RAM is upregulated during T cell activation and is required for RNA cap formation and gene expression.

On T cell activation, upregulation of gene expression produces the protein required for the differentiation and proliferation of effector cell populations. RAM (RNMT-Activating Mini protein/RAMAC/Fam103a1), the cofactor of the RNA cap methyltransferase RNMT (RNA guanosine N-7 cap methyltransferase), is upregulated following activation. Formation of the RNA cap protects RNA during synthesis and guides RNA processing and translation. Using conditional gene deletion, we found that Ram expression stabilizes RNMT protein in T cells and is required for its upregulation on activation. When the Ram gene is deleted in naïve T cells, there are major impacts on activation-induced RNA cap formation and gene expression. Activated T cell proliferation is dependent on increased ribosome production; in Ram knockout T cells, activation-induced expression of ribosomal protein genes and snoRNAs is most severely reduced. Consistent with these changes, Ram deletion resulted in reduced protein synthesis, and reduced growth and proliferation of CD4 T cells. Deletion of Ram results in a similar but milder phenotype to Rnmt deletion, supporting the role of RAM as a RNMT cofactor.

The RNA cap methyltransferases RNMT and CMTR1 co-ordinate gene expression during neural differentiation.

Regulation of RNA cap formation has potent impacts on gene regulation, controlling which transcripts are expressed, processed and translated into protein. Recently, the RNA cap methyltransferases RNA guanine-7 methyltransferase (RNMT) and cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 1 (CMTR1) have been found to be independently regulated during embryonic stem (ES) cell differentiation controlling the expression of overlapping and distinct protein families. During neural differentiation, RNMT is repressed and CMTR1 is up-regulated. RNMT promotes expression of the pluripotency-associated gene products; repression of the RNMT complex (RNMT-RAM) is required for repression of these RNAs and proteins during differentiation. The predominant RNA targets of CMTR1 encode the histones and ribosomal proteins (RPs). CMTR1 up-regulation is required to maintain the expression of histones and RPs during differentiation and to maintain DNA replication, RNA translation and cell proliferation. Thus the co-ordinate regulation of RNMT and CMTR1 is required for different aspects of ES cell differentiation. In this review, we discuss the mechanisms by which RNMT and CMTR1 are independently regulated during ES cell differentiation and explore how this influences the co-ordinated gene regulation required of emerging cell lineages.

CMTR1 is recruited to transcription start sites and promotes ribosomal protein and histone gene expression in embryonic stem cells.

CMTR1 (cap methyltransferase 1) catalyses methylation of the first transcribed nucleotide of RNAPII transcripts (N1 2'-O-Me), creating part of the mammalian RNA cap structure. In addition to marking RNA as self, N1 2'-O-Me has ill-defined roles in RNA expression and translation. Here, we investigated the gene specificity of CMTR1 and its impact on RNA expression in embryonic stem cells. Using chromatin immunoprecipitation, CMTR1 was found to bind to transcription start sites (TSS) correlating with RNAPII levels, predominantly binding at histone genes and ribosomal protein (RP) genes. Repression of CMTR1 expression resulted in repression of RNAPII binding at the TSS and repression of RNA expression, particularly of histone and RP genes. In correlation with regulation of histones and RP genes, CMTR1 repression resulted in repression of translation and induction of DNA replication stress and damage. Indicating a direct role for CMTR1 in transcription, addition of recombinant CMTR1 to purified nuclei increased transcription of the histone and RP genes. CMTR1 was found to be upregulated during neural differentiation and there was an enhanced requirement for CMTR1 for gene expression and proliferation during this process. We highlight the distinct roles of the cap methyltransferases RNMT and CMTR1 in target gene expression and differentiation.

Identification and Characterization of the Interaction Between the Methyl-7-Guanosine Cap Maturation Enzyme RNMT and the Cap-Binding Protein eIF4E.

The control of RNA metabolism is an important aspect of molecular biology with wide-ranging impacts on cells. Central to processing of coding RNAs is the addition of the methyl-7 guanosine (m7G) "cap" on their 5' end. The eukaryotic translation initiation factor eIF4E directly binds the m7G cap and through this interaction plays key roles in many steps of RNA metabolism including nuclear RNA export and translation. eIF4E also stimulates capping of many transcripts through its ability to drive the production of the enzyme RNMT which methylates the G-cap to form the mature m7G cap. Here, we found that eIF4E also physically associated with RNMT in human cells. Moreover, eIF4E directly interacted with RNMT in vitro. eIF4E is only the second protein reported to directly bind the methyltransferase domain of RNMT, the first being its co-factor RAM. We combined high-resolution NMR methods with biochemical studies to define the binding interfaces for the RNMT-eIF4E complex. Further, we found that eIF4E competes for RAM binding to RNMT and conversely, RNMT competes for binding of well-established eIF4E-binding partners such as the 4E-BPs. RNMT uses novel structural means to engage eIF4E. Finally, we observed that m7G cap-eIF4E-RNMT trimeric complexes form, and thus RNMT-eIF4E complexes may be employed so that eIF4E captures newly capped RNA. In all, we show for the first time that the cap-binding protein eIF4E directly binds to the cap-maturation enzyme RNMT.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase.

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.

Upregulation of RNA cap methyltransferase RNMT drives ribosome biogenesis during T cell activation.

The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation.

A novel RNA pol II CTD interaction site on the mRNA capping enzyme is essential for its allosteric activation.

Recruitment of the mRNA capping enzyme (CE/RNGTT) to the site of transcription is essential for the formation of the 5' mRNA cap, which in turn ensures efficient transcription, splicing, polyadenylation, nuclear export and translation of mRNA in eukaryotic cells. The CE GTase is recruited and activated by the Serine-5 phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. Through the use of molecular dynamics simulations and enhanced sampling techniques, we provide a systematic and detailed characterization of the human CE-CTD interface, describing the effect of the CTD phosphorylation state, length and orientation on this interaction. Our computational analyses identify novel CTD interaction sites on the human CE GTase surface and quantify their relative contributions to CTD binding. We also identify, for the first time, allosteric connections between the CE GTase active site and the CTD binding sites, allowing us to propose a mechanism for allosteric activation. Through binding and activity assays we validate the novel CTD binding sites and show that the CDS2 site is essential for CE GTase activity stimulation. Comparison of the novel sites with cocrystal structures of the CE-CTD complex in different eukaryotic taxa reveals that this interface is considerably more conserved than previous structures have indicated.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

The eukaryotic translation initiation factor eIF4E elevates steady-state m7G capping of coding and noncoding transcripts.

Methyl-7-guanosine (m7G) "capping" of coding and some noncoding RNAs is critical for their maturation and subsequent activity. Here, we discovered that eukaryotic translation initiation factor 4E (eIF4E), itself a cap-binding protein, drives the expression of the capping machinery and increased capping efficiency of ∼100 coding and noncoding RNAs. To quantify this, we developed enzymatic (cap quantification; CapQ) and quantitative cap immunoprecipitation (CapIP) methods. The CapQ method has the further advantage that it captures information about capping status independent of the type of 5' cap, i.e., it is not restricted to informing on m7G caps. These methodological advances led to unanticipated revelations: 1) Many RNA populations are inefficiently capped at steady state (∼30 to 50%), and eIF4E overexpression increased this to ∼60 to 100%, depending on the RNA; 2) eIF4E physically associates with noncoding RNAs in the nucleus; and 3) approximately half of eIF4E-capping targets identified are noncoding RNAs. eIF4E's association with noncoding RNAs strongly positions it to act beyond translation. Coding and noncoding capping targets have activities that influence survival, cell morphology, and cell-to-cell interaction. Given that RNA export and translation machineries typically utilize capped RNA substrates, capping regulation provides means to titrate the protein-coding capacity of the transcriptome and, for noncoding RNAs, to regulate their activities. We also discovered a cap sensitivity element (CapSE) which conferred eIF4E-dependent capping sensitivity. Finally, we observed elevated capping for specific RNAs in high-eIF4E leukemia specimens, supporting a role for cap dysregulation in malignancy. In all, levels of capping RNAs can be regulated by eIF4E.

Direct High-Throughput Screening Assay for mRNA Cap Guanine-N7 Methyltransferase Activity.

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5' cap biosynthesis, involving RNA cap guanine-N7 methyltransferase (N7-MTase). N7-MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7-MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7-MTase activity assay based on small-molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3'-O position of adenosine acted as an artificial substrate with the properties of a turn-off probe for all three tested N7-MTases (human, parasite, and viral). Using this compound, a N7-MTase inhibitor assay adaptable to high-throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.

CAP-MAP: cap analysis protocol with minimal analyte processing, a rapid and sensitive approach to analysing mRNA cap structures.

Eukaryotic messenger RNA (mRNA) is modified by the addition of an inverted guanosine cap to the 5' triphosphate. The cap guanosine and initial transcribed nucleotides are further methylated by a series of cap methyltransferases to generate the mature cap structures which protect RNA from degradation and recruit proteins involved in RNA processing and translation. Research demonstrating that the cap methyltransferases are regulated has generated interest in determining the methylation status of the mRNA cap structures present in cells. Here, we present CAP-MAP: cap analysis protocol with minimal analyte processing, a rapid and sensitive method for detecting cap structures present in mRNA isolated from tissues or cultured cells.

Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM: insights from accelerated molecular dynamics simulations.

The RNA guanine-N7 methyltransferase (RNMT) in complex with RNMT-activating miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5' end of nascent RNA polymerase II transcripts. The mRNA cap protects the primary transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the network community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme's active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.

Oncogenic PIK3CA mutations increase dependency on the mRNA cap methyltransferase, RNMT, in breast cancer cells.

Basic mechanisms in gene expression are currently being investigated as targets in cancer therapeutics. One such fundamental process is the addition of the cap to pre-mRNA, which recruits mediators of mRNA processing and translation initiation. Maturation of the cap involves mRNA cap guanosine N-7 methylation, catalysed by RNMT (RNA guanine-7 methyltransferase). In a panel of breast cancer cell lines, we investigated whether all are equivalently dependent on RNMT for proliferation. When cellular RNMT activity was experimentally reduced by 50%, the proliferation rate of non-transformed mammary epithelial cells was unchanged, whereas a subset of breast cancer cell lines exhibited reduced proliferation and increased apoptosis. Most of the cell lines which exhibited enhanced dependency on RNMT harboured oncogenic mutations in PIK3CA, which encodes the p110α subunit of PI3Kα. Conversely, all cell lines insensitive to RNMT depletion expressed wild-type PIK3CA. Expression of oncogenic PIK3CA mutants, which increase PI3K p110α activity, was sufficient to increase dependency on RNMT. Conversely, inhibition of PI3Kα reversed dependency on RNMT, suggesting that PI3Kα signalling is required. Collectively, these findings provide evidence to support RNMT as a therapeutic target in breast cancer and suggest that therapies targeting RNMT would be most valuable in a PIK3CA mutant background.

CAPAM: The mRNA Cap Adenosine N6-Methyltransferase.

The mRNA cap is a structure that protects mRNA from degradation and recruits processing and translation factors. A new mRNA capping enzyme has been identified, PCIF1/CAPAM, which methylates adenosine when it is the first transcribed nucleotide. This discovery is crucial for understanding the function of cap adenosine methylation.

mRNA cap regulation in mammalian cell function and fate.

In this review we explore the regulation of mRNA cap formation and its impact on mammalian cells. The mRNA cap is a highly methylated modification of the 5' end of RNA pol II-transcribed RNA. It protects RNA from degradation, recruits complexes involved in RNA processing, export and translation initiation, and marks cellular mRNA as "self" to avoid recognition by the innate immune system. The mRNA cap can be viewed as a unique mark which selects RNA pol II transcripts for specific processing and translation. Over recent years, examples of regulation of mRNA cap formation have emerged, induced by oncogenes, developmental pathways and during the cell cycle. These signalling pathways regulate the rate and extent of mRNA cap formation, resulting in changes in gene expression, cell physiology and cell function.

DHX15 regulates CMTR1-dependent gene expression and cell proliferation.

CMTR1 contributes to mRNA cap formation by methylating the first transcribed nucleotide ribose at the O-2 position. mRNA cap O-2 methylation has roles in mRNA stabilisation and translation, and self-RNA tolerance in innate immunity. We report that CMTR1 is recruited to serine-5-phosphorylated RNA Pol II C-terminal domain, early in transcription. We isolated CMTR1 in a complex with DHX15, an RNA helicase functioning in splicing and ribosome biogenesis, and characterised it as a regulator of CMTR1. When DHX15 is bound, CMTR1 activity is repressed and the methyl-transferase does not bind to RNA pol II. Conversely, CMTR1 activates DHX15 helicase activity, which is likely to impact several nuclear functions. In HCC1806 breast carcinoma cell line, the DHX15-CMTR1 interaction controls ribosome loading of a subset of mRNAs and regulates cell proliferation. The impact of the CMTR1-DHX15 interaction is complex and will depend on the relative expression of these enzymes and their interactors, and the cellular dependency on different RNA processing pathways.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription.

Regulation and function of CMTR1-dependent mRNA cap methylation.

mRNA is modified co-transcriptionally at the 5' end by the addition of an inverted guanosine cap structure which can be methylated at several positions. The mRNA cap recruits proteins involved in gene expression and identifies the transcript as being cellular or 'self' in the innate immune response. Methylation of the first transcribed nucleotide on the ribose 2'-O position is a prevalent cap modification which has roles in splicing, translation and provides protection against the innate immune response. In this review, we discuss the regulation and function of CMTR1, the first transcribed nucleotide ribose 2'-O methyltransferase, and the molecular interactions which mediate methylated 2'-O ribose function. WIREs RNA 2017, 8:e1450. doi: 10.1002/wrna.1450 For further resources related to this article, please visit the WIREs website.

c-Myc co-ordinates mRNA cap methylation and ribosomal RNA production.

The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)-RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT-RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT-RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT-RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT-RAM is suppressed, indicating that RNMT-RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT-RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts.

Regulation of mRNA capping in the cell cycle.

The mRNA cap structure, which is added to nascent RNA pol II transcripts, recruits the protein complexes required for pre-mRNA transcript processing, mRNA export and translation initiation. The enzymes which catalyze mRNA cap synthesis are regulated by cellular signaling pathways which impact on their expression, localization and activity. Here we discuss the recent observation that the mRNA cap methyltransferase, RNMT, is phosphorylated on Thr-77 by CDK1-cyclin B1, which regulates its activity and the proteins with which it interacts. RNMT Thr-77 phosphorylation provides a burst of mRNA cap methyltransferase activity during early G1 phase at a time when transcription is reactivated following completion of the cell cycle. This co-ordination of transcription and mRNA capping makes an important contribution to gene expression in the cell; preventing RNMT Thr-77 phosphorylation inhibits cell proliferation. Here we discuss these findings and how mRNA cap synthesis may be regulated in other scenarios.