N6-methyladenosine, or m6A, is the most common and abundant eukaryotic RNA modification, accounting for
over 80% of all RNA methylation. It can be found mainly in mRNA, but is also observed in non-coding species like
tRNA, rRNA, and miRNA. Through interactions with various binding proteins called “readers”, m6A affects virtually
every facet of ribonucleic acid biology: structure, splicing, localization, translation, stability, and turnover [1].
Aside from this central role in RNA metabolism, m6A is a factor in other physiological processes such as cell
differentiation, immunity, inflammation, and the circadian clock [2]. Abnormal m6A methylation has been implicated in
diverse pathologies: diabetes, obesity, neurodegeneration, and cancer, to name a few. The formation of m6A RNA
appears to be a co-transcriptional event occurring early on in the RNA lifecycle and is mediated by a multi-protein
methyltransferase complex.
Mapping the m6A epitranscriptome
Several methods are available for epitranscriptome-wide m6A mapping:
MeRIP-seq.
The development of methylated RNA immunoprecipitation sequencing (MeRIP-seq) was a landmark in the field of
epitranscriptomics as it was the first method to detect m6A on a transcriptome-wide level [3]. MeRIP-seq couples m6A
RNA immunoprecipitation with NGS, providing for the high-throughput localization of modified sites from enriched
m6A-containing RNA fragments that have been precipitated by a specific antibody, reverse-transcribed, and sequenced.
The fragment sizes generated during the random fragmentation step preceding immunoprecipitation limit the precise
mapping of the m6A site to within a ~200 nt stretch. The choice of antibody allows MeRIP-seq to be adapted toward the
study of other modified RNA types (e.g., 5hmC RNA [4]).
Schematic of the MeRIP-seq protocol [3].
miCLIP.
m6A individual-nucleotide resolution crosslinking and immunoprecipitation (miCLIP) was developed to address the
disadvantages associated with MeRIP-seq approaches regarding the mapping of m6A RNA sites at individual-nucleotide
resolution [5]. The key feature of this method is the UV-crosslinking of immunoprecipitated RNA fragments to the
capture antibody. Antibody remnants at the crosslinking site on the RNA after Proteinase K treatment induce signature
mutations (truncations and C→T transitions) during cDNA synthesis that can be identified by sequencing and used to
more precisely map the specific m6A location. These antibody-induced mutational signatures have also been
successfully applied to the mapping of m6Am RNA modifications.
PA-m6A-seq.
Photo-crosslinking-assisted m6A sequencing (PA-m6A-seq) is an alternative UV-based strategy that was fashioned for
high-resolution (~23 nts) transcriptome-wide m6A mapping [6]. This method employs a photoreactive ribonucleoside
crosslinker to induce a signature mutation for localizing m6A, akin to miCLIP. The uridine analogue 4-thiouridine
(4SU) is incorporated into sample RNA. Full-length, unfragmented, 4SU-labeled RNA molecules are then
immunoprecipitated with an anti-m6A antibody, and UV irradiation is applied to establish covalent crosslinks between
m6A-bound antibody and neighboring 4SU. Crosslinked RNA is digested with RNase T1 to yield ~30 nt-long fragments that
are further processed for library preparation and sequencing, whereby crosslink-generated T→C transitions adjacent to
the m6A sites can be identified.
eTAM-seq.
Evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq) is the latest addition to the researcher’s m6A
profiling toolbox [7]. eTAM-seq detects and quantifies m6A via global adenosine deamination, a process that converts
all unmethylated A into I, which is subsequently read as G, while m6A remains unaffected. The A-to-I conversion is
facilitated by a distinctive variant (TadA8.20) of the E. coli TadA enzyme that maintains RNA integrity and
thus minimizes sample loss to a greater extent when compared with harsher chemical deamination. Given the novelty of
this enzyme-assisted sequencing technology and lack of in-depth characterization, several concerns still require
attention, including time-consuming protocol; sensitivity (eTAM-seq is less sensitive to lowly methylated A sites);
enzyme accessibility (eTAM-seq may not be applicable for highly structured RNA, and has high sequence bias); enzyme
specificity (the TadA8.20 deaminase may not convert non-m6A adenosine modifications); and high cost. eTAM-seq is
currently not available commercially.
Cutting through the background noise
Cleavage under targets and release using nuclease (CUT&RUN) is a recent innovation in the study of protein/DNA
interactions, designed to tackle the limitations of the popular ChIP-seq technique, namely, the need for a large
amount of starting material, long protocol time, and limited resolution. This novel method, which is performed in situ
on intact cells without fixation, entails cleavage of chromatin at specific sites by a unique fusion protein and the
subsequent, direct capture of protein-DNA complexes. The fusion protein consists of: 1) a cleavage domain (e.g.,
micrococcal nuclease, or MNase); and 2) a protein A/G (pAG) domain that binds to an antibody against the protein of
interest.
Schematic of the CUT&RUN protocol [8].
During the procedure, the antibody specifically captures the target protein. The pAG-MNase fusion protein then binds
to the antibody and cleaves the chromatin at the protein-DNA interaction site, resulting in the release of the
protein-DNA complex from the chromatin. The CUT&RUN method eliminates interference from crosslinking and yields
shorter-length DNA fragments, allowing for reduced background signals.
MeRIP with CUT&RUN for increased sensitivity
Established methods used for epitranscriptome-wide m6A mapping like MeRIP-seq, PA-m6A-seq, and miCLIP have either
been widely used but are unable to achieve high resolution in m6A profiling; or improve the profiling resolution but
suffer from poor reproducibility and a complicated process. In particular, they are time-consuming (>2 days) and
costly.
To address these issues, EpigenTek has pioneered an improved method: cleavage under target and recover
using nuclease for m6A enrichment (CUT&RUN
m6A MeRIP) [9-18]. This innovative approach, the first of its kind, combines the advantages of
MeRIP-seq and miCLIP with CUT&RUN technology for higher enrichment, lower input, reduced background, and a faster,
more streamlined procedure. CUT&RUN m6A MeRIP uses a state-of-the-art RNA cleavage enzyme mix to simultaneously
fragment immunocaptured RNA and cleave/remove any RNA sequences in both ends of the target m6A-containing sequences
without affecting RNA regions occupied by the antibody. Short RNA fragments are consequently generated only bound
with anti-m6A antibody. True target m6A-enriched regions can therefore be reliably identified, and high-resolution
mapping achieved.
The m6A peak distribution from samples processed with CUT&RUN m6A MeRIP
correlates well with expected regions as shown in published data (Inset; see Ref 3).
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