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IRES-mediated translation of the pro-apoptotic Bcl2 family member PUMA.

ABSTRACT: 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.

SUBMITTER: Shaltouki A

PROVIDER: S-EPMC4718066 | biostudies-other | 2013

REPOSITORIES: biostudies-other

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Project description:BackgroundLithium is considered by many as the gold standard medication in the management of bipolar disorder (BD). However, the clinical response to lithium is heterogeneous, and the molecular basis for this difference in response is unknown. In the present study, we sought to determine how the peripheral blood gene expression profiles of patients with bipolar disorder (BD) changed over time following intitiation of treatment with lithium, and whether differences in those profiles over time were related to the clinical response.MethodsIllumina Sentrix Beadchip (Human-6v2) microarrays containing?>?48,000 transcript probes were used to measure levels of expression of gene-expression in peripheral blood from 20 depressed subjects with BD prior to and every two weeks during 8?weeks of open-label treatment with lithium.Changes in gene-expression were compared between treatment responders (defined as a decrease in the Hamilton Depression Rating Scale of 50% or more) and non-responders. Pathway analysis was conducted using GeneGO Metacore software.Results127 genes showed a differential response in responders vs. non-responders. Pathway analysis showed that regulation of apoptosis was the most significantly affected pathway among these genes. Closer examination of the time-course of changes among BCL2 related genes showed that in lithium-responders, one month after starting treatment with lithium, several anti-apoptotic genes including Bcl2 and insulin receptor substrate 2 (IRS2) were up-regulated, while pro-apoptotic genes, including BCL2-antagonist/killer 1 (BAK1) and BCL2-associated agonist of cell death (BAD), were down-regulated. In contrast, in lithium non-responders, BCL2 and IRS2 were down-regulated, while BAK1 and BAD up-regulated at the one-month time-point.ConclusionsThese results suggest that differential changes in the balance of pro- and anti- apoptotic gene-expression following treatment with lithium may explain some of the heterogeneity in clinical response in BD patients.

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IRES-mediated translation of the pro-apoptotic Bcl2 family member PUMA.

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