How CUT&RUN Helps Improve Methylated RNA Immunoprecipitation

Often referred to as “the fifth RNA base”, 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.

Currently, several methods are used 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, allowing 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 limits 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 as shown in Ref 3.

miCLIP.  m6A individual-nucleotide resolution crosslinking and immunoprecipitation (miCLIP) was designed 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.

Schematic of the miCLIP protocol as shown in Ref 5.

PA-m6A-seq.  Photo-crosslinking-assisted m6A sequencing (PA-m6A-seq) is an alternative UV-based strategy that was fashioned for high-resolution (~23 nt) 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.

Schematic of the PA-m6A-seq protocol as shown in Ref 6.

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 developed a new method: cleavage under target and recover using nuclease for m6A enrichment (CUT&RUN m6A MeRIP) [7-16].  This innovative approach combines the advantages of MeRIP-seq and miCLIP with EpigenTek’s proprietary EpiQuik 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).

Workflow of EpigenTek’s CUT&RUN m6A MeRIP assay.

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