The m6A pathway facilitates sex determination in Drosophila

Lijuan Kan,Dong Yan,Eric C Lai,Wu-Min Deng,Jeffrey Vedanayagam,Anya V Grozhik,Nan Pang,Shu Kondo,Samie R Jaffrey,Ching-Jung Lin,Yi-Chun Huang,Deepak P Patil,Brian Joseph,Vladimir Despic,Jian Guo,Peter C Dedon,Kok-Seong Lim

doi:10.1038/ncomms15737

Abstract

The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m6A pathway. We first apply miCLIP to map m6A across embryogenesis, characterize its m6A ‘writer’ complex, validate its YTH ‘readers’ CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway.

Highlights

The conserved modification N6-methyladenosine (m6A) modulates messenger RNA (mRNA) processing and activity
Methylated mRNA was described over 40 years ago2,3, understanding the biological roles of m6A during most of this time was limited by lack of knowledge of molecular mechanisms that generate and interpret this modification, uncertainty on identities of methylated transcripts, and paucity of genetic mutants in specific m6A pathway components that could reveal in vivo requirements of this nucleoside modification4
The m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) libraries were sequenced to depths of B14–50 million mapped reads (Supplementary Data 1) and subjected to our pipeline for identification of cross-linking–induced mutation sites (CIMSs)

Summary

Introduction

The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. Methylated mRNA was described over 40 years ago, understanding the biological roles of m6A during most of this time was limited by lack of knowledge of molecular mechanisms that generate and interpret this modification, uncertainty on identities of methylated transcripts, and paucity of genetic mutants in specific m6A pathway components that could reveal in vivo requirements of this nucleoside modification. The m6A writers, erasers and readers comprise a system for epitranscriptomic regulation, akin to chromatin-based epigenetic systems This framework is bolstered by transcriptomewide mappings of methylated regions, catalogues that were refined by identification of methylated adenosines. Diverse functional consequences of m6A and/or m6A factors have been reported, including by promoting mRNA splicing, pri-miRNA processing, affecting RNA structure, facilitating mRNA degradation, promoting expression and/or canonical translation, and promotion of cap-independent translation

Methods

Results

Conclusion