(715c) Delineating a Key Problem in Protein-RNA Biology: Recognition of m6a By RRM Containing Proteins

Protein-RNA complexes play critical roles in cellular processes and in diseases, and thus, it is important to study interactions between specific proteins with natural or modified RNAs and understand their role in biology and health^[1,2,3]. RNA modifications, such as N6-methyladenosine (m⁶A), can significantly influence these interactions by changing the binding properties of RNA to proteins. m⁶A is one of the most prevalent mRNA modifications and has been extensively studied for its recognition by the YTH domain^[1,2]. However, the mechanisms by which RRM (RNA recognition motif) domain-containing proteins recognize m⁶A-modified RNAs still remain unclear. HNRNPA2B1, a RRM domain-containing protein, is of particular interest due to the overlapping of its targetome with the m⁶A motif, suggesting its potential role as a m⁶A reader. The RRM1 domain of HNRNPA2B1 has been proposed to preferentially bind with m⁶A-modified RNAs^[4]. Additional in vitro studies indicate that m⁶A can lower its binding affinity to HNRNPA2B1^[5], raising questions about how this protein interacts with both post-transcriptionally modified and unmodified RNAs. Using a combination of computational and experimental methods, we investigated how m⁶A affects RNA binding to HNRNPA2B1 in a sequence-dependent manner. Our studies provide novel insights into the structural and functional mechanisms of RRM associated m⁶A recognition, comparing it with the well-established YTH protein readers. Furthermore, we explored the m⁶A binding properties of RBM45, another RRM domain containing protein, and provide in-depth mechanistic insights into the difference between modified and unmodified adenine, in line with experiments^[6,7]. Our work highlights the understanding of post-transcriptional regulation and underscores the intricate interplay between RNA modifications and protein recognition mechanisms. Additionally, it demonstrates the complexity of RRM recognition of m⁶A-modified RNAs and reveals distinct principles underlying their interactions compared to YTH readers.

[1] Miller LG, Demny M, Tamamis P, Contreras LM. Characterization of epitranscriptome reader proteins experimentally and in silico: Current knowledge and future perspectives beyond the YTH domain. Comput Struct Biotechnol J. 2023;21:3541-3556.

[2] Höfler S, Duss O. Interconnections between m⁶A RNA modification, RNA structure, and protein-RNA complex assembly. Life Sci Alliance. 2023;7(1):e202302240.

[3] Miller LG, Kim W, Schowe S, Taylor K, Han R, Jain V, Park R, Sherman M, Fang J, Ramirez H, Ellington A, Tamamis P, Resendiz MJE, Zhang YJ, Contreras L. Selective 8-oxo-rG stalling occurs in the catalytic core of polynucleotide phosphorylase (PNPase) during degradation. Proc Natl Acad Sci U S A. 2024;121(46):e2317865121.

[4] Alarcón CR, Goodarzi H, Lee H, Liu X, Tavazoie S, Tavazoie SF. HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events. Cell. 2015;162(6):1299-308.

[5] Wu B, Su S, Patil DP, Liu H, Gan J, Jaffrey SR, Ma J. Molecular basis for the specific and multivariant recognitions of RNA substrates by human hnRNP A2/B1. Nat Commun. 2018;9(1):420.

[6] Chen X, Yang Z, Wang W, Qian K, Liu M, Wang J, Wang M. Structural basis for RNA recognition by the N-terminal tandem RRM domains of human RBM45. Nucleic Acids Res. 2021;49(5):2946-2958.

[7] Chen X, Wei Q, Yang Z, Chen X, Guo S, Jiang M, Wang M. Structural basis for RNA recognition by the C-terminal RRM domain of human RBM45. J Biol Chem. 2024;300(9):107640.