Identification and characterization of RNA guanine-quadruplex binding proteins

Abstract

Guanine quadruplex (G-quadruplex) motifs in the 5′ untranslated region (5′-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophysical characterization and the inhibitory activity on translation was shown by luciferase reporter assays. In experiments with whole cell extracts from different eukaryotic cell lines, G-quadruplex-binding proteins were isolated by pull-down assays and subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The binding partners of the RNA G-quadruplexes we discovered included several heterogenous nuclear ribonucleoproteins, ribosomal proteins, and splicing factors, as well as other proteins that have previously not been described to interact with nucleic acids. While most of the proteins were specific for either of the investigated G-quadruplexes, some of them bound to both motifs. Selected candidate proteins were subsequently produced by recombinant expression and dissociation constants for the interaction between the proteins and RNA G-quadruplexes in the low nanomolar range were determined by surface plasmon resonance spectroscopy. The present study may thus help to increase our understanding of the mechanisms by which G-quadruplexes regulate translation.

Human proteins that interact with RNA/DNA hybrids

“…However, not all proteins that bind G-quadruplexes bind RNA/DNA hybrids. Most likely, proteins can distinguish between different types of non-B structures; for example, TPM4 (von Hacht et al 2014) and BLM helicase (Li et al 2001;Chatterjee et al 2014) that bind G-quadruplexes did not bind to either hybrid although they are expressed in our B-cell lysates.…”

DNA and RNA Quadruplex-Binding Proteins

“…The binding partners of the RNA G-quadruplexes include several hnRNPs [69]. The hnRNP A1 protein has been demonstrated to accompany the mRNA into the cytoplasm and is also present during translation.…”

G4IPDB: A database for G-quadruplex structure forming nucleic acid interacting proteins

“…The interaction of RNA G- quadruplex structures with several ribosomal proteins has been revealed in 43 S pre-initiation complex that scans mRNA for start codon. The presence of G-quadruplex motif at 5′-UTR of this RNA prevents this recognition of the start codon and causes pathogenicity in the cell16. As these proteins have been found to be involved in several cellular processes, thus their study would lead to insights for the betterment of therapeutics development for various diseases.…”

“…To the best of our knowledge, till date there is no such database available that is solely dedicated to the proteins interacting with nucleic acids forming G-quadruplex structures. Herein, we report the first database that provides detailed information for various proteins that binds to G-quadruplex structures forming DNA and/or RNA such as NCL1618, RHAU1920, BLM Helicase21, UP122, TPP123, IGF224, FMRP25, SRSF116, NOA126, etc. These comprehensive details available on a single source would allow the database users to get all the relevant information in one click that ease drug discovery process in a rational manner.…”