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// Yingxiao Chen 1 , Yue-Gang Wang 1 , Yuhuang Li 1 , Xiao-Xin Sun 1 , Mu-Shui Dai 1 1 Department of Molecular and Medical Genetics, School of Medicine, and The OHSU Knight Cancer Institute, Oregon Health and Science University, Portland, OR 97239, USA Correspondence to: Mu-Shui Dai, email: daim@ohsu.edu Keywords: deubiquitinating enzymes, MDMX, Otub1, p53, apoptosis Received: November 04, 2016 Accepted: December 20, 2016 Published: December 27, 2016 ABSTRACT Otub1 regulates p53 stability and activity via non-canonical inhibition of UbcH5, the MDM2 cognate ubiquitin-conjugating enzyme (E2). However, whether Otub1 regulates MDMX stability and activity is not clear. Here we report that Otub1 also suppresses MDM2-mediated MDMX ubiquitination in cells and in vitro , independently of its deubiquitinating enzyme activity. Consequently, overexpression of Otub1 markedly stabilized MDMX and increased its levels, whereas knockdown of Otub1 reduced the levels of MDMX. Interestingly, MDMX induced by Otub1 can localize to mitochondria in addition to the cytosol, enhance p53 phosphorylation at S46 (p53S46P) and promote mitochondria-mediated apoptotic pathway. Knockdown of MDMX reduced Otub1-induced p53S46P, which was shown to be critical for p53’s mitochondrial function and apoptotic activity. Furthermore, Otub1 promotes UV-irradiation-induced p53S46P and apoptosis, which can be significantly inhibited by MDMX depletion. Together, these results suggest that Otub1 stabilizes MDMX and promotes p53S46P and mitochondria-mediated apoptosis, providing an alternative mechanism of Otub1’s role in apoptosis.

A number of ribosomal proteins (RPs) have been shown to play a critical role in coordinating ribosome biogenesis with cell growth and proliferation by suppressing MDM2 to induce p53 activation. While how the MDM2-p53 pathway is regulated by multiple RPs is unclear, it remains to be interesting to identify additional RPs that can regulate this pathway. Here we report that ribosomal protein L4 (RPL4) directly interacts with MDM2 at the central acidic domain and suppresses MDM2-mediated p53 ubiquitination and degradation, leading to p53 stabilization and activation. Interestingly, overexpression of RPL4 promotes the binding of MDM2 to RPL5 and RPL11 and forms a complex with RPL5, RPL11 and MDM2 in cells. Conversely, knockdown of RPL4 also induces p53 levels and p53-dependent cell cycle arrest. This p53-dependent effect requires both RPL5 and RPL11, suggesting that depletion of RPL4 triggers ribosomal stress. Together, our results reveal that balanced levels of RPL4 are critical for normal cell growth and proliferation via regulating the MDM2-p53 loop.

// Yuhuang Li 1 , Kishore B. Challagundla 1 , Xiao-Xin Sun 1 , Qinghong Zhang 2 , Mu-Shui Dai 1 1 Departments of Molecular & Medical Genetics, School of Medicine and the OHSU Knight Cancer Institute, Oregon Health & Science University, Portland, OR 97239, USA 2 Department of Dermatology, University of Colorado, Denver, Aurora, CO 80045, USA Correspondence to: Mu-Shui Dai, e-mail: daim@ohsu.edu Keywords: miR-130a, L11, c-Myc, microRNA, UV irradiation Received: September 17, 2014 Accepted: November 08, 2014 Published: December 31, 2014 ABSTRACT The oncoprotein c-Myc is essential for cell growth and proliferation while its deregulated overexpression is associated with most human cancers. Thus tightly regulated levels and activity of c-Myc are critical for maintaining normal cell homeostasis. c-Myc is down-regulated in response to several types of stress, including UV-induced DNA damage. Yet, mechanism underlying UV-induced c-Myc reduction is not completely understood. Here we report that L11 promotes miR-130a targeting of c-myc mRNA to repress c-Myc expression in response to UV irradiation. miR-130a targets the 3’-untranslated region (UTR) of c-myc mRNA. Overexpression of miR-130a promotes the Ago2 binding to c-myc mRNA, significantly reduces the levels of both c-Myc protein and mRNA and inhibits cell proliferation. UV treatment markedly promotes the binding of L11 to miR-130a, c-myc mRNA as well as Ago2 in cells. Inhibiting miR-130a significantly suppresses UV-mediated c-Myc reduction. We further show that L11 is relocalized from the nucleolus to the cytoplasm where it associates with c-myc mRNA upon UV treatment. Together, these results reveal a novel mechanism underlying c-Myc down-regulation in response to UV-mediated DNA damage, wherein L11 promotes miR-130a-loaded miRISC to target c-myc mRNA.