Nonspecific and specific interactions of Y-box-binding protein 1 (YB-1) with mRNA and posttranscriptional regulation of protein synthesis in animal cells

Y-box-binding protein 1 (YB-1) is an animal multifunctional DNA/RNA-binding protein that is involved in reproduction, storing, and expression of genetic information. YB-1 accompanies mRNA throughout its life, from synthesis to degradation, and has a high specific and nonspecific affinity for RNA. In the nucleus YB-1 regulates mRNA processing. In the cytoplasm YB-1 is responsible for global and selective regulation of protein synthesis, as well as the mRNA life. This review focuses on the role of YB-1 in regulating translation. The possible mechanisms of the positive and negative effects of YB-1 on this process are considered. The recent original data are described, supporting the role of YB-1 as a major structural component of mRNP. Data about specific interactions of YB-1 with RNA are summarized for the first time.     

Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein

In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha. The region of VHL mediating interaction with HIF-1 alpha overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1 alpha and subsequent degradation. Interestingly, the VHL binding site within HIF-1 alpha overlapped with one of the minimal transactivation domains. Protection of HIF-1 alpha against degradation by VHL was a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1 alpha during this process. Finally, stabilization of HIF-1 alpha protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.     

The interaction of the von Hippel-Lindau tumor suppressor and heterochromatin protein 1

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor is associated with renal carcinoma, hemangioblastoma and pheochromocytoma. The VHL protein is a component of a ubiquitin ligase complex that ubiquitinates and degrades hypoxia inducible factor-α (HIF-α). Degradation of HIF-α by VHL is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in tumor suppression and other biological processes. Using GST-VHL pull-down experiment and mass spectrometry, we detected an interaction between VHL and heterochromatin protein 1 (HP1). We identified a conserved HP1-binding motif (PXVXL) in the β domain of VHL, which is disrupted in a renal carcinoma-associated P81S mutant. We show that the VHL P81S mutant displays reduced binding to HP1, yet retains the ability to interact with elongin B, elongin C, and cullin 2 and is fully capable of degrading HIF-α. We also demonstrate that HP1 increases the chromatin association of VHL. These results suggest a role for the VHL-HP1 interaction in VHL chromatin targeting.     

The von Hippel-Lindau tumour-suppressor protein interaction with protein kinase Cdelta

The VHL (von Hippel-Lindau) tumour-suppressor protein forms a multi-protein complex [VCB (pVHL-elongin C-elongin B)-Cul-2 (Cullin-2)] with elongin C, elongin B, Cul-2 and Rbx1, acting as a ubiquitin-ligase (E3) and directing proteasome-dependent degradation of targeted proteins. The alpha-subunit of Hif1alpha (hypoxia-inducible factor 1alpha) is the principal substrate for the VCB-Cul-2 complex; however, other substrates such as aPKC (atypical protein kinase C) have been reported. In the present study, we show with FRET (fluorescence resonance energy transfer) analysis measured by FLIM (fluorescence lifetime imaging microscopy) that PKCdelta and pVHL (VHL protein) interact directly in cells. This occurs through the catalytic domain of PKCdelta (residues 432-508), which appears to interact with two regions of pVHL, residues 113-122 and 130-154. Despite this robust interaction, analysis of the PMA-induced proteasome-dependent degradation of PKCdelta in different RCC (renal cell carcinoma) lines (RCC4, UMRC2 and 786 O) shows that there is no correlation between the degradation of PKCdelta and the presence of active pVHL. Thus, in contrast with aPKC, PKCdelta is not a conventional substrate of the ubiquitin-ligase complex, VCB-Cul-2, and the observed interaction between these two proteins must underlie a distinct signalling output.     

AUF1 cell cycle variations define genomic DNA methylation by regulation of DNMT1 mRNA stability

DNA methylation is a major determinant of epigenetic inheritance. DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division, and deregulated expression of DNMT1 leads to cellular transformation. We show herein that AU-rich element/poly(U)-binding/degradation factor 1 (AUF1)/heterogeneous nuclear ribonucleoprotein D interacts with an AU-rich conserved element in the 3' untranslated region of the DNMT1 mRNA and targets it for destabilization by the exosome. AUF1 protein levels are regulated by the cell cycle by the proteasome, resulting in cell cycle-specific destabilization of DNMT1 mRNA. AUF1 knock down leads to increased DNMT1 expression and modifications of cell cycle kinetics, increased DNA methyltransferase activity, and genome hypermethylation. Concurrent AUF1 and DNMT1 knock down abolishes this effect, suggesting that the effects of AUF1 knock down on the cell cycle are mediated at least in part by DNMT1. In this study, we demonstrate a link between AUF1, the RNA degradation machinery, and maintenance of the epigenetic integrity of the cell.     

14-3-3sigma is a p37 AUF1-binding protein that facilitates AUF1 transport and AU-rich mRNA decay

Short-lived cytokine mRNAs contain an AU-rich destabilizing element (ARE). AUF1 proteins bind the ARE, undergo shuttling, and promote cytoplasmic ARE-mRNA decay through a poorly understood mechanism. We therefore identified AUF1-interacting proteins that may play a role in ARE-mRNA decay. We used mass-spectrometry to identify 14-3-3sigma protein as an AUF1-interacting protein. 14-3-3sigma binds selectively and strongly to p37 AUF1 and to a lesser extent to the p40 isoform, the two isoforms most strongly associated with ARE-mRNA decay, but not to the two larger isoforms, p42 and p45. The 14-3-3sigma interaction site on p37 was mapped to a region found only in the two smaller AUF1 isoforms and which overlaps a putative nuclear localization signal (NLS). Stable overexpression of 14-3-3sigma significantly increased cytoplasmic accumulation of p37 AUF1 and reduced the steady-state level and half-life of a reporter ARE-mRNA. siRNA silencing of AUF1 eliminated the effect of 14-3-3sigma overexpression. 14-3-3sigma therefore binds to p37 AUF1, retains it in the cytoplasm probably by masking its NLS, and enhances rapid turnover of ARE-mRNAs.     

Identification and characterization of proteins that selectively interact with isoforms of the mRNA binding protein AUF1 (hnRNP D)

The mRNAs that encode certain cytokines and proto-oncogenes frequently contain a typical AU-rich motif that is located in their 3'-untranslated region. The protein AUF1 is the first factor identified that binds to AU-rich regions and mediates the fast degradation of the target mRNAs. AUF1 exists as four different isoforms (p37, p40, p42 and p45) that are generated by alternative splicing. The fact that AUF1 does not degrade mRNA itself had led to the suggestion that other AUF1 interacting proteins might be involved in the process of selective mRNA degradation. Here we used the yeast two-hybrid system in order to identify proteins that bind to AUF1. We detected AUF1 itself, as well as the ubiquitin-conjugating enzyme E2I and three RNA binding proteins: NSEP-1, NSAP-1 and IMP-2, as AUF1 interacting proteins. We confirmed all interactions in vitro and mapped the protein domains that are involved in the interaction with AUF1. Gel-shift assays with the recombinant purified proteins suggest that the interacting proteins and AUF1 can bind simultaneously to an AU-rich RNA oligonucleotide. Most interestingly, the AUF1 interacting protein NSEP-1 showed an endoribonuclease activity in vitro. These data suggest the possibility that the identified AUF1 interacting proteins might be involved in the regulation of mRNA stability mediated by AUF1.     

The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1

The von Hippel-Lindau disease gene (VHL) is the causative gene for most adult renal cancers. However, the mechanism by which VHL protein functions as a renal tumor suppressor remains largely unknown. To identify low occupancy VHL protein partners with potential relevance to renal cancer, we screened a human kidney library against human VHL p30 using a yeast two-hybrid approach. Jade-1 (gene for Apoptosis and Differentiation in Epithelia) encodes a previously uncharacterized 64-kDa protein that interacts strongly with VHL protein and is most highly expressed in kidney. Jade-1 protein is short-lived and contains a candidate destabilizing (PEST) motif and plant homeodomains that are not required for the VHL interaction. Jade-1 is abundant in proximal tubule cells, which are clear-cell renal cancer precursors, and expression increases with differentiation. Jade-1 is expressed in cytoplasm and the nucleus diffusely and in speckles, where it partly colocalizes with VHL. VHL reintroduction into renal cancer cells increases endogenous Jade-1 protein abundance up to 10-fold. Furthermore, VHL increases Jade-1 protein half-life up to 3-fold. Thus, direct protein stabilization is identified as a new VHL function. Moreover, Jade-1 protein represents a novel candidate regulatory factor in VHL-mediated renal tumor suppression.     

Folding of A+U-rich RNA elements modulates AUF1 binding. Potential roles in regulation of mRNA turnover

In mammals, A+U-rich elements (AREs) are potent cis-acting determinants of rapid cytoplasmic mRNA turnover. Recognition of these sequences by AUF1 is associated with acceleration of mRNA decay, likely involving recruitment or assembly of multi-subunit trans-acting complexes. Previously, we demonstrated that AUF1 deletion mutants formed tetramers on U-rich RNA substrates by sequential addition of protein dimers (Wilson, G. M., Sun, Y., Lu, H., and Brewer, G. (1999) J. Biol. Chem. 274, 33374-33381). Here, we show that binding of the full-length p37 isoform of AUF1 to these RNAs proceeds via a similar mechanism, allowing delineation of equilibrium binding constants for both stages of tetramer assembly. However, association of AUF1 with the ARE from tumor necrosis factor (TNFalpha) mRNA was significantly inhibited by magnesium ions. Further fluorescence and hydrodynamic experiments indicated that Mg(2+) induced or stabilized a conformational change in the TNFalpha ARE. Based on the solution of parameters describing both the protein-RNA and Mg(2+)-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg(2+) and AUF1 binding to the TNFalpha ARE. These studies provide the first evidence that some AREs may adopt higher order RNA structures that regulate their interaction with trans-acting factors and indicate that mRNA structural remodeling has the potential to modulate the turnover rates of some ARE-containing mRNAs.