Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 20
Filter
Add more filters










Publication year range
1.
Int J Mol Sci ; 25(3)2024 Feb 01.
Article in English | MEDLINE | ID: mdl-38339016

ABSTRACT

Y-box-binding proteins (YB proteins) are multifunctional DNA- and RNA-binding proteins that play an important role in the regulation of gene expression. The high homology of their cold shock domains and the similarity between their long, unstructured C-terminal domains suggest that Y-box-binding proteins may have similar functions in a cell. Here, we consider the functional interchangeability of the somatic YB proteins YB-1 and YB-3. RNA-seq and Ribo-seq are used to track changes in the mRNA abundance or mRNA translation in HEK293T cells solely expressing YB-1, YB-3, or neither of them. We show that YB proteins have a dual effect on translation. Although the expression of YB proteins stimulates global translation, YB-1 and YB-3 inhibit the translation of their direct CLIP-identified mRNA targets. The impact of YB-1 and YB-3 on the translation of their mRNA targets is similar, which suggests that they can substitute each other in inhibiting the translation of their mRNA targets in HEK293T cells.


Subject(s)
DNA-Binding Proteins , Protein Biosynthesis , Humans , HEK293 Cells , RNA, Messenger/genetics , RNA, Messenger/metabolism , DNA-Binding Proteins/metabolism , Y-Box-Binding Protein 1/genetics , Y-Box-Binding Protein 1/metabolism
2.
Nucleic Acids Res ; 51(2): 908-918, 2023 01 25.
Article in English | MEDLINE | ID: mdl-36583341

ABSTRACT

Polyribosomes, the groups of ribosomes simultaneously translating a single mRNA molecule, are very common in both, prokaryotic and eukaryotic cells. Even in early EM studies, polyribosomes have been shown to possess various spatial conformations, including a ring-shaped configuration which was considered to be functionally important. However, a recent in situ cryo-ET analysis of predominant regular inter-ribosome contacts did not confirm the abundance of ring-shaped polyribosomes in a cell cytoplasm. To address this discrepancy, here we analyzed the cryo-ET structure of polyribosomes in diluted lysates of HeLa cells. It was shown that the vast majority of the ribosomes were combined into polysomes and were proven to be translationally active. Tomogram analysis revealed that circular polyribosomes are indeed very common in the cytoplasm, but they mostly possess pseudo-regular structures without specific inter-ribosomal contacts. Although the size of polyribosomes varied widely, most circular polysomes were relatively small in size (4-8 ribosomes). Our results confirm the recent data that it is cellular mRNAs with short ORF that most commonly form circular structures providing an enhancement of translation.


Subject(s)
Protein Biosynthesis , Ribosomes , Humans , HeLa Cells , Polyribosomes/metabolism , Ribosomes/genetics , Ribosomes/metabolism , RNA, Messenger/metabolism , Molecular Conformation
3.
Data Brief ; 42: 108187, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35516002

ABSTRACT

N6-methyladenosine (m6A) is the most abundant, highly dynamic mRNA modification that regulates mRNA splicing, stability, and translation. The m6A epigenetic mark is erased by RNA demethylases ALKBH5 (AlkB Homolog 5) and FTO (Fat mass and obesity-associated protein). The ALKBH5 and FTO RNA demethylases recognize m6A in similar nucleotide contexts. Therefore, these proteins can partially substitute for each other. To assess the impact of total m6A demethylation failure we performed high-throughput sequencing of cytoplasmic RNA from ALKBH5 and FTO double knockout and wild type HEK293T cells. The RNA-Seq libraries were sequenced on Illumina NextSeq 500, trimmed, and mapped to the human genome. The consequent read counting and differential expression analysis in the R environment detected 5871 differentially expressed and 166 alternatively spliced genes comparing double knockout against wild type HEK293T cells. Raw data are deposited in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE198050.

4.
Biochemistry (Mosc) ; 87(Suppl 1): S1-S191, 2022 Jan.
Article in English | MEDLINE | ID: mdl-35501982

ABSTRACT

Lev Ovchinnikov was a true man of Science. Until the end of his life, he retained not only loyalty to strict scientific principles, but also a benevolent attitude towards the people around him. He devoted his scientific career to the study of mRNP and regulation of protein biosynthesis. He created a unique scientific school that received international recognition.

5.
Biochemistry (Mosc) ; 87(Suppl 1): S48-S167, 2022 Jan.
Article in English | MEDLINE | ID: mdl-35501986

ABSTRACT

YB proteins are DNA/RNA binding proteins, members of the family of proteins with cold shock domain. Role of YB proteins in the life of cells, tissues, and whole organisms is extremely important. They are involved in transcription regulation, pre-mRNA splicing, mRNA translation and stability, mRNA packaging into mRNPs, including stress granules, DNA repair, and many other cellular events. Many processes, from embryonic development to aging, depend on when and how much of these proteins have been synthesized. Here we discuss regulation of the levels of YB-1 and, in part, of its homologs in the cell. Because the amount of YB-1 is immediately associated with its functioning, understanding the mechanisms of regulation of the protein amount invariably reveals the events where YB-1 is involved. Control over the YB-1 abundance may allow using this gene/protein as a therapeutic target in cancers, where an increased expression of the YBX1 gene often correlates with the disease severity and poor prognosis.


Subject(s)
Protein Biosynthesis , Y-Box-Binding Protein 1 , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Mammals/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Y-Box-Binding Protein 1/metabolism
6.
Nucleic Acids Res ; 49(17): 10061-10081, 2021 09 27.
Article in English | MEDLINE | ID: mdl-34469566

ABSTRACT

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.


Subject(s)
Inverted Repeat Sequences/genetics , Peptide Chain Initiation, Translational/genetics , RNA, Messenger/genetics , Stress Granules/metabolism , Y-Box-Binding Protein 1/metabolism , Adenosine Triphosphate/metabolism , Arsenites/toxicity , Base Pairing/genetics , Cell Line, Tumor , HeLa Cells , Humans , Ribosomes/metabolism
7.
Int J Mol Sci ; 22(7)2021 Apr 01.
Article in English | MEDLINE | ID: mdl-33915887

ABSTRACT

The methylation of adenosine in the N6 position (m6A) is a widely used modification of eukaryotic mRNAs. Its importance for the regulation of mRNA translation was put forward recently, essentially due to the ability of methylated mRNA to be translated in conditions of inhibited cap-dependent translation initiation, e.g., under stress. However, the peculiarities of translation initiation on m6A-modified mRNAs are not fully known. In this study, we used toeprinting and translation in a cell-free system to confirm that m6A-modified mRNAs can be translated in conditions of suppressed cap-dependent translation. We show for the first time that m6A-modified mRNAs display not only decreased elongation, but also a lower efficiency of translation initiation. Additionally, we report relative resistance of m6A-mRNA translation initiation in the absence of ATP and inhibited eIF4A activity. Our novel findings indicate that the scanning of m6A-modified leader sequences is performed by a noncanonical mechanism.


Subject(s)
Protein Biosynthesis , RNA, Messenger/metabolism , HEK293 Cells , Humans , Methylation
8.
Commun Biol ; 4(1): 359, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33742080

ABSTRACT

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved ß-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.


Subject(s)
Cytoplasmic Granules/metabolism , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism , Uterine Cervical Neoplasms/metabolism , Y-Box-Binding Protein 1/metabolism , Binding Sites , Cell Proliferation , Cytoplasmic Granules/genetics , Cytoplasmic Granules/pathology , Female , HeLa Cells , Humans , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Protein Binding , Protein Interaction Domains and Motifs , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins/genetics , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Y-Box-Binding Protein 1/genetics
9.
Int J Mol Sci ; 23(1)2021 Dec 31.
Article in English | MEDLINE | ID: mdl-35008856

ABSTRACT

YB-1 is a multifunctional DNA- and RNA-binding protein involved in cell proliferation, differentiation, and migration. YB-1 is a predominantly cytoplasmic protein that is transported to the nucleus in certain conditions, including DNA-damaging stress, transcription inhibition, and viral infection. In tumors, YB-1 nuclear localization correlates with high aggressiveness, multidrug resistance, and a poor prognosis. It is known that posttranslational modifications can regulate the nuclear translocation of YB-1. In particular, well-studied phosphorylation at serine 102 (S102) activates YB-1 nuclear import. Here, we report that Akt kinase phosphorylates YB-1 in vitro at serine 209 (S209), which is located in the vicinity of the YB-1 nuclear localization signal. Using phosphomimetic substitutions, we showed that S209 phosphorylation inhibits YB-1 nuclear translocation and prevents p-S102-mediated YB-1 nuclear import.


Subject(s)
Cell Nucleus/metabolism , Phosphoserine/metabolism , Y-Box-Binding Protein 1/metabolism , Amino Acid Sequence , Animals , HeLa Cells , Humans , Mice , NIH 3T3 Cells , Phosphorylation , Protein Binding , Protein Transport , Proto-Oncogene Proteins c-akt/metabolism , RNA/metabolism , Serum , Y-Box-Binding Protein 1/chemistry
10.
Biomolecules ; 10(4)2020 04 11.
Article in English | MEDLINE | ID: mdl-32290447

ABSTRACT

Y-box binding proteins (YB proteins) are DNA/RNA-binding proteins belonging to a large family of proteins with the cold shock domain. Functionally, these proteins are known to be the most diverse, although the literature hardly offers any molecular mechanisms governing their activities in the cell, tissue, or the whole organism. This review describes the involvement of YB proteins in RNA-dependent processes, such as mRNA packaging into mRNPs, mRNA translation, and mRNA stabilization. In addition, recent data on the structural peculiarities of YB proteins underlying their interactions with nucleic acids are discussed.


Subject(s)
Protein Biosynthesis/genetics , RNA Stability/genetics , Ribonucleoproteins/metabolism , Y-Box-Binding Protein 1/metabolism , Animals , Cytoplasmic Granules/metabolism , Humans , Protein Binding , Y-Box-Binding Protein 1/chemistry
11.
Nucleic Acids Res ; 47(6): 3127-3141, 2019 04 08.
Article in English | MEDLINE | ID: mdl-30605522

ABSTRACT

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.


Subject(s)
Nucleoproteins/chemistry , RNA-Binding Proteins/ultrastructure , Y-Box-Binding Protein 1/ultrastructure , Amino Acid Sequence/genetics , Cytoskeleton/genetics , Cytoskeleton/ultrastructure , Escherichia coli/genetics , Humans , Nucleoproteins/genetics , Nucleoproteins/ultrastructure , Protein Binding/genetics , Protein Biosynthesis/genetics , Protein Folding , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Ribosomes/chemistry , Ribosomes/genetics , Y-Box-Binding Protein 1/chemistry , Y-Box-Binding Protein 1/genetics
12.
Cells ; 9(1)2019 Dec 31.
Article in English | MEDLINE | ID: mdl-31906126

ABSTRACT

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution.


Subject(s)
Cell Nucleus/metabolism , Gene Expression Regulation , Serine/metabolism , Transcription, Genetic , Y-Box-Binding Protein 1/metabolism , Animals , Cell Line, Tumor , Humans , In Situ Hybridization , Mice , Phosphorylation , RNA Polymerase II/metabolism , RNA, Messenger/genetics
13.
Biochem Biophys Res Commun ; 480(4): 629-634, 2016 Nov 25.
Article in English | MEDLINE | ID: mdl-27794479

ABSTRACT

The DNA/RNA-binding protein YB-1 (Y-box binding protein 1) performs multiple functions both in the cytoplasm and the nucleus of the cell. Generally localized to the cytoplasm, under certain conditions YB-1 is translocated to the nucleus. Here we report for the first time a transport factor that mediates YB-1 nuclear import - transportin-1. The YB-1/transportin-1 complex can be isolated from HeLa cell extract. Nuclear import of YB-1 and its truncated form YB-1 (1-219) in in vitro transport assay was diminished in the presence of a competitor substrate and ceased in the presence of transportin-1 inhibitor M9M. Inhibitors of importin ß1 had no effect on YB-1 transport. Furthermore, transport of YB-1 (P201A/Y202A) and YB-1 (1-219) (P201A/Y202A) bearing inactivating mutations in the transportin-1-dependent nuclear localization signal was practically abolished. Together, these results indicate that transportin-1 mediates YB-1 nuclear translocation.


Subject(s)
Cell Nucleus/metabolism , Y-Box-Binding Protein 1/metabolism , beta Karyopherins/metabolism , Active Transport, Cell Nucleus/physiology , Binding Sites , HeLa Cells , Humans , Protein Binding , Y-Box-Binding Protein 1/chemistry , beta Karyopherins/chemistry
14.
PLoS One ; 9(8): e104513, 2014.
Article in English | MEDLINE | ID: mdl-25116735

ABSTRACT

The multifunctional eukaryotic protein YB-1 (Y-box binding protein 1) plays a role in DNA reparation, transcription regulation, splicing, and mRNA translation, thereby participating in many crucial events in cells. Its effect is dependent mostly on its amount, and hence, on regulation of its synthesis. Published data on regulation of synthesis of YB-1 mediated by its mRNA 5' UTR, and specifically on the 5' UTR length and the presence of TOP-like motifs in this region, are contradictory. Here we report that 5' UTRs of major forms of human, rabbit, and mouse YB-1 mRNAs are about 140 nucleotides long and contain no TOP-like motifs mentioned in the literature. Also, we have found that YB-1 specifically interacts with the 5' UTR of its own mRNA within a region of about 100 nucleotides upstream from the start codon. Apart from YB-1, translation of YB-1 mRNA in a cell free system gives an additional product with an extended N-terminus and lower electrophoretic mobility. The start codon for synthesis of the additional product is AUC at position -(60-58) of the same open reading frame as that for the major product. Also, in the cell there is an alternative YB-1 mRNA with exon 1 replaced by a part of intron 1; YB-1 synthesized in vitro from this mRNA contains, instead of its N-terminal A/P domain, 10-11 amino acids encoded by intron 1.


Subject(s)
Alternative Splicing , Y-Box-Binding Protein 1/genetics , 5' Untranslated Regions , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , HEK293 Cells , Humans , MCF-7 Cells , Mice , Molecular Sequence Data , NIH 3T3 Cells , Protein Binding , Protein Biosynthesis , RNA, Messenger/chemistry , RNA, Messenger/genetics , Rabbits , Y-Box-Binding Protein 1/chemistry , Y-Box-Binding Protein 1/metabolism
15.
Wiley Interdiscip Rev RNA ; 5(1): 95-110, 2014.
Article in English | MEDLINE | ID: mdl-24217978

ABSTRACT

The Y-box binding protein 1 (YB-1, YBX1) is a member of the family of DNA- and RNA-binding proteins with an evolutionarily ancient and conserved cold shock domain. It falls into a group of intrinsically disordered proteins that do not follow the classical rule 'one protein-one function' but introduce a novel principle stating that a disordered structure suggests many functions. YB-1 participates in a wide variety of DNA/RNA-dependent events, including DNA reparation, pre-mRNA transcription and splicing, mRNA packaging, and regulation of mRNA stability and translation. At the cell level, the multiple activities of YB-1 are manifested as its involvement in cell proliferation and differentiation, stress response, and malignant cell transformation. WIREs RNA 2014, 5:95-110. doi: 10.1002/wrna.1200 CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.


Subject(s)
DNA/metabolism , RNA, Messenger/metabolism , Y-Box-Binding Protein 1/metabolism , Animals , Apoptosis , Cell Differentiation , Cell Proliferation , DNA/genetics , DNA Repair , Humans , Protein Biosynthesis , Protein Conformation , RNA, Messenger/genetics , Stress, Physiological , Transcriptional Activation , Y-Box-Binding Protein 1/chemistry , Y-Box-Binding Protein 1/genetics
16.
RNA Biol ; 9(12): 1473-87, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23134843

ABSTRACT

YB-1 is a multifunctional cold shock domain containing protein that is involved virtually in all DNA- and mRNA-dependent cellular events. Its amount is regulated at the level of both transcription and translation. We showed previously that translation of poly A(-) YB-1 mRNA in vitro is selectively controlled by two proteins, YB-1 and PABP, through their specific and competitive binding to a regulatory element (RE) within 3' UTR of this mRNA. Here, we describe effects of these two proteins on translation of poly A(+) as compared with poly A(-) YB-1 mRNA in a rabbit reticulocyte cell-free translation system. We have found that YB-1 inhibits translation of both poly A(+) and poly A(-) YB-1 mRNAs at the same comparatively low YB-1/mRNA ratio. PABP has no positive effect on translation of poly A(+) YB-1 mRNA, although it has a stimulating effect on translation of poly A(-) YB-1 mRNA. A positive PABP effect on translation of poly A(+) YB-1 mRNA arose after removal of a portion of the sequence between RE and the poly(A) tail and disappeared after its replacement by another non-specific sequence of the same length. We also report that the RE fragment forms a complex with the poly(A) fragment in the presence of rabbit reticulocyte lysate (RRL) proteins. For its formation PABP is necessary but not sufficient. These results are in agreement with the proposed model implying formation of a mini-loop at 3' UTR of YB-1 mRNA that includes RE, RRL proteins and the poly(A) tail.


Subject(s)
Poly(A)-Binding Protein I/metabolism , Polyadenylation , Y-Box-Binding Protein 1/metabolism , 3' Untranslated Regions , Animals , Base Sequence , Binding Sites , Cell-Free System , Humans , Molecular Sequence Data , Plasmids/genetics , Plasmids/metabolism , Poly(A)-Binding Protein I/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Regulatory Sequences, Ribonucleic Acid , Reticulocytes/metabolism , Transcription, Genetic , Y-Box-Binding Protein 1/genetics
17.
PLoS One ; 7(12): e52527, 2012.
Article in English | MEDLINE | ID: mdl-23285076

ABSTRACT

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.


Subject(s)
Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Y-Box-Binding Protein 1/biosynthesis , Animals , Cell Division/drug effects , Cell Line , Cells, Cultured , Gene Expression Regulation/drug effects , Humans , Indoles/pharmacology , Mice , Organ Specificity/drug effects , Organ Specificity/genetics , Polyribosomes/drug effects , Polyribosomes/metabolism , Protein Biosynthesis/drug effects , Purines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rabbits , Ribonucleoproteins/metabolism , Signal Transduction/drug effects , Y-Box-Binding Protein 1/genetics , Y-Box-Binding Protein 1/metabolism
18.
RNA Biol ; 8(5): 883-92, 2011.
Article in English | MEDLINE | ID: mdl-21788731

ABSTRACT

YB-1 is a DNA- and RNA-binding protein that regulates expression of many important genes. Its deficiency or excess may pose threats, including malignant cellular transformation and metastasis, which explains the necessity of strict control over its amount at every level. As we showed previously, the 3' untranslated region (UTR) of YB-1 mRNA contains a regulatory element specifically binding to YB-1 and PABP (PABPC1). Also, we showed that YB-1 selectively inhibits YB-1 mRNA translation, while PABP stimulates it in a poly(A) tail-independent manner. It was suggested that regulation of YB-1 mRNA translation involves competition between PABP and YB-1 for binding to the regulatory element. Here we offer cogent evidence for this model and add novel details to the mechanism of regulation of YB-1 synthesis. In experiments on regulatory element deletion we showed that it is this element that is responsible for a specific effect of YB-1 and PABP on YB-1 mRNA translation. Mutations eliminating only specific YB-1 affinity for this element suppressed the inhibitory effect of YB-1 and concurrently dramatically decreased the PABP stimulating effect. Mutations reducing only specific PABP affinity for this element, as well as spatial separation of the YB-1- and PABP binding sites, did not affect the YB-1 inhibitory action but completely abolished the positive PABP effect. Together, these results unambiguously prove direct inhibitory action of YB-1 on its mRNA translation, while the positive effect of PABP is realized through displacing YB-1 from the regulatory element.


Subject(s)
Poly(A)-Binding Proteins/metabolism , Protein Biosynthesis/genetics , Protein Sorting Signals/genetics , Y-Box-Binding Protein 1/metabolism , 3' Untranslated Regions/genetics , Binding Sites , DNA-Binding Proteins/genetics , Poly(A)-Binding Proteins/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Y-Box-Binding Protein 1/genetics
19.
Mol Cell Biol ; 25(8): 3317-23, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15798215

ABSTRACT

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.


Subject(s)
3' Untranslated Regions/metabolism , CCAAT-Enhancer-Binding Proteins/genetics , Peptide Chain Initiation, Translational/genetics , Poly(A)-Binding Proteins/metabolism , Regulatory Sequences, Ribonucleic Acid/genetics , Transcription Factors/genetics , 3' Untranslated Regions/genetics , Animals , Base Sequence , Binding Sites , Binding, Competitive , CCAAT-Enhancer-Binding Proteins/metabolism , CCAAT-Enhancer-Binding Proteins/physiology , Molecular Sequence Data , NFI Transcription Factors , Peptide Chain Initiation, Translational/physiology , RNA, Messenger/metabolism , Rats , Regulatory Sequences, Ribonucleic Acid/physiology , Reticulocytes/metabolism , Ribosomes/metabolism , Transcription Factors/metabolism , Transcription Factors/physiology , Y-Box-Binding Protein 1
20.
J Biol Chem ; 278(20): 18191-8, 2003 May 16.
Article in English | MEDLINE | ID: mdl-12646583

ABSTRACT

The major protein of cytoplasmic mRNPs from rabbit reticulocytes, YB-1, is a member of an ancient family of proteins containing a common structural feature, cold-shock domain. In eukaryotes, this family is represented by multifunctional mRNA/Y-box DNA-binding proteins that control gene expression at different stages. To address possible post-transcriptional regulation of YB-1 gene expression, we examined effects of exogenous 5'- and 3'-untranslatable region-containing fragments of YB-1 mRNA on its translation and stability in a cell-free system. The addition of the 3' mRNA fragment as well as its subfragment I shut off protein synthesis at the initiation stage without affecting mRNA stability. UV cross-linking revealed four proteins (69, 50, 46, and 44 kDa) that specifically interacted with the 3' mRNA fragment; the inhibitory subfragment I bound two of them, 69- and 50-kDa proteins. We have identified these proteins as PABP (poly(A)-binding protein) (69 kDa) and YB-1 (50 kDa) and demonstrated that titrating out of PABP by poly(A) strongly and specifically inhibits YB-1 mRNA cap(+)poly(A)(-) translation in a cell-free system. Thus, PABP is capable of positively affecting YB-1 mRNA translation in a poly(A) tail-independent manner.


Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , DNA-Binding Proteins , Poly(A)-Binding Proteins/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Transcription Factors , Animals , Blotting, Northern , Cell-Free System , Codon, Terminator , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation , Globins/metabolism , Immunoblotting , NFI Transcription Factors , Nuclear Proteins , Plasmids/metabolism , Precipitin Tests , Protein Binding , Rabbits , Reticulocytes/metabolism , Sucrose/pharmacology , Temperature , Time Factors , Ultraviolet Rays , Y-Box-Binding Protein 1
SELECTION OF CITATIONS
SEARCH DETAIL
...