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1.
Cell Cycle ; 13(7): 1187-200, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24553115

RESUMEN

At the end of the growth phase, mouse antral follicle oocytes acquire full developmental competence. In the mouse, this event is marked by the transition from the so-called non-surrounded nucleolus (NSN) chromatin configuration into the transcriptionally quiescent surrounded nucleolus (SN) configuration, which is named after a prominent perinucleolar condensed chromatin ring. However, the SN chromatin configuration alone is not sufficient for determining the developmental competence of the SN oocyte. There are additional nuclear and cytoplamic factors involved, while a little is known about the changes occurring in the cytoplasm during the NSN/SN transition. Here, we report functional analysis of maternal ELAVL2 an AU-rich element binding protein. Elavl2 gene encodes an oocyte-specific protein isoform (denoted ELAVL2°), which acts as a translational repressor. ELAVL2° is abundant in fully grown NSN oocytes, is ablated during the NSN/SN transition and remains low during the oocyte-to-embryo transition (OET). ELAVL2° overexpression during meiotic maturation causes errors in chromosome segregation, indicating the significance of naturally reduced ELAVL2° levels in SN oocytes. On the other hand, during oocyte growth, prematurely reduced Elavl2 expression results in lower yields of fully grown and meiotically matured oocytes, suggesting that Elavl2 is necessary for proper oocyte maturation. Moreover, Elavl2 knockdown showed stimulating effects on translation in fully grown oocytes. We propose that ELAVL2 has an ambivalent role in oocytes: it functions as a pleiotropic translational repressor in efficient production of fully grown oocytes, while its disposal during the NSN/SN transition contributes to the acquisition of full developmental competence.


Asunto(s)
Proteína 2 Similar a ELAV/metabolismo , Meiosis/fisiología , Oocitos/metabolismo , Animales , Línea Celular , Proteína 2 Similar a ELAV/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Oocitos/citología , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
2.
RNA ; 19(12): 1632-8, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24141620

RESUMEN

The mechanisms of gene expression regulation by miRNAs have been extensively studied. However, the regulation of miRNA function and decay has long remained enigmatic. Only recently, 3' uridylation via LIN28A-TUT4/7 has been recognized as an essential component controlling the biogenesis of let-7 miRNAs in stem cells. Although uridylation has been generally implicated in miRNA degradation, the nuclease responsible has remained unknown. Here, we identify the Perlman syndrome-associated protein DIS3L2 as an oligo(U)-binding and processing exoribonuclease that specifically targets uridylated pre-let-7 in vivo. This study establishes DIS3L2 as the missing component of the LIN28-TUT4/7-DIS3L2 pathway required for the repression of let-7 in pluripotent cells.


Asunto(s)
Exorribonucleasas/fisiología , MicroARNs/metabolismo , Precursores del ARN/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Células Madre Embrionarias/enzimología , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Ratones , MicroARNs/genética , Unión Proteica , Precursores del ARN/genética , Estabilidad del ARN , ARN Interferente Pequeño/genética
3.
Methods Mol Biol ; 942: 291-314, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23027058

RESUMEN

Double-stranded RNA (dsRNA) is involved in different biological processes. At least three different pathways can respond to dsRNA in mammals. One of these pathways is RNA interference (RNAi) where long dsRNA induces sequence-specific degradation of transcripts carrying sequences complementary to dsRNA. Long dsRNA is also a potent trigger of the interferon pathway, a sequence-independent response that leads to global suppression of translation and global RNA degradation. In addition, dsRNA can be edited by adenosine deamination, which may result in nuclear retention and degradation of dsRNA or in alteration of RNA coding potential. Here, we provide a technical review summarizing different strategies of long dsRNA usage. While the review is largely focused on long dsRNA-induced RNAi in mammalian cells, it also provides helpful information on both the in vitro production and in vivo expression of dsRNAs. We present an overview of currently available vectors for dsRNA expression and provide the latest update on oocyte-specific transgenic RNAi approaches.


Asunto(s)
Ingeniería Genética/métodos , ARN Bicatenario/biosíntesis , ARN Bicatenario/genética , Animales , Línea Celular , Vectores Genéticos/genética , Humanos , Oocitos/metabolismo , Interferencia de ARN , ARN Bicatenario/química
4.
J Biol Chem ; 283(50): 35186-98, 2008 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-18854321

RESUMEN

In response to environmental stress, the translation machinery of cells is reprogrammed. The majority of actively translated mRNAs are released from polysomes and driven to specific cytoplasmic foci called stress granules (SGs) where dynamic changes in protein-RNA interaction determine the subsequent fate of mRNAs. Here we show that the DEAH box RNA helicase RHAU is a novel SG-associated protein. Although RHAU protein was originally identified as an AU-rich element-associated protein involved in urokinase-type plasminogen activator mRNA decay, it was not clear whether RHAU could directly interact with RNA. We have demonstrated that RHAU physically interacts with RNA in vitro and in vivo through a newly identified N-terminal RNA-binding domain, which was found to be both essential and sufficient for RHAU localization in SGs. We have also shown that the ATPase activity of RHAU plays a role in the RNA interaction and in the regulation of protein retention in SGs. Thus, our results show that RHAU is the fourth RNA helicase detected in SGs, after rck/p54, DDX3, and eIF4A, and that its association with SGs is dynamic and mediated by an RHAU-specific RNA-binding domain.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , ARN Helicasas/química , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Reactivos de Enlaces Cruzados/farmacología , Escherichia coli/metabolismo , Factor 4A Eucariótico de Iniciación/química , Recuperación de Fluorescencia tras Fotoblanqueo , Células HeLa , Humanos , Cinética , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido
5.
Exp Cell Res ; 314(6): 1378-91, 2008 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-18279852

RESUMEN

RHAU (RNA helicase associated with AU-rich element) is a DExH protein originally identified as a factor accelerating AU-rich element-mediated mRNA degradation. The discovery that RHAU is predominantly localized in the nucleus, despite mRNA degradation occurring in the cytoplasm, prompted us to consider the nuclear functions of RHAU. In HeLa cells, RHAU was found to be localized throughout the nucleoplasm with some concentrated in nuclear speckles. Transcriptional arrest altered the localization to nucleolar caps, where RHAU is closely localized with RNA helicases p68 and p72, suggesting that RHAU is involved in transcription-related RNA metabolism in the nucleus. To see whether RHAU affects global gene expression transcriptionally or posttranscriptionally, we performed microarray analysis using total RNA from RHAU-depleted HeLa cell lines, measuring both steady-state mRNA levels and mRNA half-lives by actinomycin D chase. There was no change in the half-lives of most transcripts whose steady-state levels were affected by RHAU knockdown, suggesting that these transcripts are subjected to transcriptional regulation. We propose that RHAU has a dual function, being involved in both the synthesis and degradation of mRNA in different subcellular compartments.


Asunto(s)
Nucléolo Celular/enzimología , ARN Helicasas DEAD-box/metabolismo , Transcripción Genética , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , Regulación Neoplásica de la Expresión Génica , Semivida , Células HeLa , Humanos , Cinética , Proteínas Mutantes/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transfección
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