Determination of enrichment factors for modified RNA in MeRIP experiments

Kaouthar Slama; Aurellia Galliot; Franziska Weichmann; Jasmin Hertler; Regina Feederle; Gunter Meister; Mark Helm

doi:10.1016/j.ymeth.2018.10.020

Determination of enrichment factors for modified RNA in MeRIP experiments

Methods. 2019 Mar 1:156:102-109. doi: 10.1016/j.ymeth.2018.10.020. Epub 2018 Oct 28.

Authors

Kaouthar Slama¹, Aurellia Galliot¹, Franziska Weichmann², Jasmin Hertler¹, Regina Feederle³, Gunter Meister², Mark Helm⁴

Affiliations

¹ Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Germany.
² Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany.
³ Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Institute for Diabetes and Obesity, Monoclonal Antibody Core Facility and Research Group, Munich, Germany.
⁴ Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Germany. Electronic address: mhelm@uni-mainz.de.

PMID: 30394295
DOI: 10.1016/j.ymeth.2018.10.020

Abstract

In the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (m⁶A). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using m⁶A-containing mRNAs against an unmodified control of identical sequence. Both types of mRNA carry ³²P labels at different nucleotides, allowing their relative quantification in a mixture after digestion to nucleotides, separation by TLC and quantitative phosphorimaging of the labels.

Keywords: Antibody; M(6)A/N6-methyladenosine; MeRIP; RNA modification; TLC.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine / analogs & derivatives*
Adenosine / chemistry
Adenosine / metabolism
Adenosine Triphosphate / chemistry
Adenosine Triphosphate / metabolism*
Cell-Free System / chemistry
Cell-Free System / metabolism
Chromatography, Thin Layer
DNA-Directed RNA Polymerases / genetics
DNA-Directed RNA Polymerases / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Humans
Immunoglobulin G / chemistry*
Immunoglobulin G / genetics
Immunoglobulin G / metabolism
Immunoprecipitation / methods*
Isotope Labeling / methods
Methylation
Models, Molecular
Phosphorus Radioisotopes
Protein Binding
Protein Interaction Domains and Motifs
Protein Structure, Secondary
RNA, Messenger / chemistry
RNA, Messenger / genetics*
RNA, Messenger / metabolism
Viral Proteins / genetics
Viral Proteins / metabolism

Substances

Immunoglobulin G
Phosphorus Radioisotopes
RNA, Messenger
Viral Proteins
Adenosine Triphosphate
N-methyladenosine
bacteriophage T7 RNA polymerase
DNA-Directed RNA Polymerases
Adenosine