Hypoxia regulates overall mRNA homeostasis by inducing Met1-linked linear ubiquitination of AGO2 in cancer cells

Hailong Zhang,Yanmin Guo,Runhui Lu,Jinke Cheng,Xiaojia Liu,Jian Huang,Ran Chen,Jianfeng He,Lian Li,Caihu Huang,Zhe Qiang,Guo-Qiang Chen,Jianxiu Yu,Yingting Cao,Yanli Wang,Jiayu Fang,Xian Zhao,Jiayi Huang,Qianqian Yang

doi:10.1038/s41467-021-25739-5

Abstract

Hypoxia is the most prominent feature in human solid tumors and induces activation of hypoxia-inducible factors and their downstream genes to promote cancer progression. However, whether and how hypoxia regulates overall mRNA homeostasis is unclear. Here we show that hypoxia inhibits global-mRNA decay in cancer cells. Mechanistically, hypoxia induces the interaction of AGO2 with LUBAC, the linear ubiquitin chain assembly complex, which co-localizes with miRNA-induced silencing complex and in turn catalyzes AGO2 occurring Met1-linked linear ubiquitination (M1-Ubi). A series of biochemical experiments reveal that M1-Ubi of AGO2 restrains miRNA-mediated gene silencing. Moreover, combination analyses of the AGO2-associated mRNA transcriptome by RIP-Seq and the mRNA transcriptome by RNA-Seq confirm that AGO2 M1-Ubi interferes miRNA-targeted mRNA recruiting to AGO2, and thereby facilitates accumulation of global mRNAs. By this mechanism, short-term hypoxia may protect overall mRNAs and enhances stress tolerance, whereas long-term hypoxia in tumor cells results in seriously changing the entire gene expression profile to drive cell malignant evolution.

Highlights

Hypoxia is the most prominent feature in human solid tumors and induces activation of hypoxia-inducible factors and their downstream genes to promote cancer progression
To investigate the role of hypoxia in modulating mRNAs loading to AGO2, RNA immunoprecipitation sequencing (RIP-Seq) of AGO2 were performed by using stable HeLa cells expressing FlagAGO2, which were separately treated with normoxia (21% O2) and hypoxia (1% O2) for 24 h. 11,084 and 13,176 of mRNA transcripts associated with AGO2 were identified under normoxia and hypoxia conditions, respectively, and there were 7638 mRNA transcripts overlapping between normoxia (68.9%) and hypoxia (58.0%) conditions (Supplementary Fig. 1a, Supplementary Data 1)
Cumulative fraction analyses showed that hypoxia significantly decreased the interactions of mRNA transcripts with AGO2 (P = 2.272E−10, Mann–Whitney U test) (Fig. 1a) and distribution plots further showed that hypoxia attenuated 2100 mRNA transcripts and increased 1528 mRNA transcripts associated with AGO2, respectively (Supplementary Fig. 1b)

Summary

Introduction

Hypoxia is the most prominent feature in human solid tumors and induces activation of hypoxia-inducible factors and their downstream genes to promote cancer progression. Dissecting definitely mechanisms of miRNA biogenesis and miRNA-mediated gene silencing, especially in response to physiological or microenvironmental stress stimuli[7], provides a promising therapeutic strategy for cancers. Multiple molecular mechanisms underlying hypoxia promoting tumor evolution are previously described, including its inducing hypoxia-inducible factors (HIFs) and other transcription factors to activate gene transcriptions, enhancing specific oncogenic mRNA translation, as well as affecting the protein stability[8,9]. In response to hypoxia, during tumorigenesis miRNA biogenesis is modulated through post-translational modifications of core processing enzymes of the miRNA pathway[12,13,14], but whether hypoxia possesses the capacity in modulating miRNA functional efficiency and its underlying molecular mechanism is still unexplored. The linear ubiquitin chain assembly complex (LUBAC), consisting of HOIP (RNF31), HOIL-1L (heme-oxidized IRP2 ubiquitin ligase 1 L) and SHAR-

Methods

Results

Conclusion