title: Proteome-wide characterization of the RNA-binding protein RALY-interactome using the in vivo-biotinylation-pulldown-quant (iBioPQ) approach.
abstract: RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY's interaction partners, termed iBioPQ, that involves in vivo biotinylation of biotin acceptor peptide (BAP)-fused protein in the presence of the prokaryotic biotin holoenzyme synthetase of BirA so that it can be purified using streptavidin-coated magnetic beads, circumventing the need for specific antibodies and providing efficient pulldowns. Protein eluates were subjected to tryptic digestion and identified using data-independent acquisition on an ion-mobility enabled high-resolution nanoUPLC-QTOF system. Using label-free quantification, we identified 143 proteins displaying at least 2-fold difference in pulldown compared to controls. Gene Ontology overrepresentation analysis revealed an enrichment of proteins involved in mRNA metabolism and translational control. Among the most abundant interacting proteins, we confirmed RNA-dependent interactions of RALY with MATR3, PABP1 and ELAVL1. Comparative analysis of pulldowns after RNase treatment revealed a protein-protein interaction of RALY with eIF4AIII, FMRP, and hnRNP-C. Our data show that RALY-containing RNPs are much more heterogeneous than previously hypothesized.
authors.affiliation: Institute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Tenzer
authors.forename: Albertomaria
authors.initials: A
authors.lastname: Moro
authors.forename: Jörg
authors.initials: J
authors.lastname: Kuharev
authors.forename: Ashwanth Christopher
authors.initials: AC
authors.lastname: Francis
authors.forename: Laura
authors.initials: L
authors.lastname: Vidalino
authors.forename: Alessandro
authors.initials: A
authors.lastname: Provenzani
authors.forename: Paolo
authors.initials: P
authors.lastname: Macchi
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay; D001710:Biotin / Q000737:chemistry*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051959:ELAV Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000067780:ELAV-Like Protein 1; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D034521:Nuclear Matrix-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D039122:Poly(A)-Binding Protein I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D020543:Proteome / Q000032:analysis*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry
chemical_list: D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D029968:Escherichia coli Proteins; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; C518587:MATR3 protein, human; D034521:Nuclear Matrix-Associated Proteins; D039122:Poly(A)-Binding Protein I; D020543:Proteome; C088730:RALY protein, human; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D001710:Biotin; D019809:Streptavidin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli