Shaltouki Atossa, Harford Terri J, Komar Anton A, Weyman Crystal M
Center for Gene Regulation in Health and Disease; Department of Biological, Geological, and Environmental Sciences; Cleveland State University; Cleveland, OH USA.
Translation (Austin). 2013 Apr 1;1(1):e24391. doi: 10.4161/trla.24391. eCollection 2013.
The proapoptotic Bcl-2 family member PUMA is a critical regulator of apoptosis. We have previously shown that PUMA plays a pivotal role in the apoptosis associated with skeletal myoblast differentiation and that a MyoD-dependent mechanism is responsible for the increased expression of PUMA in these cells. Herein, we report that the increased expression of PUMA under these conditions involves regulation at the level of translation. Specifically, we have found that the increase in PUMA protein levels occurs under conditions of decreased total protein synthesis, eIF2-alpha phosphorylation and hypophosphorylation of eIF4E-BP, suggesting that PUMA translation is proceeding via an alternative initiation mechanism. Polyribosome analysis of PUMA mRNA further corroborated this suggestion. A combination of in vitro and ex vivo (cellular) approaches has provided evidence suggesting that PUMA mRNA 5'UTR harbors an Internal Ribosome Entry Site (IRES) element. Using mono- and bi-cistronic reporter constructs, we have delineated an mRNA fragment that allows for cap-independent translation in vitro and ex vivo (in skeletal myoblasts) in response to culture in differentiation media (DM), or in response to treatment with the DNA-damaging agent, etoposide. This mRNA fragment also supports translation in HeLa and 293T cells. Thus, our data has revealed a novel IRES-mediated regulation of PUMA expression in several cell types and in response to several stimuli. These findings contribute to our understanding and potential manipulation of any developmental or therapeutic scenario involving PUMA.
促凋亡的Bcl-2家族成员PUMA是细胞凋亡的关键调节因子。我们之前已经表明，PUMA在与骨骼肌成肌细胞分化相关的细胞凋亡中起关键作用，并且一种依赖MyoD的机制负责这些细胞中PUMA表达的增加。在此，我们报告在这些条件下PUMA表达的增加涉及翻译水平的调控。具体而言，我们发现PUMA蛋白水平的增加发生在总蛋白合成减少、eIF2-α磷酸化以及eIF4E-BP低磷酸化的条件下，这表明PUMA的翻译是通过一种替代起始机制进行的。对PUMA mRNA的多核糖体分析进一步证实了这一推测。体外和体内（细胞）方法的结合提供了证据，表明PUMA mRNA 5'UTR含有一个内部核糖体进入位点（IRES）元件。使用单顺反子和双顺反子报告基因构建体，我们确定了一个mRNA片段，该片段在体外和体内（在骨骼肌成肌细胞中）能够响应分化培养基（DM）培养或DNA损伤剂依托泊苷处理进行不依赖帽的翻译。该mRNA片段也支持在HeLa和293T细胞中的翻译。因此，我们的数据揭示了一种在几种细胞类型中以及对几种刺激响应时由IRES介导的PUMA表达调控。这些发现有助于我们理解和潜在操控任何涉及PUMA的发育或治疗情况。
