Abstract LB-187: Enhanced mapping of N6-methyladenosine (m6A) RNA modification sites with m6A-eCLIP

Abstract

Abstract N6-methyladenosine (m6A) is the most prevalent epitranscriptomic RNA modification found in eukaryotes and has been shown to regulate many stages of RNA biology including splicing, secondary/tertiary structure, localization, stability, and translation. Adenosine modifications are actively added, recognized, and removed by a series of “writer”, “reader”, and “eraser” proteins, making m6A a dynamic and reversible system for regulating RNAs. Further, there are now clear links between m6A writer, reader, and eraser proteins as well as m6A state of specific mRNAs to cancer pathogenesis, indicating that both global and specific dysregulation of m6A can have targeted effects that lead to disease. Thus, there is a need for methods to robustly profile m6A-modification states in both cell lines as well as tissues, and in normal as well as diseased or modified states. Advancements in high-throughput sequencing have led to the development of methods to map m6A transcriptome-wide, but there remains a need for improved methods that are scalable across conditions and treatments and with low abundance samples, such as blood or patient tissue. To address this need, we adapted enhanced crosslinking and immunoprecipitation (eCLIP) to create an optimized protocol for profiling m6A modification sites with 20-50-fold less starting RNA material (m6A-eCLIP). Using m6A-eCLIP, we recover traditional m6A motif enrichment and positioning enrichment at stop codons, validating the accuracy of the approach. Further, the improved handling and efficiency enabled us to profile m6A status across cell types and tissues, showing that m6A-eCLIP can be a robust method that is widely applicable for epitranscriptomic profiling across a range of conditions. Citation Format: Ines Rabano, Alexander A. Shishkin, Kylie Shen, Heather M. Foster, Peter Chu, Eric L. Van Nostrand, Gene W. Yeo. Enhanced mapping of N6-methyladenosine (m6A) RNA modification sites with m6A-eCLIP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-187.