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Supplemental material, sj-pdf-1-jbx-10.1177_24725552211000652 for Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry by Lesley-Anne Pearson, Charlotte J. Green, De Lin, Alain-Pierre Petit, David W. Gray, Victoria H. Cowling and Euan A. F. Fordyce in SLAS Discovery

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.

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.

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 PI3 Kα. Conversely, all cell lines insensitive to RNMT depletion expressed wild-type PIK3CA. Expression of oncogenic PIK3CA mutants, which increase PI3 K p110α activity, was sufficient to increase dependency on RNMT. Conversely, inhibition of PI3 Kα reversed dependency on RNMT, suggesting that PI3 Kα 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.

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., Highlights • The mRNA cap is a methylated modification of the 5′ terminus of mRNA. • RNA processing and translation factors are recruited to the mRNA cap. • The mRNA cap protects transcripts from degradation and defines mRNA as “self”. • Formation of the mRNA cap is regulated by cellular signalling pathways. • mRNA cap regulation results in changes in gene expression and cell function.

Summary 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., Graphical Abstract, Highlights • CDK1-cyclin B1 phosphorylates the mRNA cap methyltransferase RNMT on T77 • RNMT T77 phosphorylation increases mRNA cap methyltransferase activity • CDK1-cyclin B1 activation of RNMT coordinates post-mitotic transcription and capping • RNMT T77 phosphorylation is rate limiting for gene expression and cell proliferation, mRNA processing and translation are dependent on the mRNA cap structure. Aregger et al. report that the cell cycle regulator CDK1-cyclin B1 phosphorylates and activates the mRNA cap methyltransferase RNMT, resulting in increased capping activity following mitosis. RNMT phosphorylation coordinates G1 phase transcription and the cap formation required for translation initiation.

Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription.

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.