Epigenetics application spotlight: miCLIP

A new antibody-based method for mapping m6A and m6Am throughout the transcriptome.

​A recent paper from the laboratory of Samie Jaffrey 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.

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The miCLIP method

1. RNA extraction from HEK293 cell lines (human) or liver nuclei (mouse) using Trizol.



2. Fragmentation of RNA to 30–130 nucleotide lengths, and incubation with anti-m6A antibody.

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3. UV cross-linking of RNA to the bound antibody.



4. Recovery of antibody-RNA complexes with protein A/G affinity purification, SDS-PAGE and nitrocellulose membrane transfer.



5. Adapter ligation, and release of RNA with proteinase K.



6. Reverse transcription of RNA to cDNA, PCR amplification and sequencing.


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7. Identification of C-T transitions or truncations and alignment against known genomic sequences. Mapping and annotation of these binding sites, identified as m6A/m6Am residues, to the transcriptome.

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:

  1. The method is unable to identify the specific location of modified residues and only determines the general location of m6A sites,
  2. Bias in bioinformatic m6A calling  – a priori assumptions are based upon known consensus sequences that harbor m6A residues so modifications outside these motifs are missed.

    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 lack of specificity and bioinformatic challenges.

    Future directions

    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 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

      Past approaches to detect and map m6A in RNA have used antibody immunoprecipitation (using m6A specific antibodies) and sequencing of ~100 nucleotide RNA fragments, termed m6A-seq or MeRIP-seq. While this method determines the general location of m6A sites, it is unable to exactly identify the specific location of modified residues.
      Bias in bioinformatic m6A ‘calling’ is a further limitation of this technology. A priori assumptions are based upon known consensus sequences that harbor m6A residues, thus m6As outside these motifs are missed.
      A further challenge is the lack of distinguishable chemical structure between m6A and unmodified adenosine bases. Unlike, DNA methylation where modified and unmodified cytosine residues may be differentiated by bisulfite treatment leading to a new base insertion in unmodified bases only, it is not possible to quantify m6A using this approach.


      References

      • Dominissini, D. et al. (2012). Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206
      • Liu, N. et al. (2015). N6-methyladenosine-dependent RNA structural switches regulate RNA–protein interactions. Nature 518, 560-564
      • Narayan, P. and Rottman, F.M. (1988). An in vitro system for accurate methylation of internal adenosine residues in messenger RNA. Science 242, 1159–1162
      • Meyer, K.D. and Jaffrey, S.R. (2014) The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat Rev Mol Cell Biol 15, 313–326
      • Sugimoto, Y. et al. (2012). Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions. Genome Biol. 13, R67
      • Wang, X., Lu, Z., Gomez, A., Hon, G.C., Yue, Y., Han, D., Fu, Y., Parisien, M., Dai, Q., Jia, G., et al. (2014). N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120