Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as a novel interactor of cellular G-quadruplex containing transcripts

Abstract

RNA G-quadruplexes (rG4s) are secondary structures in mRNAs known to influence RNA post-transcriptional mechanisms thereby impacting neurodegenerative disease and cancer. A detailed knowledge of rG4–protein interactions is vital to understand rG4 function. Herein, we describe a systematic affinity proteomics approach that identified 80 high-confidence interactors that assemble on the rG4 located in the 5′-untranslated region (UTR) of the NRAS oncogene. Novel rG4 interactors included DDX3X, DDX5, DDX17, GRSF1 and NSUN5. The majority of identified proteins contained a glycine-arginine (GAR) domain and notably GAR-domain mutation in DDX3X and DDX17 abrogated rG4 binding. Identification of DDX3X targets by transcriptome-wide individual-nucleotide resolution UV-crosslinking and affinity enrichment (iCLAE) revealed a striking association with 5′-UTR rG4-containing transcripts which was reduced upon GAR-domain mutation. Our work highlights hitherto unrecognized features of rG4 structure–protein interactions that highlight new roles of rG4 structures in mRNA post-transcriptional control.

Integrative analysis reveals RNA G-Quadruplexes in UTRs are selectively constrained and enriched for functional associations

“…Additionally, some RNA G4 binding proteins that have been characterized in the literature were not significantly enriched for pG4 binding in the ENCODE CLIP-seq data, likely due to their propensity to also bind non-pG4 containing UTR regions. DDX3X, for example, has been shown to bind RNA G4 sequences 33 , yet it was not significantly enriched for G4 binding in our analysis for this reason.…”

“…Binding of tristetraprolin (TTP) leads to exosome complex recruitment and mRNA degradation while binding of HuR stabilizes mRNA with AU-rich 3' UTR32 . In the context of G-quadruplexes, several studies have identified proteins which tend to associate preferentially with DNA and RNA G-quadruplexes using proteomics-based approaches[33][34][35] . Using CLIP-seq data for over 150 proteins published by ENCODE, we uncovered 40 proteins whose binding sites are enriched for pG4 sequences, and identify regulatory modules associating a set of RNA binding proteins, including FAM120A, FASTKD2, and CSTF2T, with pG4 gene targets involved in viral mRNA expression.…”

The DDX5/Dbp2 subfamily of DEAD‐box RNA helicases

“…Exon exclusion may be manifested by reduced hnRNP H/F binding to these G-rich regions that are otherwise exposed in the presence of DDX5 and/or DDX17 (Dardenne et al, 2014). Consistently, DDX5 and DDX17 were found to bind the RNA G-quadruplexes of the NRAS oncogene in vivo (Herdy et al, 2018). However, we do not know if DDX5/DDX17 actively unwind G-quadruplexes or if they are biologically connected to G-quadruplexes.…”

Combining 1,3‐Ditriazolylbenzene and Quinoline to Discover a New G‐Quadruplex‐Interactive Small Molecule Active against Cancer Stem‐Like Cells

“…An emerging and promising new approach to develop more selective anticancer drugs has fourstranded G-quadruplex (G4) DNA structures formed by certain guaninerich-sequences as targets. [1,7,8] Moreover,t he enrichment of theses tructures in promoter regions of, in particular, proto-oncogenesh as been revealed by informaticsa nalysiso ft he human genome [9] and supported by antibody-based G4 chromatin immunoprecipitation and highthroughput sequencing. The G-quartets can be linked by interveningl oop sequences so that they can stack on top of each other to form aG 4, which is additionally stabi-lized in solutionb ym onovalent cations coordinated witht he O6 atoms of guanines.…”

“…[1] There is now good evidence that G4s can be formed in cells, [4,5] where they have important regulatory roles such as in telomerem aintenance, [6] DNA replication, epigenetic responses, and control of gene expression. [1,7,8] Moreover,t he enrichment of theses tructures in promoter regions of, in particular, proto-oncogenesh as been revealed by informaticsa nalysiso ft he human genome [9] and supported by antibody-based G4 chromatin immunoprecipitation and highthroughput sequencing. [10] The discovery that in many cases they negatively control transcription has led to the exploitation of these nucleic acid structures as potential targets for selective anticancer drugs.…”