RAM/Fam103a1 Is Required for mRNA Cap Methylation

Gonatopoulos-Pournatzis, Thomas; Dunn, Sianadh; Bounds, Rebecca; Cowling, Victoria H.

doi:10.1016/j.molcel.2011.08.041

Cited by 83 publications

(156 citation statements)

References 25 publications

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“…Additionally, siRNA-mediated knockdown of RNMT greatly reduced the cap methyltransferase activity of cytoplasmic extracts, suggesting that RNMT is the predominant, if not only cap methyltransferase in the cytoplasm of mammalian cells. Nuclear RNMT exists as a heterodimer with RNMT-activating miniprotein (RAM, Gonatopoulos-Pournatzis et al , 2011), and we demonstrated that cytoplasmic RNMT also binds to RAM. Reduced cytoplasmic cap methyltransferase activity upon RAM knockdown indicated that RAM is a required cofactor for cytoplasmic RNMT.…”

Section: [Background]mentioning

confidence: 86%

RNA Cap Methyltransferase Activity Assay

Trotman

Schoenberg

2018

BIO-PROTOCOL

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Methyltransferases that methylate the guanine-N7 position of the mRNA 5′ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments.

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Section: [Background]mentioning

confidence: 86%

RNA Cap Methyltransferase Activity Assay

Trotman

Schoenberg

2018

BIO-PROTOCOL

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“…9,10 RNMT has a catalytic core, which is homologous in structure and function to other eukaryotic cap methyltransferases. 11 In addition, RNMT contains an N-terminal noncatalytic domain, which is conserved in mammals but not in other eukaryotes (Fig.…”

Section: Regulation Of Mrna Cap Methylationmentioning

confidence: 99%

“…11 RAM also has a high-affinity RNA binding domain which may increase the rate of recruitment of transcripts in general or may have specificity for particular transcript motifs. 10,13 In human cells, RNMT-RAM is recruited via the N-terminal domain of RNMT to RNA pol II proximal to transcription initiation sites. 14,15 The RNMT N-terminal domain also impacts on cap methyltransferase activity.…”

Section: Regulation Of Mrna Cap Methylationmentioning

confidence: 99%

Regulation of mRNA capping in the cell cycle

Aregger

Cowling

2016

RNA Biology

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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.

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“…The human RNMT structure was released by the Structural Genomics Consortium (SGC; PDB: 3BGV), and has considerable similarity to Ecm1 (RMSD: 1.25 Å) (22). RNMT also has an activating subunit, RAM (RNMT-activating miniprotein) (23). Although homologues of RNMT are present in all eukaryotes, RAM is only present in vertebrates leading to interest in its mechanism of action and cellular function.…”

Section: Introductionmentioning

confidence: 99%

“…RAM consists of an N-terminal RNMT activation domain (residues 1–55), a central RNA binding domain (residues 56–90), and a C-terminal PY nuclear localisation domain (residues 91–118) (23,24). The RAM RNA binding domain is required for cell viability but not for RNMT activation, and therefore may select specific critical transcripts for enhanced methylation.…”

Section: Introductionmentioning

confidence: 99%

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

Varshney

Petit

Bueren-Calabuig

et al. 2016

Nucleic Acids Research

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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.

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RAM/Fam103a1 Is Required for mRNA Cap Methylation

Cited by 83 publications

References 25 publications

RNA Cap Methyltransferase Activity Assay

RNA Cap Methyltransferase Activity Assay

Regulation of mRNA capping in the cell cycle

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

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