An acidic protein, YBAP1, mediates the release of YB-1 from mRNA and relieves the translational repression activity of YB-1

Eukaryotic Y-box proteins are nucleic acid-binding proteins implicated in a wide range of gene regulatory mechanisms. They contain the cold shock domain, which is a nucleic acid-binding structure also found in bacterial cold shock proteins. The Y-box protein YB-1 is known to be a core component of messenger ribonucleoprotein particles (mRNPs) in the cytoplasm. Here we disrupted the YB-1 gene in chicken DT40 cells. Through the immunoprecipitation of an epitope-tagged YB-1 protein, which complemented the slow-growth phenotype of YB-1-depleted cells, we isolated YB-1-associated complexes that likely represented general mRNPs in somatic cells. RNase treatment prior to immunoprecipitation led to the identification of a Y-box protein-associated acidic protein (YBAP1). The specific association of YB-1 with YBAP1 resulted in the release of YB-1 from reconstituted YB-1-mRNA complexes, thereby reducing the translational repression caused by YB-1 in the in vitro system. Our data suggest that YBAP1 induces the remodeling of YB-1-mRNA complexes.     

Polysome-associated proteins in herpes simplex virus-infected cells

In the cytoplasm of eucaryotic cells, mRNA is associated with proteins. These mRNA-protein complexes, termed messenger ribonucleoprotein (mRNP) particles, are divided into two functional classes. The first class contains free (non-ribosome-associated) mRNPs which have been termed informosomes by others. The second class of mRNPs, those associated with polysomes, are actively engaged in protein synthesis and are termed polysomal mRNPs. The experiments described in this paper examined the proteins associated with polyribosomes in uninfected and herpes simplex virus type 1-infected cells. The data indicate that after infection with herpes simplex virus type 1, specific changes occur in the proteins which normally are found associated with these polysomal mRNPs. These changes include both the appearance of new and possibly virus-specific proteins and the loss of normal host-specific proteins. The relationship of these changes to the patterns of protein synthesis in these cells is also discussed.     

Analysis of the control of citrulline synthesis in isolated rat-liver mitochondria

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross-link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA-protein complexes (mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28 000, 32 000, 46 000, 65 000 and 150 000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130 000 and 43 000 was higher in free mRNPs than in polysomal mRNP complexes. To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat-shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre-existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA-binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes. Heat-shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr = 81 000, 65 000 and 25 000 (heat-shock polypeptides). Whether this altered pattern of protein synthesis following heat-shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat-shock treatment. A [35S]methionine-labeled polypeptide of Mr = 80 000 was detected in mRNPs of heat-shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80 000-Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat-shock mRNAs. The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

YB-1 Synthesis Is Regulated by mTOR Signaling Pathway

YB-1 is a eukaryotic protein with numerous intra- and extracellular functions based on its ability to interact with RNA, DNA, and many proteins. In spite of achievements in studying its functions, regulation of YB-1 synthesis in the cell remains poorly understood. In the current study Western and Northern blotting were used to determine the amounts of YB-1 and YB-1 mRNA in rabbit organs and several cell lines. As found, in the majority of studied eukaryotic cells a considerable proportion of YB-1 mRNA was stored in free mRNPs, i.e., was poorly translated. Also, we demonstrated that YB-1 synthesis depended on conditions that determined the rate of cell division. Specific suppression of YB-1 synthesis resulted from inhibition of the mTOR signaling pathway with inhibitor PP242, but not rapamycin. Experiments on reporter constructs showed that dependence of YB-1 mRNA translation on activity of the mTOR signaling pathway was dictated by 5′ untranslated regions of this mRNA, irrelatively of the TOP-like sequences at the beginning of 5′ UTR.     

The function of proteins that interact with mRNA

Specific proteins are associated with mRNA in the cytoplasm of eukaryotic cells. The complement of associated proteins depends upon whether the mRNA is an integral component of the polysomal complex being translated, or, alternatively, whether it is part of the non-translated free mRNP fraction. By subjecting cells to ultraviolet irradiation in vivo to cross-link proteins to mRNA, mRNP proteins have been shown to be associated with specific regions of the mRNA molecule. Examination of mRNP complexes containing a unique mRNA has suggested that not all mRNA contain the same family of associated RNA binding proteins. The function of mRNA associated proteins may include a role in providing stability for mRNA, and/or in modulating translation. With the recent demonstrations that both free and polysomal mRNPs are associated with the cytoskeletal framework, specific mRNP proteins may play a role in determining the subcellular localization of specific mRNPs.     

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.     

A member of the Y-box protein family interacts with an upstream element in the α1(I) collagen gene

Transforming growth factor beta (TGF-β) stimulates protein complex formation on a TGF-β response element (TAE) found in the distal portion (−1624) of the collagen alpha 1(I) promoter. To identify the fibroblast proteins in this complex, an expression library constructed from human embryonic lung fibroblasts mRNA was screened using a tetramer of TAE. Y-box binding protein (YB-1), was identified as a protein in the TAE–protein complex. The protein expressed by phage clones formed a specific complex with labeled TAE but not mutated TAE (mTAE) similar to the complex formed with nuclear protein. Nuclear protein–TAE complexes isolated from native gels contained YB-1 by Western analysis. TGF-β treatment increased the amount of YB-1 protein in nuclear extracts, decreased its amount in cytoplasm, but did not alter the steady state levels of YB-1 mRNA. A full-length YB-1 protein expressed in human lung fibroblasts was primarily located in the nucleus with punctate staining in cytoplasmic regions. The expression of YB-1 decreased in the cytoplasm after 2 h of TGF-β treatment. Therefore, the increased binding activity seen in TGF-β-stimulated nuclear extracts was due primarily to relocalization of YB-1 from the cytoplasm to the nuclear compartment. Co-transfection of YB-1 cDNA with a collagen promoter–reporter construct caused a dose-dependent activation of collagen promoter activity in rat fibroblasts whereas the promoter with a mutation in the TAE element was not sensitive to YB-1 co-expression. In conclusion, we have identified YB-1 as a protein that interacts with a TGF-β response element in the distal region of the collagen alpha 1(I) gene. YB-1 protein activates the collagen promoter and translocates into the nucleus during TGF-β addition to fibroblasts, suggesting a role for this protein in TGF-β signaling.     

Atomic force microscopy reveals binding of mRNA to microtubules mediated by two major mRNP proteins YB-1 and PABP

A significant fraction of mRNAs is known to be associated in the form of mRNPs with microtubules for active transport. However, little is known about the interaction between mRNPs and microtubules and most of previous works were focused on molecular motor:microtubule interactions. Here, we have identified, via high resolution atomic force microscopy imaging, a significant binding of mRNA to microtubules mediated by two major mRNP proteins, YB-1 and PABP. This interaction with microtubules could be of critical importance for active mRNP traffic and for mRNP granule formation. A similar role may be fulfilled by other cationic mRNA partners.     

YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5′UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.     

Messenger ribonucleoprotein particles in developing sea urchin embryos

Messenger RNA entering polysomes during early development of the sea urchin embryo consists of both oogenetic and newly transcribed sequences. Newly transcribed mRNA enters polysomes rapidly while oogenetic mRNA enters polysomes from a pool of stable, nontranslatable messenger ribonucleoprotein particles (mRNPs) derived from the unfertilized egg. Protein content may relate to differences in the regulation of newly transcribed and oogenetic mRNAs. Oogenetic poly(A)⁺ mRNA was found to be present in both polysomal and subpolysomal fractions of cleavage stage and early blastula stage embryos. This mRNA was found to be present in subpolysomal mRNPs with a density of 1.45 g/cm³ in Cs₂SO₄. Poly(A)⁺ mRNPs released from polysomes of embryos cultured in the presence of actinomycin D sedimented in a broad peak centered at 55 S and contained RNA of 21 S. The density of these particles was sensitive to the method of release; puromycin-released mRNPs had a density of 1.45 g/cm³, while EDTA caused a shift in density to 1.55 g/cm³, indicating a partial loss of protein. The results with newly synthesized mRNAs contrast sharply. Newly transcribed mRNA in subpolysomal mRNPs had a density of 1.55–1.66 g/cm³, a density approaching that of deproteinized RNA. Messenger RNA released from polysomes either by EDTA or puromycin was examined to determine the possible existence of polysomal mRNPs. When [³H]uridine-labeled mRNA was released from late cleavage stage embryo polysomes by either technique, and centrifuged on sucrose gradients, two broad peaks were found. One peak centered at 30 S contained 21 S mRNA while the other at 15 S contained 9 S histone mRNA. When these fractions were fixed with formaldehyde, they banded on Cs₂SO₄ gradients at a density of 1.60–1.66 g/cm³, very similar to that of pure RNA. We conclude that the newly transcribed mRNA may be present in stable mRNPs containing up to 10% protein in either subpolysomal or polysomal fractions. These mRNPs are clearly distinguishable from the protein-rich mRNPs containing oogenetic mRNAs.     

Molecular Composition of Staufen2-Containing Ribonucleoproteins in Embryonic Rat Brain

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (α- and β-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs.     

The Y-Box Binding Protein 1 Suppresses Alzheimer’s Disease Progression in Two Animal Models

The Y-box binding protein 1 (YB-1) is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-1₁₋₂₁₉, suppress impairment of spatial memory in olfactory bulbectomized (OBX) mice with Alzheimer’s type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain β-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric β-amyloid (1–42) inhibited formation of β-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with β-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer’s disease.     

A comparative study by sucrose gradient electrophoresis of messenger ribonucleoprotein complexes.

The recently developed method of sucrose gradient electrophoresis has been used in the investigation of mRNA containing particles prepared in an undenatured state. The particles containing messenger RNA migrate as a homogeneous fraction with a mobility different from that of ribosomal praticles. Informosomes and polysomal mRNPs have about the same mobility. On the contrary artificial complexes, formed by the reaction of cytoplasmic binding factor with RNA, migrate as a heterogeneous fraction. The particles carrying mRNA are drastically and irreversibly affected by a treatment with EDTA. Sodium deoxycholate removes some proteins but seems also to denature them. After treatment by high salt or Sodium deoxycholate the mRNPs migrate as a homogeneous fraction showing that all particles are equally affected.     

YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1

Under cell stress, global protein synthesis is inhibited to preserve energy. One mechanism is to sequester and silence mRNAs in ribonucleoprotein complexes known as stress granules (SGs), which contain translationally silent mRNAs, preinitiation factors, and RNA-binding proteins. Y-box binding protein 1 (YB-1) localizes to SGs, but its role in SG biology is unknown. We now report that YB-1 directly binds to and translationally activates the 5′ untranslated region (UTR) of G3BP1 mRNAs, thereby controlling the availability of the G3BP1 SG nucleator for SG assembly. YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro. YB-1 and G3BP1 expression are highly correlated in human sarcomas, and elevated G3BP1 expression correlates with poor survival. Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models. Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.