Dataset Information

Pooled scanning of protein variants identifies novel RNA-binding mutants

ABSTRACT: Binding to RNA has been observed for an ever-increasing number of proteins, which often have other functions. The contributions of RNA binding to protein function are best discerned by studying “separation-of-function” mutants that hamper interaction with RNA without affecting other aspects of protein function. To design these mutants, we need precise knowledge of the residues that contribute to the affinity of the protein to its RNA ligands. Here, we present RBR-scan: a technology to simultaneously measure RNA-binding affinity of a large number of protein variants. We fused individual variants with unique peptide barcodes optimized for detection by mass spectrometry (MS), purified protein pools from single bacterial culture, and assayed proteins in parallel for RNA binding. Mutations in the MS2 coat protein known to impair RNA-binding were correctly identified, as well as a previously unreported mutant, which we validated with orthogonal biochemical methods. We used RBR-scan to discover novel RNA-binding mutants in the cancer-associated splicing regulator SRSF2. Together, our results demonstrate that RBR-scan is a powerful and scalable platform for linking RNA-binding affinity to protein sequence, offering a novel strategy to decode the functional consequences of protein–RNA interactions.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Julia Tasca

LAB HEAD: Roberto Bonasio

PROVIDER: PXD062465 | Pride | 2025-06-20

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20211229_JT10_MS2CP_Barcodes_DDA_AllQuantifiedPeptides.tsv	Tabular
	MS2_IN_Exp1_Rep1.raw	Raw
	MS2_IN_Exp1_Rep2.raw	Raw
	MS2_IN_Exp2_Rep1.raw	Raw
	MS2_IN_Exp2_Rep2.raw	Raw

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Publications

Pooled scanning of protein variants identifies novel RNA-binding mutants.

Tasca Julia A JA Doherty John F JF Shields Emily J EJ Mudiyanselage Shachinthaka Dissanayaka SD Reich Lauren N LN Sarma Kavitha K Garcia Benjamin A BA Bonasio Roberto R

bioRxiv : the preprint server for biology 20250403

Binding to RNA has been observed for an ever-increasing number of proteins, which often have other functions. The contributions of RNA binding to protein function are best discerned by studying separation-of-function mutants that hamper interaction with RNA without affecting other aspects of protein function. To design these mutants, we need precise knowledge of the residues that contribute to the affinity of the protein for its RNA ligands. Here, we present RBR-scan: a technology to simultaneou ...[more]

PMID: 40236020

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