CDK1-Cyclin B1 Activates RNMT, Coordinating mRNA Cap Methylation with G1 Phase Transcription.

The creation of translation-competent mRNA is dependent on RNA polymerase II transcripts being modified by addition of the 7-methylguanosine (m7G) cap. The factors that mediate splicing, nuclear export, and translation initiation are recruited to the transcript via the cap. The cap structure is formed by several activities and completed by RNMT (RNA guanine-7 methyltransferase), which catalyzes N7 methylation of the cap guanosine. We report that CDK1-cyclin B1 phosphorylates the RNMT regulatory domain on T77 during G2/M phase of the cell cycle. RNMT T77 phosphorylation activates the enzyme both directly and indirectly by inhibiting interaction with KPNA2, an RNMT inhibitor. RNMT T77 phosphorylation results in elevated m7G cap methyltransferase activity at the beginning of G1 phase, coordinating mRNA capping with the burst of transcription that occurs following nuclear envelope reformation. RNMT T77 phosphorylation is required for the production of cohort of proteins, and inhibiting T77 phosphorylation reduces the cell proliferation rate.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Transcription-independent functions of MYC: regulation of translation and DNA replication.

MYC is a potent oncogene that drives unrestrained cell growth and proliferation. Shortly after its discovery as an oncogene, the MYC protein was recognized as a sequence-specific transcription factor. Since that time, MYC oncogene research has focused on the mechanism of MYC-induced transcription and on the identification of MYC transcriptional target genes. Recently, MYC was shown to control protein expression through mRNA translation and to directly regulate DNA replication, thus initiating exciting new areas of oncogene research.

Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes.

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses.

RAM/Fam103a1 is required for mRNA cap methylation.

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.

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.

Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM.

Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.

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.

Myc and mRNA capping.

c-Myc is upregulated in response to growth factors and transmits the signal to proliferate by altering the gene expression landscape. When genetic alterations result in growth factor-independent c-Myc expression, it can become an oncogene. The majority of human tumour types exhibit a degree of c-Myc deregulation, resulting in unrestrained cell proliferation. c-Myc binds proximal to the promoter region of genes and recruits co-factors including histone acetyltransferases and RNA pol II kinases, which promote transcription. c-Myc also promotes formation of the cap structure at the 5' end of mRNA. The cap is 7-methylguanosine linked to the first transcribed nucleotide of RNA pol II transcripts via a 5' to 5' triphosphate bridge. The cap is added to the first transcribed nucleotide by the capping enzymes, RNGTT and RNMT-RAM. During the early stages of transcription, the capping enzymes are recruited to RNA pol II phosphorylated on Serine-5 of the C-terminal domain. The mRNA cap protects transcripts from degradation during transcription and recruits factors which promote RNA processing including, splicing, export and translation initiation. The proportion of transcripts with a cap structure is increased by elevating c-Myc expression, resulting in increased rates of translation. c-Myc promotes capping by promoting RNA pol II phosphorylation and by upregulating the enzyme SAHH which neutralises the inhibitory bi-product of methylation reactions, SAH. c-Myc-induced capping is required for c-Myc-dependent gene expression and cell proliferation. Targeting capping may represent a new therapeutic opportunity to inhibit c-Myc function in tumours. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

RAM function is dependent on Kapß2-mediated nuclear entry.

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 Kapß2 (karyopherin ß2) nuclear transport protein.

Cap-binding complex (CBC).

The 7mG (7-methylguanosine cap) formed on mRNA is fundamental to eukaryotic gene expression. Protein complexes recruited to 7mG mediate key processing events throughout the lifetime of the transcript. One of the most important mediators of 7mG functions is CBC (cap-binding complex). CBC has a key role in several gene expression mechanisms, including transcription, splicing, transcript export and translation. Gene expression can be regulated by signalling pathways which influence CBC function. The aim of the present review is to discuss the mechanisms by which CBC mediates and co-ordinates multiple gene expression events.

Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites.

Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5' phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation.