Asunto(s)
Sarampión/prevención & control , Negativa a la Vacunación/estadística & datos numéricos , Australia/epidemiología , Erradicación de la Enfermedad/tendencias , Humanos , Sarampión/tratamiento farmacológico , Sarampión/epidemiología , Nueva Zelanda/epidemiología , Negativa a la Vacunación/tendenciasRESUMEN
INTRODUCTION: Survival in small cell lung cancer (SCLC) is limited by the development of chemoresistance. Factors associated with chemoresistance in vitro have been difficult to validate in vivo. Both Bcl-2 and ß(1)-integrin have been identified as in vitro chemoresistance factors in SCLC but their importance in patients remains uncertain. Tissue microarrays (TMAs) are useful to validate biomarkers but no large TMA exists for SCLC. We designed an SCLC TMA to study potential biomarkers of prognosis and then used it to clarify the role of both Bcl-2 and ß(1)-integrin in SCLC. METHODS: A TMA was constructed consisting of 184 cases of SCLC and stained for expression of Bcl-2 and ß(1)-integrin. The slides were scored and the role of the proteins in survival was determined using Cox regression analysis. A meta-analysis of the role of Bcl-2 expression in SCLC prognosis was performed based on published results. RESULTS: Both proteins were expressed at high levels in the SCLC cases. For Bcl-2 (n=140), the hazard ratio for death if the staining was weak in intensity was 0.55 (0.33-0.94, P=0.03) and for ß(1)-integrin (n=151) was 0.60 (0.39-0.92, P=0.02). The meta-analysis showed an overall hazard ratio for low expression of Bcl-2 of 0.91(0.74-1.09). CONCLUSIONS: Both Bcl-2 and ß(1)-integrin are independent prognostic factors in SCLC in this cohort although further validation is required to confirm their importance. A TMA of SCLC cases is feasible but challenging and an important tool for biomarker validation.
Asunto(s)
Integrina beta1/análisis , Neoplasias Pulmonares/mortalidad , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Carcinoma Pulmonar de Células Pequeñas/mortalidad , Análisis de Matrices Tisulares , Anciano , Femenino , Humanos , Neoplasias Pulmonares/química , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Carcinoma Pulmonar de Células Pequeñas/químicaRESUMEN
Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.
Asunto(s)
Citoplasma/metabolismo , Poliadenilación , Polinucleotido Adenililtransferasa/fisiología , Proteínas de Unión al ARN/fisiología , Factores de Transcripción/fisiología , Proteínas de Xenopus , Animales , Dominio Catalítico , Femenino , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Xenopus laevis , Factores de Escisión y Poliadenilación de ARNmAsunto(s)
Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacocinética , Hidromorfona/administración & dosificación , Hidromorfona/farmacocinética , Enfermedades Renales/metabolismo , Adulto , Analgésicos Opioides/sangre , Área Bajo la Curva , Femenino , Semivida , Humanos , Hidromorfona/sangre , Masculino , Persona de Mediana EdadAsunto(s)
Regiones no Traducidas 3'/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Mutación de Línea Germinal , Proteínas de Unión al ARN/genética , Animales , División Celular/genética , Femenino , Regulación de la Expresión Génica , Masculino , Células Madre/citologíaAsunto(s)
Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/química , ARN/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Clonación Molecular , Biblioteca de Genes , Vectores Genéticos , Conformación de Ácido Nucleico , Plásmidos , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , beta-Galactosidasa/genéticaRESUMEN
Cytoplasmic polyadenylation element binding (CPEB) proteins bind to and regulate the translation of specific mRNAs. CPEBs from Xenopus, Drosophila, and Spisula participate in oogenesis. In this report, we examine the biological roles of all identifiable CPEB homologs in a single organism, Caenorhabditis elegans. We find four homologs in the C. elegans genome: cbp-1, cpb-2, cpb-3, and fog-1. Surprisingly, two homologs, CPB-1 and FOG-1, have key functions in spermatogenesis and are dispensable for oogenesis. CPB-2 and CPB-3 also appear not to be required for oogenesis. CPB-1 is essential for progression through meiosis: cpb-1(RNAi) spermatocytes fail to undergo the meiotic cell divisions. CPB-1 protein is present in the germ line just prior to overt spermatogenesis; once sperm differentiation begins, CPB-1 disappears. CPB-1 physically interacts with FBF, another RNA-binding protein and 3' UTR regulator. In addition to its role in controlling the sperm/oocyte switch, we find that FBF also appears to be required for spermatogenesis, consistent with its interaction with CPEB. A second CPEB homolog, FOG-1, is required for specification of the sperm fate. The fog-1 gene produces fog-1(L) and fog-1(S) transcripts. The fog-1(L) RNA is enriched in animals making sperm and is predicted to encode a larger protein; fog-1(S) RNA is enriched in animals making oocytes and is predicted to encode a smaller protein. The relative abundance of the two mRNAs is controlled temporally during germ-line development and by the sex determination pathway in a fashion that suggests that the fog-1(L) species encodes the active form. In sum, our results demonstrate that, in C. elegans, two CPEB proteins have distinct functions in the germ line, both in spermatogenesis: FOG-1 specifies the sperm cell fate and CPB-1 executes that decision.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Espermatogénesis/fisiología , Proteínas de Xenopus , Factores de Escisión y Poliadenilación de ARNm , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/fisiología , Masculino , Datos de Secuencia Molecular , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Translational stimulation of mRNAs during early development is often accompanied by increases in poly(A) tail length. Poly(A)-binding protein (PAB) is an evolutionarily conserved protein that binds to the poly(A) tails of eukaryotic mRNAs. We examined PAB's role in living cells, using both Xenopus laevis oocytes and Saccharomyces cerevisiae, by tethering it to the 3'-untranslated region of reporter mRNAs. Tethered PAB stimulates translation in vivo. Neither a poly(A) tail nor PAB's poly(A)-binding activity is required. Multiple domains of PAB act redundantly in oocytes to stimulate translation: the interaction of RNA recognition motifs (RRMs) 1 and 2 with eukaryotic initiation factor-4G correlates with translational stimulation. Interaction with Paip-1 is insufficient for stimulation. RRMs 3 and 4 also stimulate, but bind neither factor. The regions of tethered PAB required in yeast to stimulate translation and stabilize mRNAs differ, implying that the two functions are distinct. Our results establish that oocytes contain the machinery necessary to support PAB-mediated translation and suggest that PAB may be an important participant in translational regulation during early development.
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Biosíntesis de Proteínas/fisiología , Proteínas de Unión al ARN/fisiología , Regiones no Traducidas 3' , Animales , Factor 4G Eucariótico de Iniciación , Oocitos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión a Poli(A) , Unión Proteica , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Xenopus laevisAsunto(s)
Proteínas/metabolismo , ARN/metabolismo , Técnicas del Sistema de Dos Híbridos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Caenorhabditis elegans/genética , ADN Complementario/metabolismo , Biblioteca de Genes , Genes Reporteros , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , ARN/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ADN , Transcripción Genética , beta-Galactosidasa/metabolismoRESUMEN
Translational control is prominent during meiotic maturation and early development. In this report, we investigate a mode of translational repression in Xenopus laevis oocytes, focusing on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. We show, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3'UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3'UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include cytoplasmic polyadenylation elements (CPEs), sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. We demonstrate that relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. Our results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition. We discuss several molecular mechanisms that might underlie this collaboration.
Asunto(s)
Ciclina B/genética , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Ciclina B1 , Citoplasma/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas In Vitro , Luciferasas/genética , Meiosis , Datos de Secuencia Molecular , Oocitos/crecimiento & desarrollo , Plásmidos/genética , Poli A/metabolismo , Biosíntesis de Proteínas , Xenopus laevisRESUMEN
The 3' untranslated region (3'UTR) of many eukaryotic mRNAs is essential for their control during early development. Negative translational control elements in 3'UTRs regulate pattern formation, cell fate, and sex determination in a variety of organisms. tra-2 mRNA in Caenorhabditis elegans is required for female development but must be repressed to permit spermatogenesis in hermaphrodites. Translational repression of tra-2 mRNA in C. elegans is mediated by tandemly repeated elements in its 3'UTR; these elements are called TGEs (for tra-2 and GLI element). To examine the mechanism of TGE-mediated repression, we first demonstrate that TGE-mediated translational repression occurs in Xenopus embryos and that Xenopus egg extracts contain a TGE-specific binding factor. Translational repression by the TGEs requires that the mRNA possess a poly(A) tail. We show that in C. elegans, the poly(A) tail of wild-type tra-2 mRNA is shorter than that of a mutant mRNA lacking the TGEs. To determine whether TGEs regulate poly(A) length directly, synthetic tra-2 3'UTRs with and without the TGEs were injected into Xenopus embryos. We find that TGEs accelerate the rate of deadenylation and permit the last 15 adenosines to be removed from the RNA, resulting in the accumulation of fully deadenylated molecules. We conclude that TGE-mediated translational repression involves either interference with poly(A)'s function in translation and/or regulated deadenylation.
Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Proteínas del Helminto/genética , Proteínas de la Membrana/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Secuencia de Bases , Femenino , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Oocitos , Secuencias Repetidas en Tándem/genética , Xenopus/embriología , Xenopus/genéticaRESUMEN
AIMS: To investigate the effects of sibutramine in combination with alcohol in a double-blind, randomised, placebo-controlled, four-way crossover study in 20 healthy volunteers. METHODS: On each study day each volunteer received either: sibutramine 20 mg+0.5 g kg-1 alcohol; sibutramine 20 mg+placebo alcohol; placebo capsules+0.5 g kg-1 alcohol; or placebo capsules+placebo alcohol. Alcohol was administered 2 h following ingestion of the study capsules. During each study day, assessments of cognitive performance were made prior to dosing, and at 3, 4.5, 6 and 10 h post dosing. Blood alcohol concentration was estimated using a breath alcometer immediately prior to each cognitive performance test session. Each study day was followed by a minimum 7 day washout period. RESULTS: Alcohol was found to produce statistically significant impairments in tests of attention (maximum impairment to speed of digit vigilance=49 ms) and episodic memory (maximum impairment to speed of word recognition=74 ms). Alcohol also increased body sway (maximum increase 17.4 units) and lowered self rated alertness (maximum decrease 13.6 mm). These effects were produced by an inferred blood alcohol level of 53.2 mg dl-1. Sibutramine was not found to potentiate any of the effects of alcohol. There was a small, yet statistically significant, interaction effect observed on the sensitivity index of the picture recognition task. In this test, the combined effects of sibutramine and alcohol were smaller than the impairments produced by alcohol alone. Sibutramine, when dosed alone, was associated with improved performance on several tasks. Sibutramine improved attention (mean speed of digit vigilance improved by 21 ms), picture recognition speed (improvement at 3=81) and motor control (tracking error at 3 h reduced by 1.58 mm). Also sibutramine improved postural stability (reducing body sway at 3 h by 14.2 units). Adverse events reported were unremarkable and consistent with the known pharmacology of sibutramine and alcohol. CONCLUSIONS: There was little evidence of a clinically relevant interaction of sibutramine with the impairment of cognitive function produced by alcohol in healthy volunteers. The single statistically significant interaction indicated a reduction, rather than a worsening, of alcohol-induced impairment when sibutramine is taken concomitantly. Sibutramine when administered alone is associated with improved performance on several tasks.
Asunto(s)
Depresores del Apetito/farmacología , Depresores del Sistema Nervioso Central/farmacología , Cognición/efectos de los fármacos , Ciclobutanos/farmacología , Etanol/farmacología , Adulto , Depresores del Apetito/efectos adversos , Astenia/inducido químicamente , Cápsulas , Depresores del Sistema Nervioso Central/efectos adversos , Cognición/fisiología , Coma/inducido químicamente , Estudios Cruzados , Ciclobutanos/efectos adversos , Mareo/inducido químicamente , Método Doble Ciego , Etanol/efectos adversos , Etanol/sangre , Femenino , Humanos , Masculino , Pruebas Neuropsicológicas , Desempeño Psicomotor/efectos de los fármacos , Resultado del TratamientoRESUMEN
BACKGROUND: The Caenorhabditis elegans FBF protein and its Drosophila relative, Pumilio, define a large family of eukaryotic RNA-binding proteins. By binding regulatory elements in the 3' untranslated regions (UTRs) of their cognate RNAs, FBF and Pumilio have key post-transcriptional roles in early developmental decisions. In C. elegans, FBF is required for repression of fem-3 mRNA to achieve the hermaphrodite switch from spermatogenesis to oogenesis. RESULTS: We report here that FBF and NANOS-3 (NOS-3), one of three C. elegans Nanos homologs, interact with each other in both yeast two-hybrid and in vitro assays. We have delineated the portions of each protein required for this interaction. Worms lacking nanos function were derived either by RNA-mediated interference (nos-1 and nos-2) or by use of a deletion mutant (nos-3). The roles of the three nos genes overlap during germ-line development. In certain nos-deficient animals, the hermaphrodite sperm-oocyte switch was defective, leading to the production of excess sperm and no oocytes. In other nos-deficient animals, the entire germ line died during larval development. This germ-line death did not require CED-3, a protease required for apoptosis. CONCLUSIONS: The data suggest that NOS-3 participates in the sperm-oocyte switch through its physical interaction with FBF, forming a regulatory complex that controls fem-3 mRNA. NOS-1 and NOS-2 also function in the switch, but do not interact directly with FBF. The three C. elegans nanos genes, like Drosophila nanos, are also critical for germ-line survival. We propose that this may have been the primitive function of nanos genes.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Trastornos del Desarrollo Sexual/metabolismo , Proteínas de Drosophila , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/metabolismo , Oocitos/citología , Oogénesis/genética , Proteínas de Unión al ARN/metabolismo , Espermatogénesis/genética , Espermatozoides/citología , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Trastornos del Desarrollo Sexual/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Proteínas del Helminto/genética , Proteínas del Helminto/fisiología , Proteínas de Insectos/química , Larva , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
The Rous sarcoma virus (RSV) Gag precursor polyprotein is the only viral protein which is necessary for specific packaging of genomic RNA. To map domains within Gag which are important for packaging, we constructed a series of Gag mutations in conjunction with a protease (PR) active-site point mutation in a full-length viral construct. We found that deletion of either the matrix (MA), the capsid (CA), or the protease (PR) domain did not abrogate packaging, although the MA domain is likely to be required for proper assembly. A previously characterized deletion of both Cys-His motifs in RSV nucleocapsid protein (NC) reduced both the efficiency of particle release and specific RNA packaging by 6- to 10-fold, consistent with previous observations that the NC Cys-His motifs played a role in assembly and RNA packaging. Most strikingly, when amino acid changes at Arg 549 and 551 immediately downstream of the distal NC Cys-His box were made, RNA packaging was reduced by more than 25-fold with no defect in particle release, demonstrating the importance of this basic amino acid region in packaging. We also used the yeast three-hybrid system to study avian retroviral RNA-Gag interactions. Using this assay, we found that the interactions of the minimal packaging region (Mpsi) with Gag are of high affinity and specificity. Using a number of Mpsi and Gag mutants, we have found a clear correlation between a reporter gene activation in a yeast three-hybrid binding system and an in vivo packaging assay. Our results showed that the binding assay provides a rapid genetic assay of both RNA and protein components for specific encapsidation.
Asunto(s)
Virus del Sarcoma Aviar/genética , Productos del Gen gag/metabolismo , ARN Viral , Ensamble de Virus , Animales , Virus del Sarcoma Aviar/metabolismo , Virus del Sarcoma Aviar/fisiología , Secuencia de Bases , Sitios de Unión , Aves , Cápside/metabolismo , Cisteína/metabolismo , Productos del Gen gag/genética , Genoma Viral , Histidina/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , Proteínas de la Matriz Viral/metabolismoRESUMEN
During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurus thymus. This report provides three lines of evidence that implicate the X. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevis oocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit of X. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevis oocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery.
Asunto(s)
Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN/fisiología , Proteínas de Xenopus , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Sustancias Macromoleculares , Datos de Secuencia Molecular , Oocitos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/fisiología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/fisiología , Xenopus laevis/genética , Factores de Escisión y Poliadenilación de ARNmRESUMEN
We have adapted the yeast three-hybrid system to identify RNA ligands for an RNA-binding protein. In this assay system, a protein-RNA interaction is detected by the reconstitution of a transcriptional activator using two hybrid proteins and a hybrid RNA. The RNA molecule is tethered to the promoter of a reporter gene by binding to a hybrid protein consisting of the bacteriophage MS2 coat protein fused to the DNA-binding protein LexA; the RNA-binding domain to be analyzed is fused to the transcriptional activation domain of the yeast Gal4 protein; and the bifunctional RNA consists of binding sites for the coat protein and for the other RNA-binding domain. We built an RNA library such that short fragments of genomic DNA from yeast were transcribed in yeast together with binding sites for the coat protein. We screened this hybrid RNA library for RNAs that bound to the yeast Snp1 protein, a homolog of the human U1-70K protein. The screen yielded as the strongest positive the fragment of U1 RNA that contains loop I, which is known to bind to Snp1 in U1 snRNP. We also identified four other RNA ligands that produced weaker three-hybrid signals, suggesting lower affinities for Snp1 as compared to U1 RNA. In addition, this search also yielded a set of RNA sequences that can activate transcription on their own when bound to a promoter through a protein interaction.
Asunto(s)
Proteínas de la Cápside , Proteínas Fúngicas/genética , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Cápside/genética , Proteínas de Unión al ADN/genética , Biblioteca de Genes , Genes Reporteros/genética , Ligandos , Regiones Promotoras Genéticas/genética , Ribonucleoproteína Nuclear Pequeña U1/genética , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3' UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3' UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3' UTR regulatory proteins, as it uncouples analysis of function from RNA binding.
Asunto(s)
Proteínas de la Cápside , Poli A/metabolismo , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Regiones no Traducidas 3'/metabolismo , Sitios de Unión/genética , Cápside/genética , Cápside/metabolismo , Extensión de la Cadena Peptídica de Translación/genética , Proteínas de Unión a Poli(A) , Unión Proteica/genética , Biosíntesis de Proteínas/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiaeRESUMEN
The translation of specific maternal mRNAs is regulated during early development. For some mRNAs, an increase in translational activity is correlated with cytoplasmic extension of their poly(A) tails; for others, translational inactivation is correlated with removal of their poly(A) tails. Recent results in several systems suggest that events at the 3' end of the mRNA can affect the state of the 5' cap structure, m7G(5')ppp(5')G. We focus here on the potential role of cap modifications on translation during early development and on the question of whether any such modifications are dependent on cytoplasmic poly(A) addition or removal. To do so, we injected synthetic RNAs into Xenopus oocytes and examined their cap structures and translational activities during meiotic maturation. We draw four main conclusions. First, the activity of a cytoplasmic guanine-7-methyltransferase increases during oocyte maturation and stimulates translation of an injected mRNA bearing a nonmethylated GpppG cap. The importance of the cap for translation in oocytes is corroborated by the sensitivity of protein synthesis to cap analogs and by the inefficient translation of mRNAs bearing nonphysiologically capped 5' termini. Second, deadenylation during oocyte maturation does not cause decapping, in contrast to deadenylation-triggered decapping in Saccharomyces cerevisiae. Third, the poly(A) tail and the N-7 methyl group of the cap stimulate translation synergistically during oocyte maturation. Fourth, cap ribose methylation of certain mRNAs is very inefficient and is not required for their translational recruitment by poly(A). These results demonstrate that polyadenylation can cause translational recruitment independent of ribose methylation. We propose that polyadenylation enhances translation through at least two mechanisms that are distinguished by their dependence on ribose modification.
Asunto(s)
Oocitos/citología , Poli A , Biosíntesis de Proteínas , Caperuzas de ARN , Animales , Diferenciación Celular , Núcleo Celular , Citoplasma , Metilación , Oogénesis , ARN Mensajero , XenopusRESUMEN
Cytoplasmic polyadenylation of specific mRNAs commonly is correlated with their translational activation during development. Here, we focus on links between cytoplasmic polyadenylation, translational activation and the control of meiotic maturation in Xenopus oocytes. We manipulate endogenous c-mos mRNA, which encodes a protein kinase that regulates meiotic maturation. We determined that translational activation of endogenous c-mos mRNA requires a long poly(A) tail per se, rather than the process of polyadenylation. For this, we injected 'prosthetic' poly(A)_synthetic poly(A) tails designed to attach by base pairing to endogenous c-mos mRNA that has had its own polyadenylation signals removed. This prosthetic poly(A) tail activates c-mos translation and restores meiotic maturation in response to progesterone. Thus the role of polyadenylation in activating c-mos mRNA differs from its role in activating certain other mRNAs, for which the act of polyadenylation is required. In the absence of progesterone, prosthetic poly(A) does not stimulate c-mos expression, implying that progesterone acts at additional steps to elevate c-Mos protein. By using a general inhibitor of polyadenylation together with prosthetic poly(A), we demonstrate that these additional steps include polyadenylation of at least one other mRNA, in addition to that of c-mos mRNA. These other mRNAs, encoding regulators of meiotic maturation, act upstream of c-Mos in the meiotic maturation pathway.