YB-1 autoregulates translation of its own mRNA at or prior to the step of 40S ribosomal subunit joining

YB-1 is a member of the numerous families of proteins with an evolutionary ancient cold-shock domain. It is involved in many DNA- and RNA-dependent events and regulates gene expression at different levels. Previously, we found a regulatory element within the 3' untranslated region (UTR) of YB-1 mRNA that specifically interacted with YB-1 and poly(A)-binding protein (PABP); we also showed that PABP positively affected YB-1 mRNA translation in a poly(A) tail-independent manner (O. V. Skabkina, M. A. Skabkin, N. V. Popova, D. N. Lyabin, L. O. Penalva, and L. P. Ovchinnikov, J. Biol. Chem. 278:18191-18198, 2003). Here, YB-1 is shown to strongly and specifically inhibit its own synthesis at the stage of initiation, with accumulation of its mRNA in the form of free mRNPs. YB-1 and PABP binding sites have been mapped on the YB-1 mRNA regulatory element. These were UCCAG/ACAA for YB-1 and a approximately 50-nucleotide A-rich sequence for PABP that overlapped each other. PABP competes with YB-1 for binding to the YB-1 mRNA regulatory element and restores translational activity of YB-1 mRNA that has been inhibited by YB-1. Thus, YB-1 negatively regulates its own synthesis, presumably by specific interaction with the 3'UTR regulatory element, whereas PABP restores translational activity of YB-1 mRNA by displacing YB-1 from this element.     

Identification of proteins specifically interacting with YB-1 mRNA 3′ UTR and the effect of hnRNP Q on YB-1 mRNA translation

In this study, proteins specifically interacting with the 3′ untranslated region (UTR) of mRNA of the multifunctional Y-box-binding protein 1 (YB-1) were identified. One of these, hnRNP Q, was shown to specifically interact with the regulatory element (RE) in YB-1 mRNA 3′ UTR and to inhibit translation of this mRNA. Its binding to the RE was accompanied by displacement from this element of the poly(A)-binding protein (PABP), a positive regulator of YB-1 mRNA translation, and by enhanced binding of the negative YB-1 mRNA translation regulator — YB-1 itself.     

Structural organization of mRNA complexes with major core mRNP protein YB-1

YB-1 is a universal major protein of cytoplasmic mRNPs, a member of the family of multifunctional cold shock domain proteins (CSD proteins). Depending on its amount on mRNA, YB-1 stimulates or inhibits mRNA translation. In this study, we have analyzed complexes formed in vitro at various YB-1 to mRNA ratios, including those typical for polysomal (translatable) and free (untranslatable) mRNPs. We have shown that at mRNA saturation with YB-1, this protein alone is sufficient to form mRNPs with the protein/RNA ratio and the sedimentation coefficient typical for natural mRNPs. These complexes are dynamic structures in which the protein can easily migrate from one mRNA molecule to another. Biochemical studies combined with atomic force microscopy and electron microscopy showed that mRNA–YB-1 complexes with a low YB-1/mRNA ratio typical for polysomal mRNPs are incompact; there, YB-1 binds to mRNA as a monomer with its both RNA-binding domains. At a high YB-1/mRNA ratio typical for untranslatable mRNPs, mRNA-bound YB-1 forms multimeric protein complexes where YB-1 binds to mRNA predominantly with its N-terminal part. A multimeric YB-1 comprises about twenty monomeric subunits; its molecular mass is about 700 kDa, and it packs a 600–700 nt mRNA segment on its surface.     

Autoregulation of poly(A)-binding protein synthesis in vitro.

The poly(A)-binding protein (PABP), in a complex with the 3'poly(A) tail of eukaryotic mRNAs, plays important roles in the control of translation and message stability. All known examples of PABP mRNAs contain an extensive A-rich sequence in their 5' untranslated regions. Studies in mammalian cells undergoing growth stimulation or terminal differentiation indicate that PABP expression is regulated at the translational level. Here we examine the hypothesis that synthesis of the PABP is autogenously controlled. We show that the endogenous inactive PABP mRNA in rabbit reticulocytes can be specifically stimulated by addition of low concentrations of poly(A) and that this stimulation is also observed with in vitro transcribed human PABP mRNA. By deleting the A-rich region from the leader of human PABP mRNA and adding it upstream of the initiator AUG in a reporter mRNA we show that the adenylate tract is sufficient and necessary for mRNA repression and poly(A)-mediated activation in the reticulocyte cell-free system. UV cross-linking experiments demonstrate that the leader adenylate tract binds PABP. Furthermore, addition of recombinant GST-PABP to the cell-free system represses translation of mRNAs containing the A-rich sequence in their 5'UTR, but has no effect on control mRNA. We thus conclude that in vitro PABP binding to the A-rich sequence in the 5' UTR of PABP mRNA represses its own synthesis.     

mRNA and DNA selection via protein multimerization: YB-1 as a case study

Translation is tightly regulated in cells for keeping adequate protein levels, this task being notably accomplished by dedicated mRNA-binding proteins recognizing a specific set of mRNAs to repress or facilitate their translation. To select specific mRNAs, mRNA-binding proteins can strongly bind to specific mRNA sequences/structures. However, many mRNA-binding proteins rather display a weak specificity to short and redundant sequences. Here we examined an alternative mechanism by which mRNA-binding proteins could inhibit the translation of specific mRNAs, using YB-1, a major translation regulator, as a case study. Based on a cooperative binding, YB-1 forms stable homo-multimers on some mRNAs while avoiding other mRNAs. Via such inhomogeneous distribution, YB-1 can selectively inhibit translation of mRNAs on which it has formed stable multimers. This novel mechanistic view on mRNA selection may be shared by other proteins considering the elevated occurrence of multimerization among mRNA-binding proteins. Interestingly, we also demonstrate how, by using the same mechanism, YB-1 can form multimers on specific DNA structures, which could provide novel insights into YB-1 nuclear functions in DNA repair and multi-drug resistance.     

A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.     

Translational control by the poly(A) binding protein: a check for mRNA integrity

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the capbinding protein eIF4E and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this "closed loop" mRNP among other effects enhance the affinity of eIF4E for the 5' cap by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP.     

A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation.

Most eukaryotic mRNAs possess a 5' cap and a 3' poly(A) tail, both of which are required for efficient translation. In yeast and plants, binding of eIF4G to poly(A)-binding protein (PABP) was implicated in poly(A)-dependent translation. In mammals, however, there has been no evidence that eIF4G binds PABP. Using 5' rapid amplification of cDNA, we have extended the known human eIF4GI open reading frame from the N-terminus by 156 amino acids. Co-immunoprecipitation experiments showed that the extended eIF4GI binds PABP, while the N-terminally truncated original eIF4GI cannot. Deletion analysis identified a 29 amino acid sequence in the new N-terminal region as the PABP-binding site. The 29 amino acid stretch is almost identical in eIF4GI and eIF4GII, and the full-length eIF4GII also binds PABP. As previously shown for yeast, human eIF4G binds to a fragment composed of RRM1 and RRM2 of PABP. In an in vitro translation system, an N-terminal fragment which includes the PABP-binding site inhibits poly(A)-dependent translation, but has no effect on translation of a deadenylated mRNA. These results indicate that, in addition to a recently identified mammalian PABP-binding protein, PAIP-1, eIF4G binds PABP and probably functions in poly(A)-dependent translation in mammalian cells.     

Translational repression by a novel partner of human poly(A) binding protein, Paip2

The eukaryotic mRNA 3' poly(A) tail acts synergistically with the 5' cap structure to enhance translation. This effect is mediated by a bridging complex, composed of the poly(A) binding protein (PABP), eIF4G, and the cap binding protein, eIF4E. PABP-interacting protein 1 (Paip1) is another factor that interacts with PABP to coactivate translation. Here, we describe a novel human PABP-interacting protein (Paip2), which acts as a repressor of translation both in vitro and in vivo. Paip2 preferentially inhibits translation of a poly(A)-containing mRNA, but has no effect on the translation of hepatitis C virus mRNA, which is cap- and eIF4G-independent. Paip2 decreases the affinity of PABP for polyadenylate RNA, and disrupts the repeating structure of poly(A) ribonucleoprotein. Furthermore, Paip2 competes with Paip1 for PABP binding. Thus, Paip2 inhibits translation by interdicting PABP function.     

Paip1 interacts with poly(A) binding protein through two independent binding motifs

The 3' poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5' cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5' and 3' termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K(d) of 1.9 nM.     

The major mRNA-associated protein YB-1 is a potent 5' cap-dependent mRNA stabilizer

mRNA silencing and storage play an important role in gene expression under diverse circumstances, such as throughout early metazoan development and in response to many types of environmental stress. Here we demonstrate that the major mRNA-associated protein YB-1, also termed p50, is a potent cap-dependent mRNA stabilizer. YB-1 addition or overexpression dramatically increases mRNA stability in vitro and in vivo, whereas YB-1 depletion results in accelerated mRNA decay. The cold shock domain of YB-1 is responsible for the mRNA stabilizing activity, and a blocked mRNA 5' end is required for YB-1-mediated stabilization. Significantly, exogenously added YB-1 destabilizes the interaction of the cap binding protein, eIF4E, with the mRNA cap structure. Conversely, sequestration of eIF4E from the cap increases the association of endogenous YB-1 with mRNA at or near the cap, and significantly enhances mRNA stability. These data support a model whereby down-regulation of eIF4E activity or increasing the YB-1 mRNA binding activity or concentration in cells activates a general default pathway for mRNA stabilization.     

Intrinsically Unstructured Sequences in the mRNA 3ʹ UTR Reduce the Ability of Poly(A) Tail to Enhance Translation

The 5? cap and 3? poly(A) tail of mRNA are known to synergistically stimulate translation initiation via the formation of the cap?eIF4E?eIF4G?PABP?poly(A) complex. Most mRNA sequences have an intrinsic propensity to fold into extensive intramolecular secondary structures that result in short end-to-end distances. The inherent compactness of mRNAs might stabilize the cap?eIF4E?eIF4G?PABP?poly(A) complex and enhance cap-poly(A) translational synergy. Here, we test this hypothesis by introducing intrinsically unstructured sequences into the 5? or 3? UTRs of model mRNAs. We found that the introduction of unstructured sequences into the 3? UTR, but not the 5? UTR, decreases mRNA translation in cell-free wheat germ and yeast extracts without affecting mRNA stability. The observed reduction in protein synthesis results from the diminished ability of the poly(A) tail to stimulate translation. These results suggest that base pair formation by the 3? UTR enhances the cap-poly(A) synergy in translation initiation.     

Poly(A) binding protein (PABP) homeostasis is mediated by the stability of its inhibitor, Paip2

The poly(A)-binding protein (PABP) is a unique translation initiation factor in that it binds to the mRNA 3' poly(A) tail and stimulates recruitment of the ribosome to the mRNA at the 5' end. PABP activity is tightly controlled by the PABP-interacting protein 2 (Paip2), which inhibits translation by displacing PABP from the mRNA. Here, we describe a close interplay between PABP and Paip2 protein levels in the cell. We demonstrate a mechanism for this co-regulation that involves an E3 ubiquitin ligase, EDD, which targets Paip2 for degradation. PABP depletion by RNA interference (RNAi) causes co-depletion of Paip2 protein without affecting Paip2 mRNA levels. Upon PABP knockdown, Paip2 interacts with EDD, which leads to Paip2 ubiquitination. Supporting a critical role for EDD in Paip2 degradation, knockdown of EDD expression by siRNA leads to an increase in Paip2 protein stability. Thus, we demonstrate that the turnover of Paip2 in the cell is mediated by EDD and is regulated by PABP. This mechanism serves as a homeostatic feedback to control the activity of PABP in cells.