Functional characterization of the putative RNA Helicase HELZ

Abstract

Aerobic organisms require oxygen to produce ATP, the energy unit of the cells. To ensure adequate ATP production, cells developed several different adaptation processes to meet variations in oxygen supply. Central in the molecular adaptation is the hypoxia-inducible factor HIF, a transcription factor that regulates the expression of several dozens of target genes that are involved in systemic as well as cellular adaptation processes. HIF belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) family of transcription factors and is composed of a constantly expressed HIF-b and an oxygen-labile HIF-a subunit [3]. The HIF-a subunit is tightly regulated by a family of HIF proly-4-hydroxylases (PHDs) that use oxygen as substrate to hydroxylate two prolines in a LxxLAP motif within the HIF-a protein enabling pVHL-mediated E3 ubiquitin ligase binding resulting in HIF-a ubiquitination and proteasomal degradation [3-5]. There are three major PHD isoforms termed PHD1, PHD2 and PHD3. PHD2 is thought to be the key regulator of HIF-a [6, 7]. In the work presented herein we attempted to find novel PHD2-interacting proteins. Using yeast 2-hybrid methodology, we discovered three novel PHD2 interactors: helicase with zinc finger domain (HELZ), zinc finger and BTB domain containing protein (ZBTB) 3 and KIAA0556. Bioinformatic analysis revealed that HELZ, similar to HIF-a, has two highly conserved LxxLAP domains. However, in contrary to HIF-α, HELZ protein levels were not regulated by the PHD function. Furthermore, hypoxia had no effect on HELZ mRNA levels as well as subcellular localization and HELZ was not involved in the regulation of HIF-transcriptional activity. To ensure proper function, cells developed adaptation mechanisms allowing rapid response to change in their physiological environment. Protein translation plays a central role in these adaption processes, since it allows an immediate response even when gene transcription is lacking. Translation initiation, the rate limiting step of protein translation, involves many proteins including initiation factors, ribosomal subunits and general RNA binding proteins. One key player in this regulation is the poly(A) binding protein (PABP) that binds to the poly(A) tail of all poly-adenylated mRNAs and brings about their circularization, a process that facilitates translation initiation [8, 9]. We found that HELZ associates with PABP via its PAM2 motif, a protein interaction motif found in various PABP interactors [10]. Furthermore, interaction with PABP suggested a role for HELZ in protein translation, in particular in translation initiation. Indeed, siRNA-mediated HELZ knock down reduced and transient overexpression of HELZ induced the translation of a heterologous luciferase gene reporter as well as general protein translation. This stimulatory function was independent of the HELZ helicase function as well as the prolines found within the HELZ LxxLAP motifs as demonstrated by site-directed mutagenesis analysis. In addition, polysomal shift analysis revealed impaired translation initiation in HELZsilenced HeLa cells supporting the notion that HELZ functions in translation initiation. Mechanistically, we were able to show that HELZ promotes cell proliferation and most strikingly, HELZ strongly impairs ribosomal protein S6 phosphorylation without affecting S6 protein expression. In summary, we suggest a role for HELZ as novel translation initiation factor that promotes cell proliferation as well as S6 phosphorylation