RNA G-quadruplex structures control ribosomal protein production

Dhaval Varshney,Shankar Balasubramanian,Sergio Martinez Cuesta,David Tannahill,Barbara Herdy,Ummi Binti Abdullah

doi:10.1038/s41598-021-01847-6

Abstract

Four-stranded G-quadruplex (G4) structures form from guanine-rich tracts, but the extent of their formation in cellular RNA and details of their role in RNA biology remain poorly defined. Herein, we first delineate the presence of endogenous RNA G4s in the human cytoplasmic transcriptome via the binding sites of G4-interacting proteins, DDX3X (previously published), DHX36 and GRSF1. We demonstrate that a sub-population of these RNA G4s are reliably detected as folded structures in cross-linked cellular lysates using the G4 structure-specific antibody BG4. The 5′ UTRs of protein coding mRNAs show significant enrichment in folded RNA G4s, particularly those for ribosomal proteins. Mutational disruption of G4s in ribosomal protein UTRs alleviates translation in vitro, whereas in cells, depletion of G4-resolving helicases or treatment with G4-stabilising small molecules inhibit the translation of ribosomal protein mRNAs. Our findings point to a common mode for translational co-regulation mediated by G4 structures. The results reveal a potential avenue for therapeutic intervention in diseases with dysregulated translation, such as cancer.

Highlights

Four-stranded G-quadruplex (G4) structures form from guanine-rich tracts, but the extent of their formation in cellular RNA and details of their role in RNA biology remain poorly defined
We demonstrate a direct role for RNA G4s in the regulation of cellular ribosomal protein production and discuss their possible role in regulating global translation
Cytoplasmic RNA-protein complexes were isolated from nitrocellulose membranes (Fig. S1c) and cDNA synthesis performed in lithium buffers to prevent reverse transcriptase (RT) stalling at G-rich r egions[5]

Summary

Introduction

Four-stranded G-quadruplex (G4) structures form from guanine-rich tracts, but the extent of their formation in cellular RNA and details of their role in RNA biology remain poorly defined. A recent in-situ chemical mapping study using azido-kethoxal in combination with RT-stalling provided evidence for the formation of RNA G4s in c ells[9]. The ability of such methods to detect G4 structures is somewhat limited as they can alter the equilibrium by chemically trapping dynamic structures in an unfolded state and the reaction conditions need to be carefully optimised to sample dynamic or lowly populated structures (see[10] for more extensive discussion). We present evidence for frequent G4 formation in the cellular transcriptome through experiments that exploit endogenous RNA binding proteins and an engineered G4 structure-specific antibody. We demonstrate a direct role for RNA G4s in the regulation of cellular ribosomal protein production and discuss their possible role in regulating global translation

Methods

Results

Conclusion