The comprehensive interactomes of human adenosine RNA methyltransferases and demethylases reveal distinct functional and regulatory features.

Helena Covelo-Molares,Ranjani Ganji,Ivana Poštulková,Lisa Gawriyski,David Potěšil,Pavlína Gregorová,Štěpánka Vaňáčová,Ales Obrdlik,Michaela Dohnálková,Markku Varjosalo

doi:10.1093/nar/gkab900

Abstract

N6-methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.

Highlights

Reversible DNA and histone modifications have a wellestablished role in the regulation of gene expression
METTL16 appears to act as a monomer [5,6,7], whereas METTL3 forms a heterodimeric complex with METTL14 (METTL3/14) [8]
For cDNA preparation, total RNA was isolated with TriPure reagent (Roche) according to manufacturer’s protocol, treated with Turbo DNase (Ambion) and 2 ␮g of RNA was used for reverse transcription (RT) with oligo dT primers and SuperScript III RT (Invitrogen)

Summary

INTRODUCTION

Reversible DNA and histone modifications have a wellestablished role in the regulation of gene expression. Mammalian cells possess dedicated cellular machinery to write, erase and read m6A and m6Am (found adjacent to the 5 cap) marks Their substrate specificity, regulation and roles in RNA metabolism, development and disease undergo intensive investigations. The possibility of crosstalk between individual MTs and DMTs and their spatial contacts has not been explored To address this question, we employed a proteomic approach and mapped the interaction networks of the key enzymes of the m6A and m6Am pathways METTL3, METTL16, PCIF1, FTO and ALKBH5 in the human cell line HEK293 T-REx Flp-In (293T). We used the proximitydependent labelling approach BioID coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) [37,38] In this method, the bait protein is fused to a promiscuous biotin ligase derived from Escherichia coli BirA (R118G, BirA*) to label proteins in vivo in an approximate radius of 10 nm [39], detecting stable and transient protein-protein interactions. This method has been successfully used to identify protein interactors of other RNA modifying proteins [40]

MATERIALS AND METHODS

RESULTS AND DISCUSSION

C C terminal METTL3 METTL14 VIRMA WTAP RBM15 HAKAI ZC3H13