Abstract
Altered expression of RNA-binding proteins modulates gene expression in association with mRNAs encoding many proto-oncogenes, cytokines, chemokines, and proinflammatory factors. Hu antigen R (HuR), a ubiquitously expressed protein, controls a range of cellular functions such as tumor progression, apoptosis, invasion, and metastasis by stabilizing the AU-rich element located at the 3'-untranslated region (UTR) of target mRNAs. Although significant progress has been made in understanding HuR regulation in gene expression, little is known about how HuR undergoes post-translational modifications and recruits target mRNAs during hypoxic stress. Here, we report that during CoCl(2)-induced hypoxic stress, HuR is significantly overexpressed and undergoes caspase-dependent cleavage in head and neck squamous cell carcinoma cells. Unexpectedly, the HuR-cleavage product 1 (HuR-CP1) was found to strongly associate with the 3'-UTR of c-myc mRNA and block mRNA translation. The binding efficiency of HuR to the 3'-UTR of c-myc mRNA was confirmed using ribonucleoprotein immunoprecipitation and site-directed mutagenesis at the AU-rich element sequences of the c-myc mRNA. Overexpression of a non-cleavable isoform, HuR-D226A, revealed a potent dominant-negative effect, repressing cleavage of endogenous HuR and promoting cell viability. Surprisingly, under hypoxia, siRNA knockdown of HuR elevated c-Myc protein expression. These findings suggest an important role for HuR in hypoxia, and we may have revealed a novel post-transcriptional mechanism that controls c-Myc expression in oral cancer progression.
Highlights
Gene expression is controlled by multiple biological networks, and post-transcriptional gene regulation determines mRNA fate in association with RNA-binding proteins and microRNAs [1, 2]
Hu antigen R (HuR)-CP1 was significantly overexpressed in tumor tissues compared with normal adjacent tissues, and more than 20 –30% of total HuR was cleaved compared with normal adjacent tissues
We report that during hypoxic stress in cultured oral cancer cells or in hypoxic human oral cancer tissues, RNP HuR is exported to the cytoplasm, undergoing caspase-mediated cleavage
Summary
Gene expression is controlled by multiple biological networks, and post-transcriptional gene regulation determines mRNA fate in association with RNA-binding proteins and microRNAs [1, 2]. HuR associates with AUand U-rich elements (AREs) in the 3Ј-UTR of target mRNAs to control transcript stability and protein translation [6]. HuR has been shown to be involved in various cancer processes such as cellular proliferation, differentiation, invasion, metastasis, apoptosis, and angiogenesis in association with target mRNAs [7, 8]. It has been shown that HuR inhibits c-myc translation by recruiting the Let-7-associated RNA microRNA-induced silencing complex (RISC) to the c-myc 3Ј-UTR under non-stress conditions [26]. The HuR cleavage product stabilizes a subset of mRNAs including c-myc and represses c-myc translation. These observations suggest that HuR cleavage at least partially regulates c-Myc expression during tumorigenesis
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