Abstract
HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52 (ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3′-UTR (untranslated region)-dependent poly(A)-tail-length-independent destabilization in HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body (processing body) components and we confirmed further that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.
Highlights
Cells respond to reduced oxygen tension by executing a transcriptional programme that is principally orchestrated by hypoxia-inducible factor 1α (HIF1A) [1]
We identified a pool of short interfering RNA (siRNA) against ubiquitin-specific protease 52 (USP52) that decrease hypoxia-dependent HREresponse activity
We were only able to deplete USP52 by approximately 50 % as judged by immunoblot (Figure 1B) and real-time PCR (Supplementary Figure S1A at http://www.biochemj.org/bj/451/bj4510185add.htm) analysis. This is consistent with previous reports in mouse NIH 3T3 cells, where siRNA was reported to reduce USP52 by a maximum of ∼ 65 % compared with control siRNA [17]
Summary
Cells respond to reduced oxygen tension by executing a transcriptional programme that is principally orchestrated by HIF1A (hypoxia-inducible factor 1α) [1]. Examination of GLUT1 and LDHA levels by immunoblot analysis of hypoxic U2OS cells revealed that USP52 depletion impairs the accumulation of GLUT1 and LDHA in response to hypoxia (Figure 2A), demonstrating that USP52 is required to potentiate the HIF1A-mediated hypoxic response.
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