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.
Highlights
Recognition of mRNA secondary structures by RNA binding proteins (RBPs) is essential for post-transcriptional control to influence mRNA processing, stability, transport and translation [1,2]
Anti-MYC, Hemagglutinin tag, DHX36, DDX5, DDX17, DHX9, DHX29 and DHX30 antibodies were purchased from Abcam, the V5-tag antibody was obtained from Source BioScience, DDX3X antibody was ordered from Santa Cruz and FXR1 antibody was purchased from Cell Signaling
Biotinylated oligonucleotides containing the RNA G4s (rG4) sequence found in the 5 -untranslated region (UTR) of NRAS were folded into a rG4 structure
Summary
Recognition of mRNA secondary structures by RNA binding proteins (RBPs) is essential for post-transcriptional control to influence mRNA processing, stability, transport and translation [1,2]. G4 structures form from guanine (G)-rich sequences in which stacks of G-quartets are stabilized by a central metal cation (Figure 1A). High-throughput sequencing combined with reverse transcriptase stalling at stabilized RNA G4s (rG4) has revealed over 13 000 loci where rG4 structures form within the human transcriptome in vitro [5,6]. Evidence supporting rG4 formation in cells includes detection of rG4s in the cytoplasm by immunofluorescence using a G4 structure-specific antibody [7,8]. RG4s are enriched in functionally important regions, including 5 - and 3 -untranslated regions (UTRs) [5,6,9,10,11]
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