mRNA ageing shapes the Cap2 methylome in mammalian mRNA.

The mRNA cap structure is a major site of dynamic mRNA methylation. mRNA caps exist in either the Cap1 or Cap2 form, depending on the presence of 2'-O-methylation on the first transcribed nucleotide or both the first and second transcribed nucleotides, respectively1,2. However, the identity of Cap2-containing mRNAs and the function of Cap2 are unclear. Here we describe CLAM-Cap-seq, a method for transcriptome-wide mapping and quantification of Cap2. We find that unlike other epitranscriptomic modifications, Cap2 can occur on all mRNAs. Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol. As a result, Cap2 is enriched on long-lived mRNAs. Large increases in the abundance of Cap1 leads to activation of RIG-I, especially in conditions in which expression of RIG-I is increased. The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind to and activate RIG-I. The slow methylation rate of Cap2 allows Cap2 to accumulate on host mRNAs, yet ensures that low levels of Cap2 occur on newly expressed viral RNAs. Overall, these results reveal an immunostimulatory role for Cap1, and that Cap2 functions to reduce activation of the innate immune response.

Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines.

The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS-CoV-2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chemically modified mRNA cap analogs contain a unique cap structure, m7 G[5']ppp[5']N (where N=G, A, C or U), present at the 5'-end of many eukaryotic cellular and viral RNAs and several non-coding RNAs. The chemical modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap analog containing N2 modification, dinucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addition, the biological applications of these novel cap analogs are delineated.

2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography.

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

High-resolution structures of the SARS-CoV-2 2'-O-methyltransferase reveal strategies for structure-based inhibitor design.

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.

Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.

A molecular dynamics simulation study decodes the Zika virus NS5 methyltransferase bound to SAH and RNA analogue.

Since 2015, widespread Zika virus outbreaks in Central and South America have caused increases in microcephaly cases, and this acute problem requires urgent attention. We employed molecular dynamics and Gaussian accelerated molecular dynamics techniques to investigate the structure of Zika NS5 protein with S-adenosyl-L-homocysteine (SAH) and an RNA analogue, namely 7-methylguanosine 5'-triphosphate (m7GTP). For the binding motif of Zika virus NS5 protein and SAH, we suggest that the four Zika NS5 substructures (residue orders: 101-112, 54-86, 127-136 and 146-161) and the residues (Ser56, Gly81, Arg84, Trp87, Thr104, Gly106, Gly107, His110, Asp146, Ile147, and Gly148) might be responsible for the selectivity of the new Zika virus drugs. For the binding motif of Zika NS5 protein and m7GTP, we suggest that the three Zika NS5 substructures (residue orders: 11-31, 146-161 and 207-218) and the residues (Asn17, Phe24, Lys28, Lys29, Ser150, Arg213, and Ser215) might be responsible for the selectivity of the new Zika virus drugs.

Hydrolytic activity of human Nudt16 enzyme on dinucleotide cap analogs and short capped oligonucleotides.

Human Nudt16 (hNudt16) is a member of the Nudix family of hydrolases, comprising enzymes catabolizing various substrates including canonical (d)NTPs, oxidized (d)NTPs, nonnucleoside polyphosphates, and capped mRNAs. Decapping activity of the Xenopus laevis (X29) Nudt16 homolog was observed in the nucleolus, with a high specificity toward U8 snoRNA. Subsequent studies have reported cytoplasmic localization of mammalian Nudt16 with cap hydrolysis activity initiating RNA turnover, similar to Dcp2. The present study focuses on hNudt16 and its hydrolytic activity toward dinucleotide cap analogs and short capped oligonucleotides. We performed a screening assay for potential dinucleotide and oligonucleotide substrates for hNudt16. Our data indicate that dinucleotide cap analogs and capped oligonucleotides containing guanine base in the first transcribed nucleotide are more susceptible to enzymatic digestion by hNudt16 than their counterparts containing adenine. Furthermore, unmethylated dinucleotides (GpppG and ApppG) and respective oligonucleotides (GpppG-16nt and GpppA-16nt) were hydrolyzed by hNudt16 with greater efficiency than were m7GpppG and m7GpppG-16nt. In conclusion, we found that hNudt16 hydrolysis of dinucleotide cap analogs and short capped oligonucleotides displayed a broader spectrum specificity than is currently known.

mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation.

Analogues of the mRNA 5'-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly different ligand conformation compared to that of the unmodified cap analogue, which likely contributes to the improved binding. Both CCl2- and CF2- analogues showed lower susceptibility to hydrolysis by the decapping scavenger enzyme (DcpS) and, when incorporated into RNA, conferred stability against major cellular decapping enzyme (Dcp2) to transcripts. Furthermore, the use of difluoromethylene cap analogues was exemplified by the development of 19F NMR assays for DcpS activity and eIF4E binding.

Diverse cap-binding properties of Drosophila eIF4E isoforms.

The majority of eukaryotic mRNAs are translated in a cap-dependent manner, which requires recognition of the mRNA 5' cap by eIF4E protein. Multiple eIF4E family members have been identified in most eukaryotic organisms. Drosophila melanogaster (Dm) has eight eIF4E related proteins; seven of them belong to Class I and one to Class II. Their biological roles with the exception of Dm eIF4E-1, Dm eIF4E-3 and Dm 4EHP, remain unknown. Here, we compare the molecular basis of Dm eIF4E's interactions with cap and eIF4G peptide by using homology modelling and fluorescence binding assays with various cap analogues. We found that despite the presence of conserved key residues responsible for cap recognition, the differences in binding different cap analogues among Class I Dm eIF4E isoforms are up to 14-fold. The highest affinity for cap analogues was observed for Dm eIF4E-3. We suggest that Dm eIF4E-3 and Dm eIF4E-5 bind the second nucleoside of the cap in an unusual manner via stacking interactions with a histidine or a phenylalanine residue, respectively. Moreover, the analysis of ternary complexes of eIF4G peptide-eIF4E-cap analogue showed cooperativity between eIF4G and cap binding only for Dm eIF4E-4, which exhibits the lowest affinity for cap analogues among all Dm eIF4Es.

Elimination of cap structures generated by mRNA decay involves the new scavenger mRNA decapping enzyme Aph1/FHIT together with DcpS.

Eukaryotic 5' mRNA cap structures participate to the post-transcriptional control of gene expression before being released by the two main mRNA decay pathways. In the 3'-5' pathway, the exosome generates free cap dinucleotides (m7GpppN) or capped oligoribonucleotides that are hydrolyzed by the Scavenger Decapping Enzyme (DcpS) forming m7GMP. In the 5'-3' pathway, the decapping enzyme Dcp2 generates m7GDP. We investigated the fate of m7GDP and m7GpppN produced by RNA decay in extracts and cells. This defined a pathway involving DcpS, NTPs and the nucleoside diphosphate kinase for m7GDP elimination. Interestingly, we identified and characterized in vitro and in vivo a new scavenger decapping enzyme involved in m7GpppN degradation. We show that activities mediating cap elimination identified in yeast are essentially conserved in human. Their alteration may contribute to pathologies, possibly through the interference of cap (di)nucleotide with cellular function.

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes.

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, ß- or γ-position of the 5',5'-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m2 (7,3'-O)GpppG. Higher expression of cancer antigens would make mRNAs containing m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2 favorable for anticancer immunization.

Construction of plasmid, bacterial expression, purification, and assay of dengue virus type 2 NS5 methyltransferase.

Dengue virus (DENV), a member of mosquito-borne flavivirus, causes self-limiting dengue fever as well as life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its positive sense RNA genome has a cap at the 5'-end and no poly(A) tail at the 3'-end. The viral RNA encodes a single polyprotein, C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5. The polyprotein is processed into 3 structural proteins (C, prM, and E) and 7 nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). NS3 and NS5 are multifunctional enzymes performing various tasks in viral life cycle. The N-terminal domain of NS5 has distinct GTP and S-adenosylmethionine (SAM) binding sites. The role of GTP binding site is implicated in guanylyltransferase (GTase) activity of NS5. The SAM binding site is involved in both N-7 and 2'-O-methyltransferase (MTase) activities involved in formation of type I cap. The C-terminal domain of NS5 catalyzes RNA-dependent RNA polymerase (RdRp) activity involved in RNA synthesis. We describe the construction of the MTase domain of NS5 in an E. coli expression vector, purification of the enzyme, and conditions for enzymatic assays of N7- and 2'O-methyltransferase activities that yield the final type I 5'-capped RNA ((7Me)GpppA2'OMe-RNA).

Potential therapeutic applications of RNA cap analogs.

Cap analogs are chemically modified derivatives of the unique cap structure present at the 5´ end of all eukaryotic mRNAs and several non-coding RNAs. Until recently, cap analogs have served primarily as tools in the study of RNA metabolism. Continuing advances in our understanding of cap biological functions (including RNA stabilization, pre-mRNA splicing, initiation of mRNA translation, as well as cellular transport of mRNAs and snRNAs) and the consequences of the disruption of these processes - resulting in serious medical disorders - have opened new possibilities for pharmaceutical applications of these compounds. In this review, the medicinal potential of cap analogs in areas, such as cancer treatment (including eIF4E targeting and mRNA-based immunotherapy), spinal muscular atrophy treatment, antiviral therapy and the improvement of the localization of nucleus-targeting drugs, are highlighted. Advances achieved to date, challenges, plausible solutions and prospects for the future development of cap analog-based drug design are described.

Solution-based approach to study binding to the eIF4E cap-binding site using CD spectroscopy.

The eukaryotic initiation factor 4E (eIF4E) is the key component of the translational initiation complex that recruits mRNA by binding to a unique "cap" structure located at the 5' end of the mRNA. Overexpression of eIF4E has been implicated in the development of cancer, potentially as a result of increasing the cellular levels of proteins involved in processes that include proliferation and regulation of apoptosis. As a result, the cap-binding site of eIF4E has become a target for the development of anti-cancer therapeutics. The structure of eIF4E bound to the cap mimic 7-methyl-GDP revealed that two tryptophans from different loops in eIF4E sandwiched the 7-methylguanine group between them. This interaction gives rise to a strong exciton coupling signal between the two tryptophans that can be visualized by CD spectroscopy. eIF4E is a challenging protein to work with because of a propensity to aggregate under conditions used in biophysical techniques. CD spectroscopy provides a gentle, solution-based approach to study binding to the cap-binding site of eIF4E. Evidence is provided that the exciton coupling signal can be used to both qualitatively and quantitatively analyze the binding of cap analogs to eIF4E.

Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis.

Insulin-like growth factor binding protein (IGFBP)-3 regulates cell proliferation and apoptosis in esophageal squamous cell carcinoma (ESCC) cells. We have investigated how the hypoxic tumor microenvironment in ESCC fosters the induction of IGFBP3. RNA interference experiments revealed that hypoxia-inducible factor (HIF)-1α, but not HIF-2α, regulates IGFBP3 mRNA induction. By chromatin immunoprecipitation and transfection assays, HIF-1α was found to transactivate IGFBP3 through a novel hypoxia responsive element (HRE) located at 57 kb upstream from the transcription start site. Metabolic labeling experiments demonstrated hypoxia-mediated inhibition of global protein synthesis. 7-Methyl GTP-cap binding assays suggested that hypoxia suppresses cap-dependent translation. Experiments using pharmacological inhibitors for mammalian target of rapamycin (mTOR) suggested that a relatively weak mTOR activity may be sufficient for cap-dependent translation of IGFBP3 under hypoxic conditions. Bicistronic RNA reporter transfection assays did not validate the possibility of an internal ribosome entry site as a potential mechanism for cap-independent translation for IGFBP3 mRNA. Finally, IGFBP3 mRNA was found enriched to the polysomes. In aggregate, our study establishes IGFBP3 as a direct HIF-1α target gene and that polysome enrichment of IGFBP3 mRNA may permit continuous translation under hypoxic conditions.

Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex.

The 5'-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5'-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2'-O positions, catalyzed by nsp14 N7-MTase and nsp16 2'-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2'-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.

Structural requirements for Caenorhabditis elegans DcpS substrates based on fluorescence and HPLC enzyme kinetic studies.

The activity of the Caenorhabditis elegans scavenger decapping enzyme (DcpS) on its natural substrates and dinucleotide cap analogs, modified with regard to the nucleoside base or ribose moiety, has been examined. All tested dinucleotides were specifically cleaved between beta- and gamma-phosphate groups in the triphosphate chain. The kinetic parameters of enzymatic hydrolysis (K(m), V(max)) were determined using fluorescence and HPLC methods, as complementary approaches for the kinetic studies of C. elegans DcpS. From the kinetic data, we determined which parts of the cap structure are crucial for DcpS binding and hydrolysis. We showed that m(3)(2,2,7)GpppG and m(3)(2,2,7)GpppA are cleaved with higher rates than their monomethylated counterparts. However, the higher specificity of C. elegans DcpS for monomethylguanosine caps is illustrated by the lower K(m) values. Modifications of the first transcribed nucleotide did not affect the activity, regardless of the type of purine base. Our findings suggest C. elegans DcpS flexibility in the first transcribed nucleoside-binding pocket. Moreover, although C. elegans DcpS accommodates bulkier groups in the N7 position (ethyl or benzyl) of the cap, both 2'-O- and 3'-O-methylations of 7-methylguanosine result in a reduction in hydrolysis by two orders of magnitude.

Plakophilin 1 stimulates translation by promoting eIF4A1 activity.

Plakophilins 1-3 (PKP1-3) are desmosomal proteins of the p120(ctn) family of armadillo-related proteins that are essential for organizing the desmosomal plaque. Recent findings identified PKPs in stress granules, suggesting an association with the translational machinery. However, a role of PKPs in controlling translation remained elusive so far. In this study, we show a direct association of PKP1 with the eukaryotic translation initiation factor 4A1 (eIF4A1). PKP1 stimulated eIF4A1-dependent translation via messenger RNA cap and encephalomyocarditis virus internal ribosomal entry site (IRES) structures, whereas eIF4A1-independent translation via hepatitis C virus IRES was not affected. PKP1 copurified with eIF4A1 in the cap complex, and its overexpression stimulated eIF4A1 recruitment into cap-binding complexes. At the molecular level, PKP1 directly promoted eIF4A1 adenosine triphosphatase activity. The stimulation of translation upon PKP1 overexpression correlated with the up-regulation of proliferation and cell size. In conclusion, these findings identify PKP1 as a regulator of translation and proliferation via modulation of eIF4A1 activity and suggest that PKP1 controls cell growth in physiological and pathological conditions.

Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities.

The Tgs proteins are structurally homologous AdoMet-dependent eukaryal enzymes that methylate the N2 atom of 7-methyl guanosine nucleotides. They have an imputed role in the synthesis of the 2,2,7-trimethylguanosine (TMG) RNA cap. Here we exploit a collection of cap-like substrates to probe the repertoire of three exemplary Tgs enzymes, from mammalian, protozoan, and viral sources, respectively. We find that human Tgs (hTgs1) is a bona fide TMG synthase adept at two separable transmethylation steps: (1) conversion of m(7)G to m(2,7)G, and (2) conversion of m(2,7)G to m(2,2,7)G. hTgs1 is unable to methylate G or m(2)G, signifying that both steps require an m(7)G cap. hTgs1 utilizes a broad range of m(7)G nucleotides, including mono-, di-, tri-, and tetraphosphate derivatives as well as cap dinucleotides with triphosphate or tetraphosphate bridges. In contrast, Giardia lamblia Tgs (GlaTgs2) exemplifies a different clade of guanine-N2 methyltransferase that synthesizes only a dimethylguanosine (DMG) cap structure and cannot per se convert DMG to TMG under any conditions tested. Methylation of benzyl(7)G and ethyl(7)G nucleotides by hTgs1 and GlaTgs2 underscored the importance of guanine N7 alkylation in providing a key pi-cation interaction in the methyl acceptor site. Mimivirus Tgs (MimiTgs) shares with the Giardia homolog the ability to catalyze only a single round of methyl addition at guanine-N2, but is distinguished by its capacity for guanine-N2 methylation in the absence of prior N7 methylation. The relaxed cap specificity of MimiTgs is revealed at alkaline pH. Our findings highlight both stark and subtle differences in acceptor specificity and reaction outcomes among Tgs family members.

Structural basis for m7G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1.

The 5'-cap of spliceosomal small nuclear RNAs, some small nucleolar RNAs and of telomerase RNA was found to be hypermethylated in vivo. The Trimethylguanosine Synthase 1 (TGS1) mediates this conversion of the 7-methylguanosine-cap to the 2,2,7-trimethylguanosine (m(3)G)-cap during maturation of the RNPs. For mammalian UsnRNAs the generated m(2,2,7)G-cap is one part of a bipartite import signal mediating the transport of the UsnRNP-core complex into the nucleus. In order to understand the structural organization of human TGS1 as well as substrate binding and recognition we solved the crystal structure of the active TGS1 methyltransferase domain containing both, the minimal substrate m(7)GTP and the reaction product S-adenosyl-L-homocysteine (AdoHcy). The methyltransferase of human TGS1 harbors the canonical class 1 methyltransferase fold as well as an unique N-terminal, alpha-helical domain of 40 amino acids, which is essential for m(7)G-cap binding and catalysis. The crystal structure of the substrate bound methyltransferase domain as well as mutagenesis studies provide insight into the catalytic mechanism of TGS1.