRNAs undergo further methylation to generate distinct cap structures. Although mRNA capping is conserved among viruses and eukaryotes, some viruses have adopted strategies for capping mRNA that are distinct from the cellular mRNA capping pathway.
PMCID: PMC3962952  PMID: 21956912
8.  Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1- and 5′ppp- mRNAs 
Nucleic Acids Research  2013;42(5):3228-3245.
Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.
PMCID: PMC3950709  PMID: 24371270
9.  RAM function is dependent on Kapβ2-mediated nuclear entry 
Biochemical Journal  2014;457(Pt 3):473-484.
Eukaryotic gene expression is dependent on the modification of the first transcribed nucleotide of pre-mRNA by the addition of the 7-methylguanosine cap. The cap protects transcripts from exonucleases and recruits complexes which mediate transcription elongation, processing and translation initiation. The cap is synthesized by a series of reactions which link 7-methylguanosine to the first transcribed nucleotide via a 5′ to 5′ triphosphate bridge. In mammals, cap synthesis is catalysed by the sequential action of RNGTT (RNA guanylyltransferase and 5′-phosphatase) and RNMT (RNA guanine-7 methyltransferase), enzymes recruited to RNA pol II (polymerase II) during the early stages of transcription. We recently discovered that the mammalian cap methyltransferase is a heterodimer consisting of RNMT and the RNMT-activating subunit RAM (RNMT-activating mini-protein). RAM activates and stabilizes RNMT and thus is critical for cellular cap methylation and cell viability. In the present study we report that RNMT interacts with the N-terminal 45 amino acids of RAM, a domain necessary and sufficient for maximal RNMT activation. In contrast, smaller components of this RAM domain are sufficient to stabilize RNMT. RAM functions in the nucleus and we report that nuclear import of RAM is dependent on PY nuclear localization signals and Kapβ2 (karyopherin β2) nuclear transport protein.
7-Methylguanosine cap formation in mammals is catalysed by RNGTT and RNMT-RAM. RAM activates the cap methyltransferase, RNMT. We define the domains of RAM required for RNMT activation and demonstrate that nuclear localization is dependent on Kapβ2 interaction with RAM PY-NLSs.
PMCID: PMC3898117  PMID: 24200467
capping; karyopherin β2 (Kapβ2); 7-methylguanosine; mRNA cap methylation; RNA guanine-7 methyltransferase (RNMT); RNMT-activating mini-protein (RAM); HA, haemagglutinin; HEK, human embryonic kidney; IF, immunofluorescence; Kapβ2, karyopherin β2; NLS, nuclear localization signal; pol II, polymerase II; RAD, RNMT-activation domain; RAM, RNMT-activating mini-protein; RNGTT, RNA guanylyltransferase and 5′-phosphatase; RNMT, RNA guanine-7 methyltransferase; WT, wild-type
10.  Hypermethylated cap 4 maximizes Trypanosoma brucei translation 
Molecular microbiology  2009;72(5):1100-1110.
Through trans-splicing of a 39-nt Spliced Leader (SL) onto each protein-coding transcript, mature kinetoplastid mRNA acquire a hypermethylated 5′-cap structure, but its function has been unclear. Gene deletions for three Trypanosoma brucei cap 2′-O-ribose methyltransferases, TbMTr1, TbMTr2, and TbMTr3, reveal distinct roles for four 2′-O-methylated nucleotides. Elimination of individual gene pairs yields viable cells, however attempts at double knockouts resulted in the generation of a TbMTr2−/−/TbMTr3−/− cell line only. Absence of both kinetoplastid-specific enzymes in TbMTr2−/−/TbMTr3−/− lines yielded substrate SL RNA and mRNA with cap 1. TbMTr1−/− translation is comparable to wildtype, while cap 3 and cap 4 loss reduced translation rates, exacerbated by the additional loss of cap 2. TbMTr1−/− and TbMTr2−/−/TbMTr3−/− lines grow to lower densities under normal culture conditions relative to wildtype cells, with growth rate differences apparent under low serum conditions. Cell viability may not tolerate delays at both the nucleolar Sm-independent and nucleoplasmic Sm-dependent stages of SL RNA maturation combined with reduced rates of translation. A minimal level of mRNA cap ribose methylation is essential for trypanosome viability, providing the first functional role for the cap 4.
PMCID: PMC2859698  PMID: 19504740
gene knockout; methyltransferase; ribose 2′-O-methylation; SL RNA; spliced leader; trans-splicing
11.  Structural analysis of human 2′-O-ribose methyltransferases involved in mRNA cap structure formation 
Nature Communications  2014;5:3004.
The 5′ cap of human messenger RNA contains 2′-O-methylation of the first and often second transcribed nucleotide that is important for its processing, translation and stability. Human enzymes that methylate these nucleotides, termed CMTr1 and CMTr2, respectively, have recently been identified. However, the structures of these enzymes and their mechanisms of action remain unknown. In the present study, we solve the crystal structures of the active CMTr1 catalytic domain in complex with a methyl group donor and a capped oligoribonucleotide, thereby revealing the mechanism of specific recognition of capped RNA. This mechanism differs significantly from viral enzymes, thus providing a framework for their specific targeting. Based on the crystal structure of CMTr1, a comparative model of the CMTr2 catalytic domain is generated. This model, together with mutational analysis, leads to the identification of residues involved in RNA and methyl group donor binding.
Human mRNA transcripts possess a 5' cap structure that is modified by methylation. Here, Smietanski et al. present the structures of human methyltransferases responsible for this reaction, revealing key differences to their viral counterparts and thereby providing a framework for targeted drug design.
PMCID: PMC3941023  PMID: 24402442
12.  Reassignment of specificities of two cap methyltransferase domains in the reovirus lambda2 protein 
Genome Biology  2001;2(9):research0038.1-research0038.6.
The reovirus λ2 protein catalyzes mRNA capping, that is, addition of a guanosine to the 5' end of each transcript in a 5'-to-5' orientation, as well as transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the N7 atom of the added guanosyl moiety and subsequently to the ribose 2'-O atom of the first template-encoded nucleotide. The structure of the human reovirus core has been solved at 3.6 Å resolution, revealing a series of domains that include a putative guanylyltransferase domain and two putative methyltransferase (MTase) domains. It has been suggested that the order of domains in the λ2 protein corresponds to the order of reactions in the pathway and that the m7G (cap 0) and the 2'-O-ribose (cap 1) MTase activities may be exerted by the MTase 1 and the MTase 2 domains, respectively.
We show that the reovirus MTase 1 domain shares a putative active site with the structurally characterized 2'-O-ribose MTases, including vaccinia virus cap 1 MTase, whereas the MTase 2 domain is structurally similar to glycine N-MTase.
On the basis of our analysis of the structural details we propose that the previously suggested functional assignments of the MTase 1 and MTase 2 domains should be swapped.
PMCID: PMC56899  PMID: 11574057
13.  Functional characterization of a 48 kDa Trypanosoma brucei cap 2 RNA methyltransferase 
Nucleic Acids Research  2006;34(19):5594-5602.
Kinetoplastid mRNAs possess a unique hypermethylated cap 4 structure derived from the standard m7GpppN cap structure, with 2′-O methylations on the first four ribose sugars and additional base methylations on the first adenine and the fourth uracil. While the enzymes responsible for m7GpppN cap 0 formations has been characterized in Trypanosoma brucei, the mechanism of cap 4 methylation and the role of the hypermethylated structure remain unclear. Here, we describe the characterization of a 48 kDa T.brucei 2′-O nucleoside methyltransferase (TbCom1). Recombinant TbCom1 transfers the methyl group from S-adenosylmethionine (AdoMet) to the 2′-OH of the second nucleoside of m7GpppNpNp-RNA to form m7GpppNpNmp-RNA. TbCom1 is also capable of converting cap 1 RNA to cap 2 RNA. The methyl transfer reaction is dependent on the m7GpppN cap, as the enzyme does not form a stable interaction with GpppN-terminated RNA. Mutational analysis establishes that the TbCom1 and vaccinia virus VP39 methyltransferases share mechanistic similarities in AdoMet- and cap-recognition. Two aromatic residues, Tyr18 and Tyr187, may participate in base-stacking interactions with the guanine ring of the cap, as the removal of each of these aromatic side-chains abolishes cap-specific RNA-binding.
PMCID: PMC1636459  PMID: 17028101
14.  Complete Cap 4 Formation Is Not Required for Viability in Trypanosoma brucei†  
Eukaryotic Cell  2006;5(6):905-915.
In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5′ ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m7G cap, ribose 2′-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2′-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A2 and is not required for subsequent steps; TbMT511 methylates C3, without which U4 methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m7G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A2, C3, and U4 methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.
PMCID: PMC1489268  PMID: 16757738
15.  Sequestration by IFIT1 Impairs Translation of 2′O-unmethylated Capped RNA 
PLoS Pathogens  2013;9(10):e1003663.
Viruses that generate capped RNA lacking 2′O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2′O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2′O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2′O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2′O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells.
Author Summary
Cellular messenger RNAs of higher eukaryotes are capped with a methylated guanine and, in addition, methylated at the 2′O position of the first ribose. Viruses unable to methylate their RNA at the 2′O position of the cap and viruses generating uncapped RNA with 5′ triphosphate groups are inhibited by an antiviral complex of different IFIT proteins. How IFIT proteins restrict viruses lacking 2′O methylation at the RNA cap remained unclear. We used a mass spectrometry-based approach to identify proteins binding to capped RNA with different methylation states. We found that IFIT1 directly binds to capped RNA and that this binding was dependent on the methylation state of the cap. Having identified IFIT1 as being central for recognition of 2′O-unmethylated viral RNA we further examined the mode of action of IFITs in vitro and in vivo. Our experiments clearly show that the antiviral mechanism of IFIT1 is based on sequestration of viral RNA lacking cap 2′O methylation, thereby selectively preventing translation of viral RNA. Our data establish IFIT1 as a general sensor for RNA 5′ end structures and provide an important missing link in our understanding of the antiviral activity of IFIT proteins.
PMCID: PMC3789756  PMID: 24098121
16.  Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo. 
Molecular and Cellular Biology  1992;12(11):4844-4851.
The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.
PMCID: PMC360417  PMID: 1406666
17.  Ribose 2′-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein▿  
Journal of Virology  2009;83(21):11043-11050.
During conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently at the ribose 2′-O position. For vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses, the two methylase activities share a binding site for the methyl donor S-adenosyl-l-methionine and are inhibited by individual amino acid substitutions within the C-terminal domain of the large (L) polymerase protein. This led to the suggestion that a single methylase domain functions for both 2′-O and G-N-7 methylations. Here we report a trans-methylation assay that recapitulates both ribose 2′-O and G-N-7 modifications by using purified recombinant L and in vitro-synthesized RNA. Using this assay, we demonstrate that VSV L typically modifies the 2′-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2′-O methylation of the substrate RNA. Amino acid substitutions in the C terminus of L that prevent all cap methylation in recombinant VSV (rVSV) partially retain the ability to G-N-7 methylate a pre-2′-O-methylated RNA, therefore uncoupling the effect of substitutions in the C terminus of the L protein on the two methylations. In addition, we show that the 2′-O and G-N-7 methylase activities act specifically on RNA substrates that contain the conserved elements of a VSV mRNA start at the 5′ terminus. This study provides new mechanistic insights into the mRNA cap methylase activities of VSV L, demonstrates that 2′-O methylation precedes and facilitates subsequent G-N-7 methylation, and reveals an RNA sequence and length requirement for the two methylase activities. We propose a model of regulation of the activity of the C terminus of L protein in 2′-O and G-N-7 methylation of the cap structure.
PMCID: PMC2772757  PMID: 19710136
18.  RNA methylation in vaccinia virus-infected chick embryo fibroblasts treated with homologous interferon. 
Nucleic Acids Research  1978;5(7):2441-2454.
Interferon-pretreatment of vaccinia-infected chick embryo fibroblasts resulted in a greater than 50% decrease in ribose methylation of the penultimate "cap" nucleotide in virus-specific mRNA. However, in contrast to results obtained with cell-free systems, in intact infected cells there was (a) no detectable reduction in methylation of the 5'-ultimate m7G of viral mRNA; (b) a virus specificity of the interferon-induced change in mRNA "CAP"-methylation seems unlikely and (c) analysis of the ribosomal and transfer RNA fractions isolated from interferon-treated and control cells revealed identical patterns of methylated nucleotides. Thus, the interferon-induced change in methylation is specific for mRNA "CAPS".
PMCID: PMC342175  PMID: 673858
19.  Specific inhibition of capped mRNA translation in vitro by m7G5'pppp5'G and m7G5'pppp5'm7G. 
Nucleic Acids Research  1979;6(11):3613-3624.
A unique set of diguanosine cap analogues containing a 5'-5' tetraphosphate linkage instead of the normal triphosphate was synthesized by chemical methylation of G5'pppp5'G. Both 7-methylguanosine products, m7G5'pppp5'G and m7G5'pppp5'm7G, acted as potent inhibitors of capped brome mosaic virus (BMV) RNA translation in the homologous wheat germ protein synthesis system. Inhibition of in vitro protein synthesis required the presence of the 7-methyl group on guanosine and was specific for capped mRNA. In comparison with the partial cap analogue, m7GTP, the methylated diguanosine tetraphosphate structures were 25-50 fold more potent inhibitors of in vitro protein synthesis. Analysis of the in vitro translation products of the four species of BMV RNA showed a differential sensitivity to inhibition by m7G5'pppp5'm7G.
PMCID: PMC327960  PMID: 493116
20.  SnoRNA-guided ribose methylation of rRNA: structural features of the guide RNA duplex influencing the extent of the reaction. 
Nucleic Acids Research  1998;26(7):1576-1587.
Eukaryotic rRNAs contain a large number of ribose-methylated nucleotides of elusive function which are confined to the universally conserved rRNA domains. Ribose methylation of these nucleotides is directed by a large family of small trans -acting guide RNAs, called box C/D antisense snoRNAs. Each snoRNA targets precisely one of the nucleotides to be methylated within the pre-rRNA sequence, through transient formation of a 10-21 bp regular RNA duplex around the modification site. In this study we have analyzed how different features of the double-stranded RNA guide structure affect the extent of site-specific ribose methylation, by co-expressing an appropriate RNA substrate and its cognate tailored snoRNA guide in transfected mouse cells. We show that an increased GC content of the duplex can make up for the inhibitory effects of a helix truncation or for the presence of helix irregularities such as a mismatched pair or a bulge nucleotide. However, some helix irregularities dramatically inhibit the reaction and are not offset by further stabilization of the duplex. Overall, the RNA duplex tolerates a much larger degree of irregularity than anticipated, even in the immediate vicinity of the methylation site, which offers new prospects in the search for additional snoRNA guides. Accordingly, a few snoRNA-like sequences of uncertain status detected in the yeast Saccharomyces cerevisiae genome now appear as likely bona fide ribose methylation guides.
PMCID: PMC147472  PMID: 9512526
21.  Enzymatic Synthesis of RNAs Capped with Nucleotide Analogues Reveals the Molecular Basis for Substrate Selectivity of RNA Capping Enzyme: Impacts on RNA Metabolism 
PLoS ONE  2013;8(9):e75310.
RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5’ ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Parameciumbursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.
PMCID: PMC3783409  PMID: 24086504
22.  Regulation of mRNA cap methylation 
Biochemical Journal  2009;425(Pt 2):295-302.
The 7-methylguanosine cap added to the 5′ end of mRNA is essential for efficient gene expression and cell viability. Methylation of the guanosine cap is necessary for the translation of most cellular mRNAs in all eukaryotic organisms in which it has been investigated. In some experimental systems, cap methylation has also been demonstrated to promote transcription, splicing, polyadenylation and nuclear export of mRNA. The present review discusses how the 7-methylguanosine cap is synthesized by cellular enzymes, the impact that the 7-methylguanosine cap has on biological processes, and how the mRNA cap methylation reaction is regulated.
PMCID: PMC2825737  PMID: 20025612
gene regulation; mRNA cap methylation; translation; transcription; CBC, cap-binding complex; CBP, cap-binding protein; CDK, cyclin-dependent kinase; CE, capping enzyme; CTD, C-terminal domain; DSIF, DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity inducing factor; eIF, eukaryotic initiation factor; NELF, negative elongation factor; P-TEFb, positive transcription elongation factor b; RNGTT, RNA guanylyltransferase and 5′ triphosphatase; RNMT, RNA (guanine-7-) methyltransferase; SAH, S-adenosylhomocysteine; SAHH, SAH hydrolase; SAM, S-adenosylmethionine; TFIIH, transcription factor IIH; UTR, untranslated region; xSAHH, Xenopus laevis SAHH
23.  Methylation status of 13S ribosomal RNA from hamster mitochondria: the presence of a novel riboside, N4-methylcytidine. 
Nucleic Acids Research  1978;5(11):4385-4397.
The ribosomal RNA ("13S" RNA) of the small ribosomal subunit of hamster cell mitochondria has been found to have a distinctive pattern of methylated residues. Each molecule contained, on the average, approximately one residue of m4Cp, m5Cp and m5Up, and two residues of m62Ap. The natural occurrence of m4Cp has not previously been reported; we propose that this nucleotide is homologous to its ribose-methylated congener, m4Cmp, which is characteristic of bacterial 16S ribosomal RNA. We detected neither m4Cp nor m4Cmp in the hamster cell cytoplasmic ribosomal RNA. This is the first documentation of a modified residue present in mitochondrial RNA but absent from the cytoplasmic RNA of the same cells.
PMCID: PMC342756  PMID: 724519
24.  Identification of 10 novel snoRNA gene clusters from Arabidopsis thaliana 
Nucleic Acids Research  2001;29(7):1623-1630.
Ten novel small nucleolar RNA (snoRNA) gene clusters, consisting of two or three snoRNA genes, respectively, were identified from Arabidopsis thaliana. Twelve of the 25 snoRNA genes in these clusters are homologous to those of yeast and mammals according to the conserved antisense sequences that guide 2′-O-ribose methylation of rRNA. The remaining 13 snoRNA genes, including two 5.8S rRNA methylation guides, are new genes identified from A.thaliana. Interestingly, seven methylated nucleotides, predicted by novel snoRNAs Z41a–Z46, are methylated neither in yeast nor in vertebrates. Using primer extension at low dNTP concentration the six methylation sites were determined as expected. These snoRNAs were recognized as specific guides for 2′-O-ribose methylation of plant rRNAs. Z42, however, did not guide the expected methylation of 25S rRNA in our assay. Thus, its function remains to be elucidated. The intergenic spacers of the gene clusters are rich in uridine (up to 40%) and most of them range in size from 35 to 100 nt. Lack of a conserved promoter element in each spacer and the determination of polycistronic transcription from a cluster by RT–PCR assay suggest that the snoRNAs encoded in the clusters are transcribed as a polycistron under an upstream promoter, and individual snoRNAs are released after processing of the precursor. Numerous snoRNA gene clusters identified from A.thaliana and other organisms suggest that the snoRNA gene cluster is an ancient gene organization existing abundantly in plants.
PMCID: PMC31268  PMID: 11266566
25.  RAM/Fam103a1 Is Required for mRNA Cap Methylation 
Molecular Cell  2011;44(4):585-596.
The 7-methylguanosine cap added to the 5′ end of mRNA is required for efficient gene expression in eukaryotes. In mammals, methylation of the guanosine cap is catalyzed by RNMT (RNA guanine-7 methyltransferase), an enzyme previously thought to function as a monomer. We have identified an obligate component of the mammalian cap methyltransferase, RAM (RNMT-Activating Mini protein)/Fam103a1, a previously uncharacterized protein. RAM consists of an N-terminal RNMT-activating domain and a C-terminal RNA-binding domain. As monomers RNMT and RAM have a relatively weak affinity for RNA; however, together their RNA affinity is significantly increased. RAM is required for efficient cap methylation in vitro and in vivo, and is indirectly required to maintain mRNA expression levels, for mRNA translation and for cell viability. Our findings demonstrate that RAM is an essential component of the core gene expression machinery.
Graphical Abstract
► RAM/Fam103a1 is a component of the human cap methyltransferase complex ► RAM is required for efficient cap methylation in vitro and in vivo ► RAM activates RNMT (cap methyltransferase), and increases RNMT affinity for RNA ► RAM is indirectly required to maintain mRNA levels and for mRNA translation
PMCID: PMC3235549  PMID: 22099306

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