Abstract
The hypoxia–inducible transcription factor (HIF) is a key component of the cellular adaptation mechanisms to hypoxic conditions. HIFα subunits are degraded by prolyl-4-hydroxylase domain (PHD) enzyme-dependent prolyl-4-hydroxylation of LxxLAP motifs that confer oxygen-dependent proteolytic degradation. Interestingly, only three non-HIFα proteins contain two conserved LxxLAP motifs, including the putative RNA helicase with a zinc finger domain HELZ. However, HELZ proteolytic regulation was found to be oxygen-independent, supporting the notion that a LxxLAP sequence motif alone is not sufficient for oxygen-dependent protein destruction. Since biochemical pathways involving RNA often require RNA helicases to modulate RNA structure and activity, we used luciferase reporter gene constructs and metabolic labeling to demonstrate that HELZ overexpression activates global protein translation whereas RNA-interference mediated HELZ suppression had the opposite effect. Although HELZ interacted with the poly(A)-binding protein (PABP) via its PAM2 motif, PABP was dispensable for HELZ function in protein translation. Importantly, downregulation of HELZ reduced translational initiation, resulting in the disassembly of polysomes, in a reduction of cell proliferation and hypophosphorylation of ribosomal protein S6.
Highlights
RNA helicases use ATP to modulate the structure of RNA, thereby altering the biologic activity of the RNA molecule or regulating access by other proteins
We concentrated on HELZ, because coincidentally the same protein has been identified in a previously performed yeast two-hybrid screen for PHD2 interactors [22], suggesting that the two conserved LxxLAP motifs might be of functional relevance for interaction with the prolyl-4-hydroxylase domain (PHD) oxygen sensors and probably oxygendependent regulation of HELZ protein stability and/or function
The specific prolyl residues within the HIFa subunits hydroxylated by PHDs share the sequence LxxLAP, in vitro studies on hydroxylase activity using substrate mutations have indicated that very few residues outside the hydroxylated proline itself are critical for hydroxylation [32,33]
Summary
RNA helicases use ATP to modulate the structure of RNA, thereby altering the biologic activity of the RNA molecule or regulating access by other proteins. The putative RNA helicase with a zinc finger domain HELZ belongs to the Upf1-like family of the superfamily I class of helicases [3,4]. Members of this family have previously been implicated in mRNA processing [5,6]. Tagged-HELZ has been shown to co-immunoprecipitate with co-transfected histone methyltransferases Smyd and Smyd and a functional role as adaptor molecule to RNA polymerase II has been suggested [7,8]. The function of HELZ, especially in RNA metabolism, remained incompletely understood
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