Efficient translation of mouse hypoxia-inducible factor-1α under normoxic and hypoxic conditions

Abstract

The heterodimeric hypoxia-inducible factor-1 (HIF-1), consisting of the subunits HIF-1alpha and HIF-1beta/ARNT, is a master transcriptional regulator of oxygen homeostasis. Under hypoxic conditions, HIF-1alpha levels very rapidly increase, mostly due to protein stabilization. However, translational regulation of HIF-1alpha has not been directly analyzed so far. Mouse HIF-1alpha exists as two mRNA isoforms (termed mHIF-1alphaI.1 and mHIF-1alphaI. 2) containing structurally different 5'-termini which might modulate translation initiation. Whereas the in vitro translation efficiency of these two mRNA isoforms was about equal, the mHIF-1alphaI.2 5'-untranslated region (5'-UTR) conferred significantly higher in vivo luciferase reporter gene activity than the mHIF-1alphaI.1 5'-UTR. Similar corresponding luciferase mRNA levels indicate translational rather than transcriptional alterations. Reporter gene expression was not affected upon exposure of transiently transfected cells to hypoxia (1% oxygen). Direct assessment of translational regulation by polysomal profile analysis of HeLaS3 cells showed that HIF-1alpha (and to a lower extent ARNT) mRNA was found mainly in the translationally active polyribosomal fractions under both normoxic and hypoxic conditions. In contrast, the association of mRNAs for beta-actin and ribosomal protein L28 with the polyribosomal fractions was substantially reduced under hypoxic conditions, suggesting decreased overall protein synthesis. Thus, efficient translation of mouse HIF-1alpha in a situation where the general translation efficiency is reduced represents a prerequisite for the very rapid accumulation of HIF-1alpha protein upon exposure to hypoxia.

Hypoxia-inducible Factor-1α mRNA Contains an Internal Ribosome Entry Site That Allows Efficient Translation during Normoxia and Hypoxia

“…Because HIF-1␣ is expressed during hypoxia and because the 5ЈUTR is long and G ϩ C rich, we investigated whether HIF-1␣ is efficiently translated during hypoxia. We show that the translation of endogenous HIF-1␣ mRNA in NIH3T3 cells is not compromised under hypoxic conditions, in agreement with a recent study of the effect of hypoxia on HIF1-␣ translation in HeLaS3 cells (Gorlach et al, 2000). Using dicistronic assays we demonstrate that the HIF-1␣ 5ЈUTR contains an IRES that has activity similar to that of the IRES in c-myc.…”

Preferential expression of the novel alternative isoform I.3 of hypoxia-inducible factor 1α in activated human T lymphocytes

“…It was shown that the mouse activation-inducible mRNA isoform I.1 produces transcriptionally active HIF-1α protein that is twelve amino acids shorter than the ubiquitous HIF-1α isoform [8,14]. In contrast, the novel human I.3 mRNA isoform encodes HIF-1α that is twenty four amino acids longer than the ubiquitous isoform ( Figure 1C).…”

Functions of the Per/ARNT/Sim Domains of the Hypoxia-inducible Factor

“…Each construct was cloned into the pcDNA3.1 vector (Invitrogen) in which the cytomegalovirus promoter was replaced by the human HIF-1␣ promoter and 5Ј-untranslated region (34) to better approximate O 2 -sensitive responses in HIF-␣ transcription/translation (35,36). The resulting constructs were cotransfected with the pPUR vector (BD Biosciences) conferring puromycin resistance.…”