Distinct 5′ UTRs regulate XIAP expression under normal growth conditions and during cellular stress

X-chromosome linked inhibitor of apoptosis, XIAP, is cellular caspase inhibitor and a key regulator of apoptosis. We and others have previously shown that XIAP expression is regulated primarily at the level of protein synthesis; the 5′ untranslated region (UTR) of XIAP mRNA contains an Internal Ribosome Entry Site (IRES) that supports cap-independent expression of XIAP protein during conditions of pathophysiological stress, such as serum deprivation or gamma irradiation. Here, we show that XIAP is encoded by two distinct mRNAs that differ in their 5′ UTRs. We further show that the dominant, shorter, 5′ UTR promotes a basal level of XIAP expression under normal growth conditions. In contrast, the less abundant longer 5′ UTR contains an IRES and supports cap-independent translation during stress. Our data suggest that the combination of alternate regulatory regions and distinct translational initiation modes is critical in maintaining XIAP levels in response to cellular stress and may represent a general mechanism of cellular adaptation

Cloning and characterization of the rat homologues of the Inhibitor of Apoptosis protein 1, 2, and 3 genes.

BACKGROUND: Inhibitor of Apoptosis (IAP) proteins are key intrinsic regulators of apoptosis induced by a variety of triggers. We isolated the rat Inhibitor of Apoptosis genes 1, 2 and 3 and characterized their tissue distribution and expression. RESULTS: Rat iap-1 encodes a protein of 67.1 kDa with 73 % and 89.2 % homology to human and mouse iap-1 respectively. Rat iap-2 encodes a protein of 66.7 kDa with 81.6 % and 89.3 % homology to human and mouse iap-2 respectively. Rat iap-3 encodes a protein of 56.1 kDa with 89.5 % and 93.1 % homology to human and mouse iap-3 respectively. We have generated rabbit polyclonal antibodies against all three rat IAP genes. Northern and Western blot analysis detected rat IAP transcripts and proteins in majority of the tissues examined. In addition, a shorter, alternatively spliced transcript corresponding to iap-2 was found in testes. CONCLUSIONS: We have identified three rat homologues of the IAP genes. The elevated expression of rat iap-1 and iap2 in testes suggests that these two genes play an important antiapoptotic role in spermatogenesis

A search for structurally similar cellular internal ribosome entry sites

Internal ribosome entry sites (IRES) allow ribosomes to be recruited to mRNA in a cap-independent manner. Some viruses that impair cap-dependent translation initiation utilize IRES to ensure that the viral RNA will efficiently compete for the translation machinery. IRES are also employed for the translation of a subset of cellular messages during conditions that inhibit cap-dependent translation initiation. IRES from viruses like Hepatitis C and Classical Swine Fever virus share a similar structure/function without sharing primary sequence similarity. Of the cellular IRES structures derived so far, none were shown to share an overall structural similarity. Therefore, we undertook a genome-wide search of human 5′UTRs (untranslated regions) with an empirically derived structure of the IRES from the key inhibitor of apoptosis, X-linked inhibitor of apoptosis protein (XIAP), to identify novel IRES that share structure/function similarity. Three of the top matches identified by this search that exhibit IRES activity are the 5′UTRs of Aquaporin 4, ELG1 and NF-kappaB repressing factor (NRF). The structures of AQP4 and ELG1 IRES have limited similarity to the XIAP IRES; however, they share trans-acting factors that bind the XIAP IRES. We therefore propose that cellular IRES are not defined by overall structure, as viral IRES, but are instead dependent upon short motifs and trans-acting factors for their function

IRES-mediated translation of cellular messenger RNA operates in eIF2α- independent manner during stress

Physiological and pathophysiological stress attenuates global translation via phosphorylation of eIF2α. This in turn leads to the reprogramming of gene expression that is required for adaptive stress response. One class of cellular messenger RNAs whose translation was reported to be insensitive to eIF2α phosphorylation-mediated repression of translation is that harboring an Internal Ribosome Entry Site (IRES). IRES-mediated translation of several apoptosis-regulating genes increases in response to hypoxia, serum deprivation or gamma irradiation and promotes tumor cell survival and chemoresistance. However, the molecular mechanism that allows IRES-mediated translation to continue in an eIF2α-independent manner is not known. Here we have used the X-chromosome linked Inhibitor of Apoptosis, XIAP, IRES to address this question. Using toeprinting assay, western blot analysis and polysomal profiling we show that the XIAP IRES supports cap-independent translation when eIF2α is phosphorylated both in vitro and in vivo. During normal growth condition eIF2α-dependent translation on the IRES is preferred. However, IRES-mediated translation switches to eIF5B-dependent mode when eIF2α is phosphorylated as a consequence of cellular stress

IRES-mediated translation of cellular messenger RNA operates in eIF2α- independent manner during stress

Physiological and pathophysiological stress attenuates global translation via phosphorylation of eIF2α. This in turn leads to the reprogramming of gene expression that is required for adaptive stress response. One class of cellular messenger RNAs whose translation was reported to be insensitive to eIF2α phosphorylation-mediated repression of translation is that harboring an Internal Ribosome Entry Site (IRES). IRES-mediated translation of several apoptosis-regulating genes increases in response to hypoxia, serum deprivation or gamma irradiation and promotes tumor cell survival and chemoresistance. However, the molecular mechanism that allows IRES-mediated translation to continue in an eIF2α-independent manner is not known. Here we have used the X-chromosome linked Inhibitor of Apoptosis, XIAP, IRES to address this question. Using toeprinting assay, western blot analysis and polysomal profiling we show that the XIAP IRES supports cap-independent translation when eIF2α is phosphorylated both in vitro and in vivo. During normal growth condition eIF2α-dependent translation on the IRES is preferred. However, IRES-mediated translation switches to eIF5B-dependent mode when eIF2α is phosphorylated as a consequence of cellular stress

The eIF4G homolog DAP5/p97 supports the translation of select mRNAs during endoplasmic reticulum stress

DAP5/p97 is a member of the eIF4G family of translation initiation factors that has been suggested to play an important role in the translation of select messenger RNA molecules. We have shown previously that the caspase-cleaved form of DAP5/p97, termed p86, is required for the induction of the endoplasmic reticulum (ER)-stress-responsive internal ribosome entry site (IRES) of the caspase inhibitor HIAP2. We show here that expression of DAP5/p97 is enhanced during ER stress by selective recruitment of DAP5/p97 mRNA into polysomes via the DAP5/p97 IRES. Importantly, enhanced translation mediated by the DAP5/p97 IRES is dependent on DAP5/p97 itself, thus providing a positive feedback loop. In addition, we show that activation of DAP5/p97 and HIAP2 IRES during ER stress requires DAP5/p97. Significantly, the induction of DAP5/p97 during ER stress is caspase-independent, whereas the induction of HIAP2 requires proteolytic processing of DAP5/p97. Thus, DAP5/p97 is a translational activator that selectively modulates translation of specific mRNAs during conditions of cellular stress in both a caspase-dependent and caspase-independent manner

An internal ribosomal entry site mediates redox-sensitive translation of Nrf2

Nrf2 plays pivotal roles in coordinating the antioxidant response and maintaining redox homeostasis. Nrf2 expression is exquisitely regulated; Nrf2 expression is suppressed under unstressed conditions but strikingly induced under oxidative stress. Previous studies showed that stress-induced Nrf2 up-regulation results from both the inhibition of Nrf2 degradation and enhanced Nrf2 translation. In the present study, we elucidate the mechanism underlying translational control of Nrf2. An internal ribosomal entry site (IRES) was identified within the 5′ untranslated region of human Nrf2 mRNA. The IRESNrf2 contains a highly conserved 18S rRNA binding site (RBS) that is required for internal initiation. This IRESNrf2 also contains a hairpin structured inhibitory element (IE) located upstream of the RBS. Deletion of this IE remarkably enhanced translation. Significantly, treatment of cells with hydrogen peroxide (H2O2) and phyto-oxidant sulforaphane further stimulated IRESNrf2-mediated translation initiation despite the attenuation of global protein synthesis. Polyribosomal profile assay confirmed that endogenous Nrf2 mRNAs were recruited into polysomal fractions under oxidative stress conditions. Collectively, these data demonstrate that Nrf2 translation is suppressed under normal conditions and specifically enhanced upon oxidant exposure by internal initiation, and provide a mechanistic explanation for translational control of Nrf2 by oxidative stress