Biology subjects

Cowling, V. H.

Publications and source records attributed to Cowling, V. H..

9 recordsLinked to original sources

bioRxiv2026

Not all TOP RNAs are created equal: 3'UTR length and TSS selection predict the translational regulation of LARP1-bound mRNAs in CD4+ T cells

Naive T cells are poised for activation and contain a pool of translationally repressed ribosomal protein (RP) mRNA prepared to induce ribosome biogenesis to support protein synthesis, cell growth and proliferation. RP mRNA are the prototypical members of a class of transcripts initiating at cytosine followed by a CU rich element called terminal oligo pyrimidine (TOP) RNAs. TOP RNAs are regulated by an RNA binding protein LARP1, which promotes transcript stabilisation and translational repression. We investigated LARP1 function in T cell activation by generating cross-linking immunoprecipitation (CLIP) datasets detailing the LARP1-RNA interactions in naive and activated CD4+ T cells and identifying novel TOP RNAs. TOP RNAs identified by this analysis were functionally diverse. RP mRNAs were typified by high stability, and translational repression in naive T cells followed by MTORC1-dependent translation increases following T cell activation. However, other TOP RNAs varied in these aspects of their regulation. Notably, TOP RNAs with longer 3'UTRs had a relaxed dependency on LARP1 for stability and a reduced dependency on MTORC1 for their translation. Transcription start site heterogeneity also impacted TOP RNA regulation by generating a mixture of transcript isoforms with different TOP motif lengths. Longer terminal oligo pyrimidine stretches were associated with a greater dependency on MTORC1 for translation. Differential regulation of TOP RNAs may allow tuneable translational responses to MTORC1 and indicates potential roles for LARP1 beyond translation regulation and stability.

cell biology↗

bioRxiv2025

RNMT is recruited to RNA by interaction with RNA G-quadraplexes

The 7-methylguanosine (m7G) cap protects RNA pol II transcripts from exonucleases and allows interaction with cap binding proteins which direct processing and translation. Duringm7G cap formation in mammals, nascent transcripts receive a guanosine cap which is methylated by RNA guanine-7 methyltransferase (RNMT). Unlike other capping enzymes which interact directly with RNA pol II, RNMT is recruited to the guanosine cap by interactions with RNA and the cap itself. RNMT is regulated during cell differentiation with significant impact on which RNAs are expressed and translated. The gene-specificity of RNMT was unexplained since RNMT in complex with activating subunit, RAM, binds to RNA without sequence preference. Through the development of improved RNA-protein detection, CLIP-ART, we report that RNMT interacts directly with RNA G4 quadraplexes (rG4). RNMT predominantly interacts with rG4s in the 5 UTR (untranslated regions) of mRNAs, indicating a mechanism for anchoring RNMT adjacent to the guanosine cap substrate. RNMT interacts with rG4s in transcripts which encode proteins involved in growth and proliferation. The RNMT-rG4 interaction is specific to the RNMT monomer, rather than RNMT-RAM, indicating a mechanism by which differential regulation of RNMT and RAM, can lead to cap methylation of specific RNAs involved in growth control. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/688685v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f546a1org.highwire.dtl.DTLVardef@f000d4org.highwire.dtl.DTLVardef@c665f5org.highwire.dtl.DTLVardef@14fcfd0_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

biochemistry↗

bioRxiv2025

CaMKII-RNMT axis directs activity-dependent control of RNA dynamics in neurons

In neurons, mRNAs are transported into axons and dendrites for local translation at synapses. Diverse proteins support the transport of specific mRNAs, however, mechanisms co-ordinating mRNA translocation are unclear. We report that that CaMKII (Ca2+/calmodulin-dependent protein kinase II) is a key regulator of RNA dynamics through activity-dependent phosphorylation of the RNA cap guanine-7 methyltransferase, RNMT. Following RNA cap methylation in the nucleus, RNMT is retained on specific mRNAs. On stimulation, RNMT translocates into the cytoplasm, increasing levels of locally translated mRNAs. In the cytoplasm, CaMKII phosphorylates RNMT Thr317 on the active site, which inhibits methyltransferase activity and targets RNMT for degradation, thus limiting cytoplasmic RNMT function. RNMT mutants protected from CaMKII-dependent degradation increase locally translated, cytoplasmic mRNAs and accelerate neuronal morphogenesis. RNMT Thr317 phospho-mimic co-ordinately decreases locally translated mRNAs in the cytoplasm and reduces differentiation. The CaMKII-RNMT relationship links neuronal activity to the spatial regulation of gene expression in neurons. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/685591v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@b97c4borg.highwire.dtl.DTLVardef@18f38eaorg.highwire.dtl.DTLVardef@1e1bcb0org.highwire.dtl.DTLVardef@12ac7c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

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.

cell biology↗

bioRxiv2021

Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the 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 fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. In order 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 a key role 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 5,000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified 4 compounds as potential inhibitors of nsp14, all of which also show antiviral capacity in a cell based model of SARS-CoV-2 infection. Three of the 4 compounds also exhibited synergistic effects on viral replication with remdesivir.

bioengineering↗

bioRxiv2020

Identification of novel RNA Polymerase II CTD interaction sites on the mRNA Capping Enzyme

1Recruitment 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 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 characterisation 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 surface and quantify their relative contributions to CTD binding. We also identify differences in the CTD binding conformation when phosphorylated at either the Serine-2 or Serine-5 positions, thus providing insights into how the CE reads the CTD code. The computational findings are then validated by binding and activity assays. These novel CTD interaction sites are compared with cocrystal structures of the CE-CTD complex in different eukaryotic taxa, leading to the conclusion that this interface is considerably more conserved than previous structures have indicated.

biophysics↗

bioRxiv2019

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 triphosphate at the 5 end. 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 cell cultures.

biochemistry↗

bioRxiv2019

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

The RNA guanine-7 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 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 dynamic network and 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 enzymes active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.

biophysics↗

bioRxiv2017

DHX15 regulates CMTR1-dependent gene expression and cell proliferation

CMTR1 contributes to mRNA cap formation by methylating the O-2 position of the 1st transcribed nucleotide ribose. mRNA cap O-2 methylation has roles in mRNA translation and self-RNA tolerance in innate immunity, however its role in cell physiology is unclear. We report that CMTR1 is recruited to Serine-5 phosphorylated RNA Pol II CTD, facilitating cotranscriptional methylation. 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 bound to DHX15, CMTR1 activity is repressed and prevented from binding to RNA pol II, thus constraining 1st nucleotide methylation to a co-transcriptional event. Conversely CMTR1 activates DHX15 helicase activity and influences its nuclear localisation, which is likely to impact on several nuclear functions. 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. In HCC1806 cells, the DHX15-CMTR1 interaction controls ribosome loading of a subset of mRNAs and impacts on cell proliferation.

cell biology↗