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

Gerhard Dürnberger,Thomas R Burkard,Andreas Schönegger,Tobias Doerks,Ines Kaupe,Keiryn L Bennett,André C Müller,Gerhard F Ecker,Evren Karayel,Kilian Huber,Giulio Superti-Furga,Tilmann Bürckstümmer,Jacques Colinge,Hans Lohninger,Roberto Giambruno,Peer Bork

doi:10.1186/gb-2013-14-7-r81

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.

Highlights

The 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
We opted for the synthesis of baits containing all possible dinucleotide combinations comprising single-stranded RNA, single-stranded DNA and double-stranded DNA (Figure 1a)
The synthetic oligonucleotides were coupled to a matrix by a 5’ biotin moiety and used to purify nucleic acid-binding protein (NABP) from the biological samples and the enriched proteins were subsequently identified by mass spectrometry (MS) (Figure 1a)

Summary

Introduction

The 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. Interactions between proteins and nucleic acids play a pivotal role in a wide variety of essential biological processes, such as transcription, translation, splicing, or chromatin remodeling, defects in which can cause multiple diseases [1]. Transcription factors that recognize specific DNA motifs constitute only part of the nucleic acid-binding proteins (NABPs), which include less sequence-specific interactors. The dataset provided new insight into the structural properties of mRNA-binding proteins, such as being enriched for short repetitive amino acid motifs and highly intrinsically disordered

Methods

Results

Conclusion