Explore the RNA Methylome with SYSY Antibodies

SYSY was the first commercial supplier of m6A specific antibodies. Our products have been used in more than 700 scientific publications, and enabled the development of entirely new methods for researching the RNA methylome. The development of monoclonal and recombinant antibody variants complements the extremely successful polyclonal anti-m6a antibody and guarantees best reproducibility and lot-to-lot consistency. The anti m6A antibodies are suitable for a broad panel of applications like MeRIP, m6A-CLIP and miCLIP sequencing, classical IP, dot blotting, and ELISA.

m6A Antibodies

Overview

• Introduction
• Exploring the spliceosome, where everything began
• Discovering the m6A RNA methylome with MeRIP Sequencing
• Advanced techniques
• Products
• Literature

Introduction

Modifications of nucleic acids on the DNA and RNA level play an important role in many different biological processes. In prokaryotes, DAM methylation of adenosine residues in DNA at the 5'-GATC-3' motif allows the cell to differentiate between the parental and the daughter strand during mismatch repair (A L Lu, D Y Chang, 1988). In eukaryotes, distinct heritable CpG methylation patterns of DNA play an important role in the differential epigenetic regulation of gene expression (Meehan et al., 1992).

RNA molecules are subject to a variety of posttranscriptional modifications. During their maturation, mRNA molecules undergo splicing, 5´-capping and 3’ -polyadenylation, all necessary to obtain fully functional mRNAs (Carter, 1994).
More recently, other chemical modifications of single nucleotide residues have been shown to be involved in the fine-tuning of protein translation and RNA processing and stability. The most important modifications are methylation of different RNA residues at distinct positions like m1A, m5C, m6Am and last but not least N6-methyl-adenosine also known as m6A, the most abundant and probably most important RNA methylation variant (Zhou et al., 2020). There are writers of m6A like the METTL methyltransferase complex and demethylating erasers like FTO (figure 1) that fine-tune the reversible state of m6A RNA modification at the methyltransferase recognition site, the so-called DRACH motif (Sergeeva et al., 2020). SYSY antibodies have been part of the game from the very beginning.

Exploring the spliceosome, where everything began

In 1987, Peter Bringmann and Reinhard Luehrmann developed the first polyclonal antibodies against m6A to investigate the relevance of this RNA modification in the eukaryotic spliceosome. These antibodies enabled the quantitative immunoisolation of m6A modified small nuclear RNAs (snRNAs) U2, U4 and U6 from small nuclear riboproteins (snRNPs) (Bringmann, Lührmann, 1987).

In 1997, SYSY included the original m6A antiserum from R. Luehrmann and P. Bringmann in its catalog and was the first commercial supplier of m6A specific antibodies.

Discovering the m6A RNA methylome with MeRIP Sequencing

Later it turned out that the N6-methyl adenosine or m6A modification is involved in many more post-transcriptional regulatory processes and the m6A methylome of the transcriptome emerged as an entirely new and rapidly growing field of research.
The key publications from Kate Meyer (Meyer et al., 2012) and Dan Dominissini (Dominissini et al., 2012 and 2013) paved the way for the worldwide success of our affinity purified version of the m6A rabbit polyclonal antibody (cat. no. 202 003). Meyer and Dominissini have developed the antibody based m6A-sequencing method, an approach based on parallel sequencing of immunoisolated RNA fragments (100 -200 nt long) carrying m6A modifications (figure 2).

This new technique, also called MeRIP sequencing, allowed scientists to uncover numerous posttranscriptional regulatory mechanisms, all of which are modulated by m6A modifications, leading to a constantly growing number of scientific publications (figure 3).

M6A peaks were found to be enriched in 3' -UTR regions, near stop codons and within long exons (7, 8). It has been shown that m6A modifications can affect every process of the RNA life cycle and have an impact on many physiological processes like stem-cell development, regulation of translation, cancer, adipogenesis and more (Linder et al., 2015).

Advanced techniques

With MeRIP sequencing, m6A residues can be mapped to a 100-200 nt long region. By allocating the m6A modification to identified DRACH motifs, the exact position of m6A residues can be narrowed down considerably. However, for single nucleotide resolution, more advanced techniques had to be developed. M6A-CLIP and miCLIP are based on m6A site specific UV-induced cross-linking of antibodies that induce predictable mutational and truncation patterns during cDNA synthesis. This allows a much more precise mapping of m6A residues (Linder et al., 2015, Ke et al., 2015).

Products

Boost your RNA Methylome research with SYSY antibodies!

Literature

A L Lu, D Y Chang, 1988: Repair of single base-pair transversion mismatches of Escherichia coli in vitro: correction of certain A/G mismatches is independent of dam methylation and host mutHLS gene functions. PMID: 3284785

Bringmann, Lührmann, 1987: Antibodies specific for N6-methyladenosine react with intact snRNPs U2 and U4/U6. PMID: 2951275

Carter, 1994: Spatial localization of pre-mRNA transcription and processing within the nucleus. PMID: 7765739

Dominissini et al., 2012: Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. PMID: 22575960

Dominissini et al., 2013: Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing. PMID: 23288318

Ke et al., 2015: A majority of m6A residues are in the last exons, allowing the potential for 3' UTR regulation. PMID: 26404942

Linder et al., 2015: Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. PMID: 26121403

Meehan et al., 1992: Transcriptional repression by methylation of CpG. PMID: 1297654

Meyer et al., 2012: Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. PMID: 22608085

Sergeeva et al., 2020: Modification of Adenosine196 by Mettl3 Methyltransferase in the 5'-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation. PMID: 32344536

Zhou et al., 2020: Principles of RNA methylation and their implications for biology and medicine. PMID: 32920520