Identification of m⁶A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

Abstract

Methylation at the N 6 position of adenosine (m 6 A) is one of the most abundant RNA modifications found in eukaryotes, however accurate detection of specific m 6 A nucleotides within transcripts has been historically challenging due to m 6 A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-Seq) have relied on m 6 A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m 6 A residues. More recently, modified cross-linking and immunoprecipitation (CLIP) based approaches that rely on inducing specific mutations during reverse transcription via UV crosslinking of the anti-m 6 A antibody to methylated RNA have been employed to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries.Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m 6 A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m 6 A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m 6 A sites at true single nucleotide resolution. As the effort to accurately identify m 6 As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m 6 A epitranscriptome.

A single N6-methyladenosine site in lncRNA HOTAIR regulates its function in breast cancer cells

“…Samples were sequenced at the Genomics and Microarray Shared Resource facility at University of Colorado Denver Cancer Center on an Illumina MiSeq or NovaSEQ6000 with 2x 150 base pair paired-end reads to generate 40 million raw reads for each sample. Computational analysis methods are described in 26 . Briefly, a custom Snakemake workflow was generated based on the original eCLIP analysis strategies 54 to map reads to the human genome.…”

m6A RNA Methylation in Systemic Autoimmune Diseases—A New Target for Epigenetic-Based Therapy?

“…Several variants for the CLIP approach have been recently established, including enhanced CLIP (eCLIP) or m6A-eCLIP (meCLIP). Compared to the basic CLIP method, the new protocols are technically simplified and yield higher numbers of identified sites [ 67 ]. The selected m6A detection methods are presented in Table 1 and reviewed in [ 53 , 68 , 69 ].…”