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A recent paper from Samie Jaffrey's laboratory at Cornell University outlines a new approach for high-resolution localization of N6-methyladenosine in eukaryotic RNA, called m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP).
Although m6A is the most abundant modified base in eukaryotic mRNA, current methods to accurately study it have limitations. New approaches to high-resolution mapping of m6A will be essential for understanding this epigenetic RNA modification.
The miCLIP method
1. RNA extraction from HEK293 cell lines (human) or liver nuclei (mouse) using Trizol.
Adapted from Linder et al. (2015) Nature Methods 12:767–772
The principle of miCLIP
By cross-linking RNA-m6A antibody-bound sites, mutagenesis at these specific sites can occur during reverse transcription of the antibody-bound RNA.
This unique mutagenesis signature, e.g., a C-T transition or a truncation, can be sequenced and used to precisely map m6A.
Challenges with existing m6A detection methods
Existing techniques, MeRIP-seq or m6A-seq, use IP with m6a specific antibodies followed by sequencing of ~100 nucleotide RNA fragments.
There are two main limitations to this approach:
Researchers solved the same issues for detection of DNA methylation by the introduction of bisulfite-sequencing, where modified and unmodified cytosine residues are differentiated by sodium bisulfite treatment. It is not yet possible to quantify m6A using a similar approach due to the lack of distinguishing chemical properties between m6A and unmodified adenosine.
Advantages and applications of miCLIP
miCLIP allows for high-resolution detection of single m6A residues and m6A clustering across the entire RNA. Using miCLIP, the researchers were able to map m6A and the related dimethylated version m6Am (N6,2′-O-dimethyladenosine), at single-nucleotide resolution in human and mouse mRNA.
Additionally, the development of miCLIP lends new insight into the m6Am modification, occurring at the 5’ transcription start site. This facilitates research into the poorly understood function of this unique epigenetic signature in RNA.
The researchers also demonstrated that miCLIP is applicable to smaller RNAs. They discovered that m6A is present in small nucleolar RNAs (snoRNAs), a class of small non-coding RNAs. This was impossible to establish with previous applications due to a lack of specificity and bioinformatic challenges.
Thorough validation of miCLIP against previous approaches suggests that this is a highly sensitive tool for the precise mapping of m6A and m6Am RNA modifications in the eukaryotic transcriptome.
This antibody-based technology has the potential to be widely applied to other epigenetic modifications occurring on RNA, should specific antibodies be available.
This advance has the potential to open up the floodgates for high-resolution epigenetic mapping and the unraveling of epigenetic control of RNA mechanisms.
Read the paper in full: Linder et al. (2015) Nature Methods