Deciphering the Recognition, Recruitment and Modification of structured lncRNAs by the methyl-transferase complex METTL3/METTL14 and its cofactors

The human METTL3/METTL14 methyltransferase complex (MTC) is responsible for the m6A methylation of a large set of RNAs, and plays a crucial role in tumorigenesis. It targets numerous long non-coding RNAs (lncRNAs), including Xist and MALAT1.

To date, studies have only considered single-stranded RNA motifs, and a critical need for structural information on the complex in interaction with lncRNA has grown over the years. METTL3 and METTL14 form a heterodimer through their catalytic domains (MTDs).

Additionally, each monomer comprises flexible regions that are essential to the complex activity but have been truncated because of their dynamics: the METTL3 tandem zinc-finger domains (ZFDs) and the RGG region of METTL14.

Moreover, two cofactors of the MTC display a significant importance for its activity in vivo but have never been studied in interaction with lncRNA: the RNA Binding Motif protein 15 (RBM15) and the Zinc Finger CCCH-Type Containing 13 protein (ZC3H13).

This project aims at understand how structured regions of lncRNAs are recognized by the functionally active MTC and its cofactors at an atomic level.

For that purpose, we will measure the binding and methylation activity of both the ZFDs and RGG region to a set of RNA targets corresponding to modified region of Xist and MALAT1 in the context of the whole MTC with or without the cofactors.

By using mutated and post-translationally modified protein, we will assess (i) the lncRNA binding cooperativity between ZFD and the MTD, (ii) the role of the RGG region methylation in lncRNA binding structure specificity, (iii) the role of the cofactors in lncRNA recognition.

Finally, combining 13C methyl-TROSY experiments, segmental labeling, and paramagnetic relaxation enhancement, we will access the dynamics of the complex in interaction with the lncRNA in solution.

We expect to bring high-impact structural information of the specific recognition, recruitment and modification of lncRNAs by the MTC.