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1.
Nucleic Acids Res ; 49(17): 10061-10081, 2021 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-34469566

RESUMEN

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.


Asunto(s)
Secuencias Invertidas Repetidas/genética , Iniciación de la Cadena Peptídica Traduccional/genética , ARN Mensajero/genética , Gránulos de Estrés/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Adenosina Trifosfato/metabolismo , Arsenitos/toxicidad , Emparejamiento Base/genética , Línea Celular Tumoral , Células HeLa , Humanos , Ribosomas/metabolismo
2.
Nucleic Acids Res ; 47(6): 3127-3141, 2019 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-30605522

RESUMEN

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.


Asunto(s)
Nucleoproteínas/química , Proteínas de Unión al ARN/ultraestructura , Proteína 1 de Unión a la Caja Y/ultraestructura , Secuencia de Aminoácidos/genética , Citoesqueleto/genética , Citoesqueleto/ultraestructura , Escherichia coli/genética , Humanos , Nucleoproteínas/genética , Nucleoproteínas/ultraestructura , Unión Proteica/genética , Biosíntesis de Proteínas/genética , Pliegue de Proteína , ARN Mensajero/química , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Ribosomas/química , Ribosomas/genética , Proteína 1 de Unión a la Caja Y/química , Proteína 1 de Unión a la Caja Y/genética
3.
Int J Mol Sci ; 23(1)2021 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-35008856

RESUMEN

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.


Asunto(s)
Núcleo Celular/metabolismo , Fosfoserina/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Secuencia de Aminoácidos , Animales , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN/metabolismo , Suero , Proteína 1 de Unión a la Caja Y/química
4.
Nucleic Acids Res ; 43(19): 9457-73, 2015 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-26271991

RESUMEN

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.


Asunto(s)
ADN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Animales , Células Cultivadas , ADN/ultraestructura , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Superhelicoidal/metabolismo , Microscopía de Fuerza Atómica , Unión Proteica , Biosíntesis de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/ultraestructura , Ratas , Proteína 1 de Unión a la Caja Y/química , Proteína 1 de Unión a la Caja Y/ultraestructura
5.
Biochem Biophys Res Commun ; 480(4): 629-634, 2016 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-27794479

RESUMEN

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.


Asunto(s)
Núcleo Celular/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Sitios de Unión , Células HeLa , Humanos , Unión Proteica , Proteína 1 de Unión a la Caja Y/química , beta Carioferinas/química
6.
J Mol Recognit ; 28(2): 117-23, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25605055

RESUMEN

Y-box binding protein 1 (YB-1) is widely known to participate in a multiple DNA and RNA processing events in the living cell. YB-1 is also regarded as a putative component of DNA repair. This possibility is supported by relocalization of YB-1 into the nucleus following genotoxic stress. Increased affinity of YB-1 for damaged DNA, especially in its single-stranded form, and its functional interaction with proteins responsible for the initiation of apurinic/apyrimidinic (AP) site repair, namely, AP endonuclease 1 and DNA glycosylase NEIL1, suggest that YB-1 could be involved in the repair of AP sites as a regulatory protein. Here we show that YB-1 has a significant inhibitory effect on the cleavage of AP sites located in single-stranded DNA and in DNA bubble structures. Such interference may be considered as a possible mechanism to prevent single-stranded intermediates of DNA replication, transcription and repair from being converted into highly genotoxic DNA strand breaks, thus allowing the cell to coordinate different DNA processing mechanisms.


Asunto(s)
ADN Glicosilasas/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN/química , ADN/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Sitios de Unión , Núcleo Celular/metabolismo , Daño del ADN , ADN Glicosilasas/genética , Reparación del ADN , Replicación del ADN , ADN de Cadena Simple , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Humanos , Especificidad por Sustrato
7.
Biochim Biophys Acta ; 1834(2): 559-67, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23220387

RESUMEN

YB-1 is a major mRNP protein participating in the regulation of transcription and translation of a wide range of eukaryotic genes in many organisms probably due to its influence on mRNA packing into mRNPs. While the functional properties of YB-1 are extensively studied, little is known about its structural properties. In the present work we focused on studying its secondary structure, rigidity of its tertiary structure, compactness, and oligomerization in vitro by using far UV-CD, DSC, one-dimensional (1)H NMR, SAXS, sedimentation and FPLC. It was shown that only the cold shock domain within the entire YB-1 chain has a well-packed tertiary structure undergoing cooperative heat and cold denaturation transitions. In contrast, the rest of the YB-1 molecule is not rigidly packed and consists of PP II-like helical secondary structure elements and coil-like regions. At the same time, the overall dimension of the protein molecule is unexpectedly small. The polypeptide chains of YB-1 have a high tendency to form oligomers at neutral pH, while the extent and structural organization of the oligomers depend on protein concentration and ionic strength varying from compact monomeric units up to high molecular weight oligomers. These oligomers in solution are unstable and dissociate upon protein concentration decrease.


Asunto(s)
Multimerización de Proteína/fisiología , Ribonucleoproteínas/química , Proteína 1 de Unión a la Caja Y/química , Animales , Concentración de Iones de Hidrógeno , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Conejos , Ribonucleoproteínas/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo
8.
EMBO J ; 28(1): 58-68, 2009 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-19078965

RESUMEN

The interaction between the poly(A)-binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA-binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB-1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB-1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP-dependent after the addition of YB-1. In this system, eIF4E binding to the cap structure is inhibited by YB-1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB-1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB-1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factor 4F Eucariótico de Iniciación/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Biosíntesis de Proteínas , Animales , Extractos Celulares , Línea Celular , Ratones , Modelos Biológicos , Conejos , Proteína 1 de Unión a la Caja Y
9.
J Mol Recognit ; 25(4): 224-33, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22434712

RESUMEN

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.


Asunto(s)
División del ADN , ADN Glicosilasas/química , Proteínas de Unión al ADN/química , Proteína de Replicación A/química , Factores de Transcripción/química , Animales , Ácido Apurínico/química , Borohidruros/química , ADN de Cadena Simple/química , Ratones , Polinucleótidos/química , Unión Proteica , Conejos , Bases de Schiff/química
10.
RNA Biol ; 9(12): 1473-87, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23134843

RESUMEN

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.


Asunto(s)
Proteína I de Unión a Poli(A)/metabolismo , Poliadenilación , Proteína 1 de Unión a la Caja Y/metabolismo , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Sistema Libre de Células , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Plásmidos/metabolismo , Proteína I de Unión a Poli(A)/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Reticulocitos/metabolismo , Transcripción Genética , Proteína 1 de Unión a la Caja Y/genética
11.
RNA Biol ; 8(5): 883-92, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21788731

RESUMEN

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.


Asunto(s)
Proteínas de Unión a Poli(A)/metabolismo , Biosíntesis de Proteínas/genética , Señales de Clasificación de Proteína/genética , Proteína 1 de Unión a la Caja Y/metabolismo , Regiones no Traducidas 3'/genética , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión a Poli(A)/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteína 1 de Unión a la Caja Y/genética
12.
Commun Biol ; 4(1): 359, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33742080

RESUMEN

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.


Asunto(s)
Gránulos Citoplasmáticos/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Sitios de Unión , Proliferación Celular , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/patología , Femenino , Células HeLa , Humanos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Proteína 1 de Unión a la Caja Y/genética
13.
J Biol Chem ; 284(52): 36569-36580, 2009 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-19843517

RESUMEN

Following exposure to various stresses (arsenite, UV, hyperthermia, and hypoxia), mRNAs are assembled into large cytoplasmic bodies known as "stress granules," in which mRNAs and associated proteins may be processed by specific enzymes for different purposes like transient storing, sorting, silencing, or other still unknown processes. To limit mRNA damage during stress, the assembly of micrometric granules has to be rapid, and, indeed, it takes only approximately 10-20 min in living cells. However, such a rapid assembly breaks the rules of hindered diffusion in the cytoplasm, which states that large cytoplasmic bodies are almost immobile. In the present work, using HeLa cells and YB-1 protein as a stress granule marker, we studied three hypotheses to understand how cells overcome the limitation of hindered diffusion: shuttling of small messenger ribonucleoprotein particles from small to large stress granules, sliding of messenger ribonucleoprotein particles along microtubules, microtubule-mediated stirring of large stress granules. Our data favor the two last hypotheses and underline that microtubule dynamic instability favors the formation of micrometric stress granules.


Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Microtúbulos/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Estrés Fisiológico/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Gránulos Citoplasmáticos/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Microtúbulos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , Ribonucleoproteínas/genética , Ovinos , Proteína 1 de Unión a la Caja Y
14.
Biomolecules ; 10(4)2020 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-32290447

RESUMEN

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.


Asunto(s)
Biosíntesis de Proteínas/genética , Estabilidad del ARN/genética , Ribonucleoproteínas/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Animales , Gránulos Citoplasmáticos/metabolismo , Humanos , Unión Proteica , Proteína 1 de Unión a la Caja Y/química
15.
Mol Cell Biol ; 26(1): 277-92, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16354698

RESUMEN

YB-1 is a broad-specificity RNA-binding protein that is involved in regulation of mRNA transcription, splicing, translation, and stability. In both germinal and somatic cells, YB-1 and related proteins are major components of translationally inactive messenger ribonucleoprotein particles (mRNPs) and are mainly responsible for storage of mRNAs in a silent state. However, mechanisms regulating the repressor activity of YB-1 are not well understood. Here we demonstrate that association of YB-1 with the capped 5' terminus of the mRNA is regulated via phosphorylation by the serine/threonine protein kinase Akt. In contrast to its nonphosphorylated form, phosphorylated YB-1 fails to inhibit cap-dependent but not internal ribosome entry site-dependent translation of a reporter mRNA in vitro. We also show that similar to YB-1, Akt is associated with inactive mRNPs and that activated Akt may relieve translational repression of the YB-1-bound mRNAs. Using Affymetrix microarrays, we found that many of the YB-1-associated messages encode stress- and growth-related proteins, raising the intriguing possibility that Akt-mediated YB-1 phosphorylation could, in part, increase production of proteins regulating cell proliferation, oncogenic transformation, and stress response.


Asunto(s)
Biosíntesis de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión a Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Animales , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Ratones , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Caperuzas de ARN/metabolismo , Ribonucleoproteínas/metabolismo
16.
Genes (Basel) ; 10(2)2019 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-30700035

RESUMEN

The mammalian target of rapamycin (mTOR) kinase is a well-known master regulator of growth-dependent gene expression in higher eukaryotes. Translation regulation is an important function of the mTORC1 pathway that controls the synthesis of many ribosomal proteins and translation factors. Housekeeping genes such as ß-actin (ACTB) are widely used as negative control genes in studies of growth-dependent translation. Here we demonstrate that translation of both endogenous and reporter ACTB mRNA is inhibited in the presence of mTOR kinase inhibitor (Torin1) and under amino acid starvation. Notably, 5'UTR and promoter of ACTB are sufficient for the mTOR-dependent translational response, and the degree of mTOR-sensitivity of ACTB mRNA translation is cell type-dependent.


Asunto(s)
Actinas/genética , ARN Mensajero/genética , Serina-Treonina Quinasas TOR/metabolismo , Actinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Naftiridinas/farmacología , Células PC-3 , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores
17.
Cells ; 9(1)2019 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-31906126

RESUMEN

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.


Asunto(s)
Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Serina/metabolismo , Transcripción Genética , Proteína 1 de Unión a la Caja Y/metabolismo , Animales , Línea Celular Tumoral , Humanos , Hibridación in Situ , Ratones , Fosforilación , ARN Polimerasa II/metabolismo , ARN Mensajero/genética
18.
FEBS Lett ; 582(19): 2875-81, 2008 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-18652827

RESUMEN

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.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Proteínas de Unión al ADN/genética , Humanos , Microscopía de Fuerza Atómica , Microtúbulos/genética , Proteínas Nucleares/genética , Ribonucleoproteínas/genética
19.
BMC Biochem ; 9: 23, 2008 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-18793384

RESUMEN

BACKGROUND: YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. RESULTS: We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes. CONCLUSION: These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Cromatografía de Afinidad , Humanos , Microscopía de Fuerza Atómica , Microtúbulos/metabolismo , Fragmentos de Péptidos/metabolismo , ARN Mensajero/metabolismo , Conejos , Ribonucleoproteínas/metabolismo , Extractos de Tejidos , Tubulina (Proteína)/aislamiento & purificación , Tubulina (Proteína)/ultraestructura , Proteína 1 de Unión a la Caja Y
20.
Mol Cell Biol ; 25(8): 3317-23, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15798215

RESUMEN

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.


Asunto(s)
Regiones no Traducidas 3'/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Iniciación de la Cadena Peptídica Traduccional/genética , Proteínas de Unión a Poli(A)/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico/genética , Factores de Transcripción/genética , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Datos de Secuencia Molecular , Factores de Transcripción NFI , Iniciación de la Cadena Peptídica Traduccional/fisiología , ARN Mensajero/metabolismo , Ratas , Secuencias Reguladoras de Ácido Ribonucleico/fisiología , Reticulocitos/metabolismo , Ribosomas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Proteína 1 de Unión a la Caja Y
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