Translational control by DHX36 binding to 5\u2032UTR G-quadruplex is essential for muscle stem-cell regenerative functions

Xiaona Chen,Chun Kit Kwok,Guang Xue,Mubarak Ishaq Umar,Wen Wang,Yuying Li,Hao Sun,Eusebio Perdiguero,Yuanchao Xue,Xiaohua Yu,Pura Muñoz‐Cánoves ,Christopher Mann ,Wei Chen,Yoshikuni Nagamine,Joan Isern,Lei Wang,Di Wang,Xi Mou,Silvia Campanario,Jie Yuan,Shiau Wei Liew,Liangqiang He,Yu Zhao,Huating Wang

doi:10.1038/s41467-021-25170-w

Abstract

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5′-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5′ UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5′ UTR of target mRNAs.

Highlights

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells
Quiescent satellite cells (SCs) were isolated by fluorescence-activated cell sorting (FACS) from Pax7-nGFP mice[25], either fixed in situ by 0.5% paraformaldehyde (PFA) prior to muscle digestion (SCT0) or from muscles digested without fixation (SCT8)[26]
Supporting the hypothesis that this translational effect is largely related to 5′ untranslated region (UTR) binding, we found that DHX36 binding to 5′ UTRs conferred a stronger effect on translational efficiency (TE) change as compared to its binding within 3′ UTRs (19) and coding sequences (CDS) (46) (Fig. 5h, i)

Summary

Introduction

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells ( called satellite cells, SCs). Additional studies showed that RBP-mediated RNA degradation played key role in SCs. proteins binding to AU-rich elements (ARE) located in the 3′ UTRs of many mRNAs, such as AUF19, TTP10, and HuR11, regulate SC quiescence maintenance, activation, and differentiation through modulating the stability of their interacting mRNAs. Still, molecular insight into SC post-transcriptional regulation remains largely unknown. DHX36, a DEAH-Box RNA and DNA helicase ( known as RHAU or G4R1)[12,13], has emerged as a key RBP/helicase capable of binding and unwinding G-quadruplex (G4) structures, which are formed by guanine-rich nucleic acids harboring the motif [GX–N1–7–GX–N1–7–GX–N1–7–GX], whereby x is 3–6 nucleotides (nt) and N corresponds to any nt. Whether DHX36 has a regulatory role in the mRNA metabolism of somatic stem cells has never been addressed

Methods

Results

Conclusion