Hypoxia-mediated Selective mRNA Translation by an Internal Ribosome Entry Site-independent Mechanism

Abstract

Although it is advantageous for hypoxic cells to inhibit protein synthesis and conserve energy, it is also important to translate mRNAs critical for adaptive responses to hypoxic stress. Because internal ribosome entry sites (IRES) have been postulated to mediate this preferential synthesis, we analyzed the 5-untranslated regions from a panel of stress-regulated mRNAs for m 7 GTP cap-independent translation and identified putative IRES elements in encephalomyocarditis virus, vascular endothelial growth factor, hypoxia-inducible factors (HIFs) 1␣ and 2␣, glucose transporter-like protein 1, p57Kip2 , La, BiP, and triose phosphate isomerase transcripts. However, when capped and polyadenylated dicistronic RNAs were synthesized in vitro and transfected into cells, cellular IRES-mediated translation accounted for less than 1% that of the level of cap-dependent translation. Moreover, hypoxic stress failed to activate cap-independent synthesis, indicating that it is unlikely that this is the primary mechanism for the maintenance of the translation of these mRNAs under low O 2 . Furthermore, although HIF-1␣ is frequently cited as an example of an mRNA that is preferentially translated, we demonstrate that under different levels and durations of hypoxic stress, changes in newly synthesized HIF-1␣ and ␤-actin protein levels mirror alterations in corresponding mRNA abundance. In addition, our data suggest that cyclin-dependent kinase inhibitor p57 Kip2 and vascular endothelial growth factor mRNAs are selectively translated by an IRES-independent mechanism under hypoxic stress.Hypoxic stress is a central component of normal development and physiology as well as the pathology of multiple human diseases (1-4). Cells adapt to low oxygen (O 2 ) levels by altering their mRNA expression and translation profiles (5, 6). Most of the transcriptional effects are mediated by a family of hypoxia inducible factors (HIFs) 2 that transactivate genes by binding to hypoxia response elements in their promoters, introns, and/or enhancers (4, 7). In addition to the transcriptional effects mediated by HIF, hypoxic stress is associated with energy starvation (8, 9) and alterations in cell cycle progression (10, 11). Furthermore, whereas global protein synthesis is attenuated under low O 2 , select mRNAs believed to be involved in the adaptive response to hypoxia are preferentially translated.Hypoxia-mediated inhibition of general protein synthesis (12) is regulated primarily by the modification of eukaryotic translation initiation factors (eIFs) at two steps; that is, regeneration of the "ternary complex" (eIF2-GTP and met-tRNA) and regulation of the m 7

Stress-resistant Translation of Cathepsin L mRNA in Breast Cancer Progression

“…This means all 5Ј UTR splice variants of CTSL are recruited to ribosomes with the same efficiency. Indeed, the biological relevance of IRES-mediated translation on cellular mRNAs has recently been doubted for some classical cellular "IRES genes" like hypoxia inducible factor and vascular endothelial growth factor (24,40). Thus, although IRES sequences proved to function in bicistronic assays, they might play a minor role in translational regulation in cancers or upon cell stress, at least in case of CTSL.…”

Alternative Ways to Think about Cellular Internal Ribosome Entry

“…A typical Northern blot exposure is simply not adequate to rule out the possibility that 1% of the total mRNA encoding the 3Ј-cistron is monocistronic. This is the appropriate level of detection to consider for most putative cellular IRESs, whose demonstrably IRES-dependent translation is only ϳ1% as efficient as cap-dependent translation of a control reporter mRNA (8,9,16). It is important to note that the popular Renilla/firefly luciferase reporter is not the only bicistronic reporter system vulnerable to cryptic promoter artifacts.…”

“…Most putative cellular IRESs are much less active than their viral counterparts when tested in assays that reliably measure translational activity (in vitro translation or in vivo translation of transfected in vitro transcribed mRNAs) (8,9,16). Although it is true that the presumed raison d'être of most cellular IRESs, to permit expression of key regulatory proteins, often of low abundance, under conditions of global inhibition of cap-dependent translation, does not require that cellular IRES-dependent initiation be nearly as efficient as viral IRES-dependent translation (which typically drives unregulated high-level expression of viral proteins), the very low level of activity of most putative cellular IRESs makes it far more difficult to rule out alternative explanations for activity in bicistronic reporter assays, mechanisms that do not involve translation at all, such as cryptic promoter activity.…”

Hypoxia suppresses conversion from proliferative arrest to cellular senescence

“…Hypoxia can cause cell cycle arrest. Simultaneously, hypoxia decreases cap-dependent translation, by inhibiting the mTOR pathway, as well as global translation by phosphorylating eukaryotic initiation eIF2a and elongation eEF2 factors (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Given that hypoxia inhibits the mTOR pathway, we suggest that hypoxia should suppress geroconversion during cell cycle arrest caused by other agents, which otherwise cause senescence.…”