Proteome-Wide Characterization of the RNA-Binding Protein RALY-Interactome Using the in Vivo-Biotinylation-Pulldown-Quant (iBioPQ) Approach

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.