The transition from transcription initiation to elongation involves phosphorylation of the large subunit (Rpb1) of RNA polymerase II on the repetitive carboxyl-terminal domain. The elongating hyperphosphorylated Rpb1 is subject to ubiquitination, particularly in response to UV radiation and DNA-damaging agents. By using computer modeling, we identified regions of Rpb1 and the adjacent subunit 6 of RNA polymerase II (Rpb6) that share sequence and structural similarity with the domain of hypoxia-inducible transcription factor 1α (HIF-1α) that binds von Hippel–Lindau tumor suppressor protein (pVHL). pVHL confers substrate specificity to the E3 ligase complex, which ubiquitinates HIF-α and targets it for proteasomal degradation. In agreement with the computational model, we show biochemical evidence that pVHL specifically binds the hyperphosphorylated Rpb1 in a proline-hydroxylation-dependent manner, targeting it for ubiquitination. This interaction is regulated by UV radiation.

Hypoxia‐inducible factors (HIFs) are ubiquitous transcription factors that mediate adaptation to hypoxia by inducing specific sets of target genes. It is well accepted that hypoxia induces accumulation and activity of HIFs by causing stabilization of their α subunits. We have demonstrated that hypoxia stimulates translation of HIF‐1α and ‐2α proteins by distributing HIF‐α mRNAs to larger polysome fractions. This requires influx of extracellular calcium, stimulation of classical protein kinase C‐α (cPKC‐α), and the activity of mammalian target of rapamycin, mTOR. The translational component contributes to α40–50% of HIF‐α proteins accumulation after3hof1%O ₂ . Hypoxia also inhibits general protein synthesis and mTOR activity; however, cPKC‐α inhibitors or rapamycin reduce mTOR activity and total protein synthesis beyond the effects of hypoxia alone. These data show that during general inhibition of protein synthesis by hypoxia, cap‐mediated translation of selected mRNAs is induced through the mTOR pathway. We propose that calcium‐induced activation of cPKC‐α hypoxia partially protects an activity of mTOR from hypoxic inhibition. These results provide an important physiologic insight into the mechanism by which hypoxia‐stimulated influx of calcium selectively induces the translation of mRNAs necessary for adaptation to hypoxia under conditions repressing general protein synthesis.—Hui, A. S., Bauer, A. L., Striet, J. B., Schnell, P. O., Czyzyk‐Krzeska, M. F. Calcium signaling stimulates translation of HIF‐α during hypoxia. FASEB J. 20, 466–475 (2006)

The stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is regulated by oxygen tension in the pheochromocytoma-derived PC12 cell line. We previously identified a pyrimidine-rich 27-base-long protein-binding sequence in the 3′-untranslated region of TH mRNA that is associated with hypoxia-inducible formation of a ribonucleoprotein complex (hypoxia-inducible protein-binding site (HIPBS)). In this study, we show that HIPBS is an mRNA stabilizing element necessary for both constitutive and hypoxia-regulated stability of TH mRNA. The mutations within this sequence that abolish protein binding markedly decrease constitutive TH mRNA stability and ablate its hypoxic regulation. A short fragment of TH mRNA that contains the wild-type HIPBS confers the increased mRNA stability to the reporter chloramphenicol acetyltransferase mRNA. However, it is not sufficient to confer hypoxic regulation. The HIPBS element binds two isoforms of a 40-kDa poly(C)-binding protein (PCBP). Hypoxia induces expression of the isoform 1, PCBP₁, but not the isoform 2, PCBP₂, in PC12 cells.

Tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine biosynthesis, is induced by hypoxia in oxygen‐sensitive cells of the carotid body and pheochromocytoma‐derived PC12 cells. TH is also regulated by the von Hippel–Lindau tumor suppressor protein (pVHL). Here, we report that induction of TH gene expression involves activation of the hypoxia‐inducible transcription factors (HIFs) that interact with a specific hypoxia‐responsive element (HRE) in the proximal region of the TH promoter. We also show that some of the effects of pVHL on activity of the TH promoter are mediated through HIFs. Low levels of pVHL are associated with decreased HIFα ubiquitination, increased accumulation of HIFα proteins, increased binding of HIFs to the HRE within the TH promoter, and increased activity of a TH promoter–reporter construct. In contrast, high levels of pVHL repress HIF accumulation and inhibit its activity in hypoxic cells. These results indicate that HIFs may play an important role in regulation of TH gene expression in oxygen‐sensitive cells and also in the development of hypercatecholaminemia in pheochromocytoma tumors.