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Deciphering the RNA recognition by Musashi-1 to design protein and RNA mutants for in vitro and in vivo applications

Perez-Rafols, A.; Perez-Ropero, G.; Cerofolini, L.; Sperotto, L.; Roca-Martinez, J.; Anahi Higuera-Rodriguez, R.; Russomanno, P.; Kaiser, W.; Vranken, W. F.; Danielson, H.; Provenzani, A. F.; Martelli, T.; Sattler, M.; Buijs, J.; Fragai, M.

2024-10-29 biophysics
10.1101/2024.10.24.619864 bioRxiv
Show abstract

RNA Recognition Motifs (RRMs) are essential post-transcriptional regulators of gene expression in eukaryotic cells. The Human Musashi-1 (MSI-1) is an RNA-binding protein that recognizes (G/A)U1-3AGU and UAG sequences in diverse RNAs through two RRMs and regulates the fate of target RNA. Here, we combined structural biology and computational approaches to analyse the binding of the RRM domains of human MSI-1 with single-stranded and structured RNAs ligands. We used our recently developed computational tool RRMScorer to design a set of mutants of the MSI-1 protein to bind novel RNA sequences to alter the binding selectivity. The in-silico predictions of the designed protein-RNA interactions are assessed by NMR and SPR. These experiments also are used to study the competition of the two RRM domains of MSI-1 for the same binding site within linear and harpin RNA. Our experimental results confirm the in-silico designed interactions, thus opening the way for the development of new biomolecules for in vitro and in vivo studies and downstream applications.

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