Novel translational control through an iron-responsive element by interaction of multifunctional protein YB-1 and IRP2.

The eukaryotic Y-box-binding protein YB-1 functions in various biological processes, including DNA repair, cell proliferation, and transcriptional and translational controls. To gain further insight into how human YB-1 plays its role in pleiotropic functions, we here used two-hybrid screenings to identify partners of this protein; the results showed that YB-1 itself, iron-regulatory protein 2 (IRP2), and five ribosomal proteins each served as partners to YB-1. We then examined the biological effect of the interaction of YB-1 and IRP2 on translational regulation. Both in vitro binding and coimmunoprecipitation assays showed the direct interaction of YB-1 and IRP2 in the presence of a high concentration of iron. RNA gel shift assays showed that YB-1 reduced the formation of the IRP2-mRNA complex when the iron-responsive element of the ferritin mRNA 5' untranslated region (UTR) was used as a probe. By using an in vitro translation assay using luciferase mRNA ligated to the ferritin mRNA 5'UTR as a reporter construct, we showed that both YB-1 and IRP2 inhibited the translation of the mRNA. However, coadministration of YB-1 and IRP2 proteins abrogated the inhibition of protein synthesis by each protein. An In vivo coimmunoprecipitation assay showed that IRP2 bound to YB-1 in the presence of iron and a proteasome inhibitor. The direct interaction of YB-1 and IRP2 provides the first evidence of the involvement of YB-1 in the translational regulation of an iron-related protein.

Characterization of the 5'-untranslated region of YB-1 mRNA and autoregulation of translation by YB-1 protein.

The eukaryotic Y-box binding protein YB-1 is involved in various biological processes, including DNA repair, cell proliferation and the regulation of transcription and translation. YB-1 protein is abundant and expressed ubiquitously in human cells, functioning in cell proliferation and transformation. Its concentration is thought to be highly regulated at both the levels of transcription and translation. Therefore, we investigated whether or not the 5'-UTR of YB-1 mRNA affects the translation of YB-1 protein, thus influencing expression levels. Luciferase mRNA ligated to the YB-1 mRNA 5'-UTR was used as a reporter construct. Ligation of the full-length YB-1 5'-UTR (331 bases) enhanced translation as assessed by in vitro and in vivo translation assays. Deletion constructs of the YB-1 5'-UTR also resulted in a higher efficiency of translation, especially in the region mapped to +197 to +331 from the major transcription start site. RNA gel shift assays revealed that the affinity of YB-1 for various 5'-UTR probe sequences was higher for the full-length 5'-UTR than for deleted 5'-UTR sequences. An in vitro translation assay was used to demonstrate that recombinant YB-1 protein inhibited translation of the full-length 5'-UTR of YB-1 mRNA. Thus, our findings provide evidence for the autoregulation of YB-1 mRNA translation via the 5'-UTR.

Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts.

Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca(2+) ([Ca(2+)](o)) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca(2+)-induced increase in fluo-3 fluorescence intensity. Elevated [Ca(2+)](o) enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E(2) in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca(2+)-induced expression of COX-2 mRNA. Elevated [Ca(2+)](o)-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca(2+)-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca(2+)-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca(2+) may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.

The pleiotropic functions of the Y-box-binding protein, YB-1.

The Y-box-binding protein (YB-1) represents the most evolutionary conserved nucleic-acid-binding protein currently known. YB-1 is a member of the cold-shock domain (CSD) protein superfamily. It performs a wide variety of cellular functions, including transcriptional regulation, translational regulation, DNA repair, drug resistance and stress responses to extracellular signals. As a result, YB-1 expression is closely associated with cell proliferation. In this review, we will begin by briefly describing the characteristics of YB-1 and will then summarize the pleiotropic functions brought about via DNA-RNA transaction and protein-protein interactions. In addition, we will discuss the diverse range of potential physiological and pathological functions of YB-1.

Binding capacity of human YB-1 protein for RNA containing 8-oxoguanine.

8-oxoguanine (8-oxo-7,8-dihydroguanine) is generated in the cellular nucleotide pool as well as in nucleic acids, by the action of oxygen radicals produced in cells. 8-oxoguanine has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms should have mechanisms for preventing the misincorporation of 8-oxoguanine-containing nucleotide into RNA and for removing 8-oxoguanine-containing RNA from processes of translation. We now report that mammalian Y box-binding protein 1 (YB-1 protein) possesses the activity to bind specifically to RNA containing 8-oxoguanine. On incubation with a purified preparation of YB-1 protein, 8-oxoguanine-containing RNA forms stable complexes with the protein while normal RNA scarcely forms such a complex. Using a series of deletion mutants which produce altered forms of YB-1 protein lacking some parts of the sequence, domains of the protein necessary for RNA binding were identified. Escherichia coli cells expressing normal or truncated forms of YB-1 protein with the binding capacity acquire resistance against paraquat, a drug that induces oxidative stress in cells, whereas cells with truncated proteins lacking such an activity do not. YB-1 protein may disturb the bacterial system in recognizing oxidatively damaged RNA, thus exerting a dominant negative effect on cell growth. We propose that YB-1 protein may discriminate the oxidized RNA molecule from normal ones, thus contributing to the high fidelity of translation in cells.