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

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Abstract

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.

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Methylated nucleotides block 5' terminus of HeLa cell messenger RNA.

C. Wei, A Gershowitz, B. Moss (1975)

Polyadenylylated [poly(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poly(A)+ RNA was calculated from the 3H/14C ratios and known degrees of methylation of 18S and 28S ribosomal RNAs. All four 2'-O-methylribonucleosides, but only two base-methylated derivatives, 7-methylguanosine (7MeG) and 6-methyladenosine (6MeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyl-labeled ribonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5' terminus of the poly(A)+ RNA and could be removed by periodate oxidation and beta elimination. Evidence for a 5' to 5' linkage of 7MeG to adjacent 2'-O-methylribonucleosides through at least two and probably three phosphates to give structures of the type 7MeG5'ppp5pNMep- and 7MeG5'ppp5'NMepNmep- was presented. The previous finding of similar sequences of methylated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5' termini may be a characteristic feature of mRNAs that function in eucaryotic cells.

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METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation

Luca Pandolfini, Isaia Barbieri, Andrew Bannister … (2019)

Summary 7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.

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Cap and cap-binding proteins in the control of gene expression.

Ivan Topisirovic, Yuri Svitkin, Nahum Sonenberg … (2015)

The 5' mRNA cap structure is essential for efficient gene expression from yeast to human. It plays a critical role in all aspects of the life cycle of an mRNA molecule. Capping occurs co-transcriptionally on the nascent pre-mRNA as it emerges from the RNA exit channel of RNA polymerase II. The cap structure protects mRNAs from degradation by exonucleases and promotes transcription, polyadenylation, splicing, and nuclear export of mRNA and U-rich, capped snRNAs. In addition, the cap structure is required for the optimal translation of the vast majority of cellular mRNAs, and it also plays a prominent role in the expression of eukaryotic, viral, and parasite mRNAs. Cap-binding proteins specifically bind to the cap structure and mediate its functions in the cell. Two major cellular cap-binding proteins have been described to date: eukaryotic translation initiation factor 4E (eIF4E) in the cytoplasm and nuclear cap binding complex (nCBC), a nuclear complex consisting of a cap-binding subunit cap-binding protein 20 (CBP 20) and an auxiliary protein cap-binding protein 80 (CBP 80). nCBC plays an important role in various aspects of nuclear mRNA metabolism such as pre-mRNA splicing and nuclear export, whereas eIF4E acts primarily as a facilitator of mRNA translation. In this review, we highlight recent findings on the role of the cap structure and cap-binding proteins in the regulation of gene expression. We also describe emerging regulatory pathways that control mRNA capping and cap-binding proteins in the cell. Copyright © 2010 John Wiley & Sons, Ltd.

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Journal

Journal ID (nlm-ta): Open Biol

Journal ID (iso-abbrev): Open Biol

Journal ID (publisher-id): RSOB

Journal ID (hwp): royopenbio

Title: Open Biology

Publisher: The Royal Society

ISSN (Electronic): 2046-2441

Publication date Collection: February 2020

Publication date (Electronic): 26 February 2020

Publication date PMC-release: 26 February 2020

Volume: 10

Issue: 2

Electronic Location Identifier: 190306

Affiliations

[1 ]Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee , Dundee DD1 5EH, UK

[2 ]FingerPrints Proteomics Facility, School of Life Sciences, University of Dundee , Dundee DD1 5EH, UK

[3 ]Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee , Dundee DD1 5EH, UK

[4 ]Centre of New Technologies, University of Warsaw , 02-097 Warsaw, Poland

[5 ]Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw , 02-097 Warsaw, Poland

Author notes

e-mail: m.a.j.ferguson@ 123456dundee.ac.uk

e-mail: v.h.cowling@ 123456dundee.ac.uk

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.4853172.

Author information

Alison Galloway http://orcid.org/0000-0001-8780-0382

Victoria H. Cowling http://orcid.org/0000-0001-7638-4870

Article

Publisher ID: rsob190306

DOI: 10.1098/rsob.190306

PMC ID: 7058934

PubMed ID: 32097574

SO-VID: 6bcb16af-57df-4fa4-b97e-58f51bdd4bf7

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Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

History

Date received : 11 December 2019

Date accepted : 30 January 2020

Funding

Funded by: Medical Research Council, http://dx.doi.org/10.13039/501100000265;

Award ID: MR/K024213/1 Senior Fellowship

Funded by: Lister Institute of Preventive Medicine, http://dx.doi.org/10.13039/501100001255;

Award ID: Prize Fellowship

Funded by: H2020 European Research Council, http://dx.doi.org/10.13039/100010663;

Award ID: 769080

Funded by: Wellcome Trust, http://dx.doi.org/10.13039/100004440;

Award ID: 097945/Z/11/Z

Funded by: Royal Society, http://dx.doi.org/10.13039/501100000288;

Award ID: Wolfson Research Merit Award

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cover-date February 2020

ScienceOpen disciplines: Life sciences

Keywords: mass spectrometry,rna cap,rna methylation,rna processing,7-methylguanosine,ribose o-2 methylation

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ScienceOpen disciplines: Life sciences

Keywords: mass spectrometry, rna cap, rna methylation, rna processing, 7-methylguanosine, ribose o-2 methylation

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

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Most cited references 50

Methylated nucleotides block 5' terminus of HeLa cell messenger RNA.

METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation

Cap and cap-binding proteins in the control of gene expression.

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