Experimental characterization of the human non-sequence-specific nucleic acid interactome

Abstract

BackgroundThe interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported.ResultsWe designed an experimental approach to achieve broad coverage of the non-sequence-specific RNA and DNA binding space, including methylated cytosine, and tested for interaction potential with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high-confidence direct binders, 139 of which were novel and 237 devoid of previous experimental evidence. We could assign specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and individual domains. The evolutionarily conserved protein YB-1, previously associated with cancer and drug resistance, was shown to bind methylated cytosine preferentially, potentially conferring upon YB-1 an epigenetics-related function.ConclusionsThe dataset described here represents a rich resource of experimentally determined nucleic acid-binding proteins, and our methodology has great potential for further exploration of the interface between the protein and nucleic acid realms.

Oncofetal HMGA2 effectively curbs unconstrained (+) and (−) DNA supercoiling

“…This binding is indicated by a moderate decrease in ss DNA extension in a few pN force range. Although the ss DNA binding activity is unexpected due to the lack of typical ss DNA binding domains such as OB fold, the interaction between ss DNA and HMGA2 was also suggested in a recent study of nucleic acid-protein interaction screen 18 . This ss DNA binding was observed at protein concentration greater than 50 nM, much higher than the k D of the replication protein A (RPA) (~1 nM) 19 .…”

The RNA‐binding protein HuR/ELAVL1 regulates IFN‐β mRNA abundance and the type I IFN response

“…An affinity purification (AP) combined with MS approach was used to identify interactors specific for the ARE of the 3′UTR of the IFN‐β mRNA (Supporting Information Fig. 2A, scheme of procedure) . In order to decrease unspecific interactions, a short biotinylated 20‐nucleotide fragment of the human IFN‐β mRNA (ARE20) and a control RNA (CTR) were chosen as baits for AP of RBPs (Fig.…”

“…APs were performed as previously described . HelaS3 cells (4 × 10 6 cells) were lysed in RNA pull down buffer (20 mM Hepes, pH 8, 100 mM NaCl, 20% v/v glycerol, 0.2 mM EDTA, 1 mM DTT, 0.01% Nonidet‐P40, 50 μg/mL yeast tRNA (Ambion), 160 U/mL RNasin) containing protease inhibitors by three cycles of freeze and thaw.…”

The mRNA-bound proteome of the early fly embryo

“…Many Drosophila RBPs, including Oskar, Staufen, and Nanos, were identified through genetic screens, but a complete understanding of the role of RNA in embryogenesis require comprehensive, transcriptome-wide methods. Previous studies have described in vitro approaches for discovering large numbers of RBPs, including screening protein arrays for RNA-binding activities (Scherrer et al 2010;Tsvetanova et al 2010) and RNA affinity chromatography of cellular extracts followed by mass spectrometry (Tsvetanova et al 2010;Dürnberger et al 2013). In vivo, UV crosslinking has been used to capture physiological protein-mRNA interactions, which are then purified by oligo(dT) affinity chromatography and analyzed by mass spectrometry.…”