Reich, L. N. | bioRxiv Science

bioRxiv2025

Pseudouridine selects RNAs for extracellular transport

RNAs move through the extracellular space to transmit information between cells, including mammalian neurons, yet how specific RNAs are channeled into these extracellular routes is unknown. Using genome-wide CRISPR screening, proteomics, and high-sensitivity transcriptomics in a neuronal model system, we identify domesticated retroviral proteins and RNA-modifying enzymes that regulate RNA loading into and transportation via extracellular vesicles. We show that the pseudouridine synthase PUS1 is a key determinant of RNA trafficking, and that its catalytic product in RNA, pseudouridine, is enriched in extracellular RNAs from transformed and primary neurons. Furthermore, the presence of pseudouridine on select RNAs is both necessary and sufficient for their extracellular export. Finally, we show that myosin light chain 6 (MYL6) is a pseudouridine-binding protein required for secretion of synthetic and endogenous RNAs. These findings reveal a biochemical code linking chemical RNA modification to extracellular transport, and establish a framework to study the function of extracellular RNAs in the nervous system and beyond.

molecular biology↗

bioRxiv2025

Pooled scanning of protein variants identifies novel RNA-binding mutants

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 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.

biochemistry↗