Abstract 2127: Dissecting the regulatory mechanisms between m6A and alternative splicing: A data-driven study
Authors: Wang Lin , Tsui Stephen Kwok Wing 
Published: September 2020

N6-methyladenosine (m6A) is the most prevalent modification in the mRNAs, which is a methylated modification on the sixth nitrogen of adenosine. Increasing evidence shows that m6A plays a critical role in many biological processes and diseases including oocyte development, diabetes and cancers. However, the regulation of m6A in the RNA metabolism, especially the relationship between m6A and alternative splicing (AS), remains largely elusive.

In this study, we make the first attempt to investigate the underlying regulation relationship between m6A and AS using public resources. We collected both RNA-seq and MeRIP-seq data from HepG2 cell lines with or without METTL14, a methyltransferase, knocked-down. We called differential m6A peaks using an R package named ExomePeak and detected differential alternative splicing events using rMATS software. We considered the AS events overlapped with differential m6A peaks as the regulatory consequence of m6A modification, where we applied GO ontology and pathway enrichment analyses.

As the results, we detected 7149 differential genes out of the total 11291 m6A sites. Each differential gene had an average of 1.58 m6A loci. Among these differential m6A genes, 5871 genes were down-regulated and only 2021 genes were up-regulated. We then identified 2465 differential AS events from 1824 unique genes. Skipped exon (SE), the most common type of AS, got 1278 unique genes from 120 events and an average of 1.3 patterns occurred for each gene. For all differentially spliced genes, there were no significant differences between the proportions of up-regulated and down-regulated genes.

Next, we intersected total differentially spliced genes with the differential m6A peaks and got 989 differentially m6a-associated spliced genes, which mainly enriched in biological processes including histone modification and intracellular receptor signaling pathway. In addition, by further analyzing the regulatory relationship between m6A and five AS modes, we chose down-regulated m6A genes separately to intersect each AS mode of five. However, we found that the three AS modes, SE, A3SS and A5SS, were more likely co-affected by the knock-down of METTL14. In order to explain this phenomenon, we performed motif analysis on these intersected sequences and compared the enriched motif with known databases. Interestingly, we found that most of the enriched motifs were transcription factor binding.

In this work, we explored the role of m6A modification in pre-mRNA splicing. Our results suggested that m6A may have a regulatory role in pre-mRNA splicing, especially ES, A3SS and A5SS modes. Moreover, functional pathway analysis and motif discovery of m6A associated alternative splicing patterns have elucidate the possible mechanisms of m6A in the regulation of biological functions.

Citation Format: Lin Wang, Stephen Kwok Wing Tsui. Dissecting the regulatory mechanisms between m6A and alternative splicing: A data-driven study [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 2127.