Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates.

Cryo Electron Microscopy and Tomography Core Facility Central European Institute of Technology Masaryk University Kamenice 753 5 625 00 Brno Czech Republic

Cryo EM Knowledge Hub ScopeM Otto Stern Weg 3 ETH Zürich 8093 Zürich Switzerland

Department of Chemistry Biochemistry and Pharmaceutical Sciences University of Bern Freiestrasse 3 3012 Bern Switzerland

Institute of Biochemistry Department of Biology ETH Zürich Hönggerbergring 64 8093 Zürich Switzerland

Proteomics and Mass Spectrometry Core Facility Department for BioMedical Research University of Bern Murtenstrasse 28 3008 Bern Switzerland

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