Dataset Information

Characterizations of G-quadruplex RNA-Protein Interactions in Living Cells

ABSTRACT: RNA guanine quadruplexes (rG4s) are non-canonical nucleic acid structures essential for various cellular functions and disease pathways. Characterizing rG4-interacting proteins (rG4IPs) is crucial for deciphering biological roles of rG4s. Building upon the RNA-protein interaction detection (RaPID) method, we introduced G4-RaPID – a tailored strategy for robustly profiling rG4IPs in living cells. Using G4-RaPID with three distinct rG4 sequences in live cells, we identified 105 candidate rG4IPs with diverse biological functions. We also validated the direct binding of recombinant hnRNPA0, CHD4, and IGF2BP1 proteins with rG4 structures in vitro. Additionally, our CLIP-Seq data revealed an enrichment of hnRNPA0 at rG4 loci. Moreover, luciferase reporter assay results showed that hnRNPA0 interacts with the rG4 in the 5UTR of NRAS mRNA to negatively regulate its translation. Together, we developed an rG4-RaPID method for characterizing rG4-protein interactions in living cells, and documented the functions of hnRNPA0-rG4 interaction in modulating the translation of NRAS mRNA.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: feng tang

LAB HEAD: Yinsheng Wang

PROVIDER: PXD070623 | Pride | 2026-06-01

REPOSITORIES: Pride

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Characterizations of G-Quadruplex RNA-Protein Interactions in Living Cells.

Tang Feng F Liang Xiaochen X Porter Douglas F DF Miao Weili W Maehlmann Kevin K Yuan Jun J Khavari Paul A PA Wang Yinsheng Y

Analytical chemistry 20260417 16

RNA guanine quadruplexes (rG4s) are noncanonical nucleic acid structures that contribute to diverse cellular functions and disease mechanisms. Defining the proteins that interact with rG4s (rG4IPs) is essential for elucidating their biological roles. Here, we build on the RNA-protein interaction detection (RaPID) platform to develop G4-RaPID, a tailored chemoproteomic strategy for the unbiased profiling of rG4IPs in living cells. Using G4-RaPID, we identified 105 candidate rG4IPs that were commo ...[more]

PMID: 41996289

Similar Datasets

Project description:RNA structures are of crucial importance for biological function and gene regulation. Guanine (G)-rich sequences in RNA can assemble to form RNA G-quadruplex (rG4) structures; specific rG4s have been shown to regulate gene expression, and are associated with human diseases. However, no transcriptome-wide method has been reported to map rG4s, limiting our understanding of the rG4 structure and its relationship with function and regulation. Here we introduce a transcriptome-wide rG4 profiling method, rG4-Seq, in which the rG4-mediated reverse transcriptase stalling is read out by next-generation sequencing at nucleotide resolution. We apply this method to human RNA and report the first global map of rG4 structures for any organism. Our analysis identifies over 3,000 rG4s, and increases to over 11,000 upon addition of a rG4 stabilizing ligand Pyridostatin (PDS). Notably, we discover that besides canonical rG4s, many rG4s are non-canonical with novel structural features such as long-loops, bulges, and 2-quartet. We also find that rG4 formation is associated with its stability, Cytosine (C)-content, and the stability of alternative RNA structures. Our data reveals that rG4s can be found in messenger RNAs (mRNAs) and long intergenic noncoding RNAs (lincRNAs). In mRNAs, rG4s are enriched in untranslated regions, and are significantly correlated with microRNA target sites and polyadenylation signals. Remarkably, we found that majority of our in vitro identified rG4s can change the in silico predicted RNA structure to uncover alternative RNA conformation.. Futhermore, the rG4s that have analogues in many ortholog genes across different species show a preferential association with RNA processing and stability. rG4-seq is a broadly applicable method that enables the RNA G4 structurome and its biological roles to be interrogated and can be readily applied in other organisms on a transcriptome-wide scale. 12 samples, 150 bp single-ended RNA-Seq libraries from cell extracts after performing RTS reaction in different buffers (Li, K and K+PDS rich buffer). Each of the 3 conditions has 4 libraries from independent biological replicates.

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	RAPID-APP-1.raw	Raw
	RAPID-APP-2.raw	Raw
	RAPID-APP-3.raw	Raw
	RAPID-CFG4-1.raw	Raw
	RAPID-CFG4-2.raw	Raw