PA es un beneficiario de una beca &quot;Pierre Durand&quot; de la Facultad de medicina de la Universidad de Laval. Este trabajo fue apoyado por las Ciencias Naturales e Ingeniería de Investigación de Canadá (MOP-CG095386) a RM El fraccionador polisomas fue adquirida a través de una subvención de la Fundación Canadiense para la Innovación (MOP-GF091050) para RMR M tiene un nuevo premio salarial investigador CIHR. Agradecemos a los Dres. E. Khandjian, I. Gallouzi, S. Di-Marco y A. Cammas para consejos útiles.

El protocolo sigue las directrices aprobado por la Junta de Revisión Ética de Laval. 1. Preparación de cultivos celulares y de manipulación del cerebro <ol><li> Las células de mamíferos y Drosophila <ol><li> Crecer HeLa células de cáncer cervical y las células embrionarias Schneider de Drosophila como recomienda la American Type Culture Collection. Trabajar con células en un pasaje bajo. </li><li> Las células de la placa con el fin de alcanzar el 80% de confluencia el día del experimento. Para obtener los mejores resultados, utilice ~ 12 x 10 6 células para cada condición experimental. Antes de hacer los extractos para el análisis polisomas, estimular traducción añadiendo medio completo fresco durante al menos 90 min. </li></ol></li><li> Aislamiento cerebro: Aislar los cerebros de los ratones de edades comprendidas entre 9 y 16 días después del nacimiento. La eutanasia implica inhalación de CO2 después de la anestesia y la dislocación cervical. Tras el sacrificio, aislar todo el cerebro y, o bien el lugar it a -80 ° C o transferir directamente en frío PBS 1X para su uso inmediato. </li></ol> 2. Preparación del gradiente de densidad sistema de fraccionamiento <ol><li> Lave el sistema de tubos con 0,1% SDS durante 5 min. </li><li> Lavar el sistema de tubos con 70% de etanol durante 5 min, después con una solución RNaseZAP durante 5 min antes de lavarlo con agua DEPC. </li><li> La bomba de aire con el fin de secar el sistema de tubos del aparato. </li></ol> 3. Preparación de los gradientes de sacarosa <ol><li> Hacer 15% y 55% de sacarosa en soluciones 20 mM de Tris-HCl, pH 7,4, 1,25 mM de MgCl 2, NaCl 150 mM, y DTT 1 mM. </li><li> Generar el 15-55% de gradiente lineal de sacarosa utilizando un Isco Modelo 160 gradiente de primera, como se describe por las instrucciones del fabricante. Sin embargo, es importante para controlar así la velocidad de la bomba peristáltica de TRIS con el fin de evitar la creación de burbujas o turbulencias en los gradientes. Mantener los gradientes a 4 ° C hasta su uso. </li><li> Durante el tiempo en el programa de fabricante de gradiente se está ejecutando, enfriar la ultracentrífuga a 4 ° C. </li></ol> 4. Preparación de células de ratón y extractos de cerebro <ol><li> Preparación de extractos celulares <ol><li> Coloque la placa (s) en las células de hielo y lavado 3 veces con PBS frío 1X. </li><li> Células de la cosecha (~ 12 x 10 6) en 1 ml de tampón de lisis (Tris-HCl a pH 20 7,4, 1,25 mM de MgCl 2, NaCl 150 mM, DTT 1 mM, 1% Nonidet P40, 5 inhibidor U / ml de RNasa, complementado con mini tabletas completas de cóctel inhibidor de la proteasa libre de EDTA), transferirlos a un tubo Eppendorf, y mezclar bien pasándolos 15x a través de una jeringa de insulina U100 1cc 28 G 1/2. Deje que el lisado celular descansar en hielo durante 15 min. </li><li> Ponga unas gotas de lisado de células en un portaobjetos para evaluar la lisis celular mediante la observación en un microscopio de contraste de fase utilizando un objetivo de 10X. Solamente los núcleos deben ser visibles y no hay membranas celulares deben ser visibles fehaciente para la lisis celular. Como control, observarlas células no lisadas. </li><li> Aclarar el lisado de células por centrifugación a 11.000 xg durante 20 min a 4 ° C y mantener el lisado soluble que contiene los polirribosomas. </li></ol></li><li> Preparación de extractos de cerebro del ratón <ol><li> Homogeneizar todo el cerebro aislado (~ 500 mg) mediante 10 golpes en un homogeneizador Dounce enfriado con hielo con 2 ml de tampón de lisis y aclarar el homogeneizado por centrifugación a 11.000 xg durante 15 min a 4 ° C. </li><li> Cargar el sobrenadante resultante sobre un cojín de 50% sacarosa y proceder con la sedimentación durante 2 horas a 200.000 xg usando un rotor de ultracentrífuga TH-641 a 4 ° C. </li><li> Resuspender el precipitado translúcida que contiene los polirribosomas en 1 ml de tampón de lisis y mezclar bien pipeteando arriba y abajo. Dejar reposar la suspensión en el hielo durante 30 minutos antes de cargar en gradientes. </li></ol></li></ol> 5. Carga de los extractos en sacarosa Degradados y Ultracentrifugación <ol><li> Medir la concentraciónde ARN presente en el extracto citoplásmico utilizando un espectrofotómetro. Con cuidado y poco a poco cargar ~ 20 OD 260 unidades de extracto en el gradiente de sacarosa 15% a 55%. Asegúrese de que no tiene marca de 2-3 mm de espacio disponible en la superficie del tubo para evitar que desborda los tubos durante la centrifugación. </li><li> Centrifugar los gradientes usando un ultra-centrífuga durante 2 horas 30 minutos a 230.000 xg utilizando un rotor de ultracentrífuga TH-641 a 4 ° C. Cuando termina la centrifugación, retire los tubos y colocarlos en hielo con cuidado de no molestar a los gradientes. </li></ol> 6. Fraccionamiento de extractos citoplasmáticos de polisomas Profiling <ol><li> Coloque gradientes de sacarosa en sistema de fraccionamiento Densidad automatizado y proceder con el fraccionamiento automatizado y recogida de las fracciones (0,5 ml cada uno), tal como se describe por el fabricante. </li><li> Recoger cada fracción (~ 500 l) en tubos Eppendorf individuales con un seguimiento continuo de la absorbancia a 254 nm. En parallel de la operación de fraccionamiento de gradiente, el perfil polyribosomal se edita en el papel gráfico. También puede utilizar una unidad de adquisición de datos conectado a la grabadora de tabla para tener una adquisición electrónica del perfil polisomas. </li><li> Al final de cada ejecución, transferir Eppendorfs recogidos en hielo seco. En este momento, ya sea tienda recogieron fracciones a -80 ° C o precipitar directamente los complejos de proteína-ARN de la siguiente manera. </li></ol> 7. Extracción de Proteínas y Análisis <ol><li> Para cada fracción recogida de gradiente de sacarosa, añadir 2 volúmenes de etanol 100% frío y dejar que los complejos proteína-ARN precipitado a -20 ° C durante la noche. </li><li> Centrifugar cada precipitado de ARN-proteína a 11.000 xg durante 20 min a 4 ° C y luego se lava con etanol al 70%. Seco y resuspender el precipitado en tampón de muestra de SDS-PAGE antes de proceder con transferencia de Western usando anticuerpos específicos (ver Figura 2). </li></ol> 8. Extracción de ARN y Análisis <ol><li> Precipitar complejos proteína-ARN como en la sección 7.1. Resuspender cada precipitado de ARN-proteína en el tampón de lisis que contiene 0,1% de SDS. Se digiere el componente de proteína del precipitado con 2 mg / ml de proteinasa K durante 30 min en un 55 ° C baño de agua. </li><li> Extraer los componentes de ARN de la precipitado mediante la adición de 1 volumen de &quot;fenol: cloroformo&quot;, 2 volúmenes de cloroformo, y 0,1 volumen de 2 M de NaOAc pH 4 y centrifugando a 10.000 xg a 4 ° C durante 20 min. El precipitado de ARN de la fase acuosa resultante de cada muestra durante la noche a -20 ° C mediante la adición de 1 volumen de isopropanol y 0,2 g / l de glucógeno. Haga girar el precipitado durante 1 hora a 10.000 xga 4 º C. Lavar el precipitado de ARN con etanol al 70% frío. </li><li> Resuspender el sedimento de ARN en un pequeño volumen de agua libre de ARNasa. Evaluar la cantidad y la calidad de ARN usando el espectrofotómetro. </li></ol>

<ol>
	<li>Gebauer, F., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+translational+control.">Molecular mechanisms of translational control.</a> Nature Reviews. Molecular Cell Biology. 5 (10), 827-835 (2004).</li><li>Jackson, R. J., Hellen, C. U. T., Pestova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+translation+initiation+and+principles+of+its+regulation.">The mechanism of eukaryotic translation initiation and principles of its regulation.</a> Nature Reviews. Molecular Cell Biology. 11 (2), 113-127 (2010).</li><li>Fournier, M. -. J., Gareau, C., Mazroui, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotherapeutic+agent+bortezomib+induces+the+formation+of+stress+granules.">The chemotherapeutic agent bortezomib induces the formation of stress granules.</a> Cancer Cell International. 10 (12), (2010).</li><li>Holcik, M., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+in+stress+and+apoptosis.">Translational control in stress and apoptosis.</a> Nature Reviews. Molecular Cell Biology. 6 (4), 318-327 (2005).</li><li>Mazroui, R., Huot, M. -. E., Tremblay, S., Filion, C., Labelle, Y., Khandjian, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+of+messenger+RNA+by+Fragile+X+Mental+Retardation+protein+into+cytoplasmic+granules+induces+translation+repression.">Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression.</a> Human Molecular Genetics. 11 (24), 3007-3017 (2002).</li><li>Mazroui, R., Huot, M. -. E., Tremblay, S., Boilard, N., Labelle, Y., Khandjian, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X+Mental+Retardation+protein+determinants+required+for+its+association+with+polyribosomal+mRNPs.">Fragile X Mental Retardation protein determinants required for its association with polyribosomal mRNPs.</a> Human Molecular Genetics. 12 (23), 3087-3096 (2003).</li><li>Farny, N. G., Kedersha, N. L., Silver, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Metazoan+stress+granule+assembly+is+mediated+by+P-eIF2alpha-dependent+and+-independent+mechanisms.">a Metazoan stress granule assembly is mediated by P-eIF2alpha-dependent and -independent mechanisms.</a> RNA. 15 (10), 1814-1821 (2009).</li><li>Gareau, C., Houssin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+fragile+x+mental+retardation+protein+recruitment+and+dynamics+in+Drosophila+stress+granules.">Characterization of fragile x mental retardation protein recruitment and dynamics in Drosophila stress granules.</a> PLoS ONE. 8 (2), (2013).</li><li>Brackett, D. M., Qing, F., Amieux, P. S., Sellers, D. L., Horner, P. J., Morris, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FMR1+transcript+isoforms:+association+with+polyribosomes;+regional+and+developmental+expression+in+mouse+brain.">FMR1 transcript isoforms: association with polyribosomes; regional and developmental expression in mouse brain.</a> PLoS ONE. 8 (3), (2013).</li><li>Khandjian, E. W., Huot, M. -. E., Tremblay, S., Davidovic, L., Mazroui, R., Bardoni, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+evidence+for+the+association+of+fragile+X+mental+retardation+protein+with+brain+polyribosomal+ribonucleoparticles.">Biochemical evidence for the association of fragile X mental retardation protein with brain polyribosomal ribonucleoparticles.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (36), 13357-13362 (2004).</li><li>Erikson, A., Winblad, B., Wallace, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+of+gene+expression+in+the+human+brain.">Translational control of gene expression in the human brain.</a> Prog Neuropsychopharmacol Biol Psychiatry. 13 (3-4), 469-479 (1989).</li><li>Stephens, S. B., Nicchitta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V+In+vitro+and+tissue+culture+methods+for+analysis+of+translation+initiation+on+the+endoplasmic+reticulum.">V In vitro and tissue culture methods for analysis of translation initiation on the endoplasmic reticulum.</a> Methods in Enzymology. 431, 47-60 (2007).</li><li>Koritzinsky, M., Wouters, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+regulation+of+messenger+RNA+translation.">Hypoxia and regulation of messenger RNA translation.</a> Methods in Enzymology. 435, 247-273 (2007).</li><li>Khandjian, E. W., Corbin, F., Woerly, S., Rousseau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fragile+X+mental+retardation+protein+is+associated+with+ribosomes.">The fragile X mental retardation protein is associated with ribosomes.</a> Nature Genetics. 12, 91-93 (1996).</li><li>Sivan, G., Kedersha, N., Elroy-Stein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+slowdown+mediates+translational+arrest+during+cellular+division.">Ribosomal slowdown mediates translational arrest during cellular division.</a> Molecular and Cellular Biology. 27 (19), 6639-6646 (2007).</li><li>Thomas, J. D., Johannes, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mRNAs+that+continue+to+associate+with+polysomes+during+hypoxia.">Identification of mRNAs that continue to associate with polysomes during hypoxia.</a> RNA. 13, 1116-1131 (2007).</li><li>Kumaraswamy, S., Chinnaiyan, P., Shankavaram, U. T., L&#252;, X., Camphausen, K., Tofilon, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+gene+translation+profiles+reveal+tumor+type+and+cancer-specific+components.">Radiation-induced gene translation profiles reveal tumor type and cancer-specific components.</a> Cancer Research. 68 (10), 3819-3826 (2008).</li><li>Fournier, M. -. J., Coudert, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+mTORC1-eIF4E+Pathway+alters+Stress+Granules+Formation.">Inactivation of the mTORC1-eIF4E Pathway alters Stress Granules Formation.</a> Molecular and Cellular Biology. 33 (11), 2285-2301 (2013).</li><li>Sanchez, G., Dury, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+function+for+the+survival+motoneuron+protein+as+a+translational+regulator.">A novel function for the survival motoneuron protein as a translational regulator.</a> Human Molecular Genetics. 22 (4), 668-684 (2013).</li><li>B&#233;chade, C., Rostaing, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+distribution+of+survival+motor+neuron+(SMN)+protein:+possible+involvement+in+nucleocytoplasmic+and+dendritic+transport.">Subcellular distribution of survival motor neuron (SMN) protein: possible involvement in nucleocytoplasmic and dendritic transport.</a> The European Journal of Neuroscience. 11 (1), 293-304 (1999).</li><li>Goulet, I., Boisvenue, S., Mokas, S., Mazroui, R., C&#244;t&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDRD3,+a+novel+Tudor+domain-containing+protein,+localizes+to+cytoplasmic+stress+granules.">TDRD3, a novel Tudor domain-containing protein, localizes to cytoplasmic stress granules.</a> Human Molecular Genetics. 17 (19), 3055-3074 (2008).</li><li>Nottrott, S., Simard, M. J., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+let-7a+miRNA+blocks+protein+production+on+actively+translating+polyribosomes.">Human let-7a miRNA blocks protein production on actively translating polyribosomes.</a> Nature Structural &amp; Molecular Biology. 13 (12), 1108-1114 (2006).</li><li>Genolet, R., Araud, T., Maillard, L., Jaquier-Gubler, P., Curran, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+analyse+the+specific+impact+of+rapamycin+on+mRNA-ribosome+association.">An approach to analyse the specific impact of rapamycin on mRNA-ribosome association.</a> BMC Medical Genomics. 1 (33), (2008).</li><li>Del Prete, M. J., Vernal, R., Dolznig, H., M&#252;llner, E. W., Garcia-Sanz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Isolation+of+polysome-bound+mRNA+from+solid+tissues+amenable+for+RT-PCR+and+profiling+experiments.">a Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments.</a> RNA. 13 (3), 414-421 (2007).</li><li>Thoreen, C. C., Chantranupong, L., Keys, H. R., Wang, T., Gray, N. S., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unifying+model+for+mTORC1-mediated+regulation+of+mRNA+translation.">A unifying model for mTORC1-mediated regulation of mRNA translation.</a> Nature. 485 (7396), 109-113 (2012).</li><li>Ingolia, N. T., Brar, G. A., Rouskin, S., Mcgeachy, A. M., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+profiling+strategy+for+monitoring+translation+in+vivo+by+deep+sequencing+of+ribosome-protected+mRNA+fragments.">The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.</a> Nature Protocols. 7 (8), 1534-1550 (2012).</li><li>Morris, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+footprints+on+a+transcriptome+landscape.">Ribosomal footprints on a transcriptome landscape.</a> Genome Biology. 10 (4), (2009).</li></ol>

Los autores no tienen nada que revelar.

El análisis del perfil de polisomas en gradientes de sacarosa permite la medición de iniciación de la traducción mediante el análisis de la densidad de polisomas aislados a partir de células o tejidos 9,11-14. Esta técnica es la mejor (si no el único) método para medir la iniciación de la traducción in vivo. Se utiliza para supervisar el estado de traslación de crecimiento de las células durante el ciclo celular 15, y para evaluar los efectos de diferentes tipos de estrés, in...

Las células eucariotas constantemente se encuentran con una amplia gama de condiciones de estrés fisiológicos y ambientales nocivos que requieren una rápida respuesta celular adaptativa. Respuesta al estrés celular requiere un equilibrio preciso entre la lucha contra la supervivencia y pro-supervivencia factores que actúan. La interrupción de este equilibrio puede tener consecuencias irreversibles que conducen al desarrollo de patologías humanas como el cáncer y las enfermedades neurodegenerativas. Durante la primera etapa de la respuesta al estrés, las células activan vías de pro-supervivencia que implican el control coordinado de los cambios en la expresión génica a nivel de la traducción del ARNm. la traducción del ARNm en eucariotas es un proceso celular complejo que implica interacciones coordinadas entre los factores de iniciación de la traducción (EIFS), proteínas de unión de ARN específica (RBDS), y moléculas de ARN 1. la traducción del ARNm se divide en tres fases distintas: la iniciación, elongación, y terminación. Aunque las tres fases están sujetas a regulaciónmecanismos reguladoras, los mecanismos de control de la traducción se dirigen principalmente la fase de iniciación de la traducción, que de este modo constituye el paso limitante de la velocidad de la síntesis de proteínas 2. Iniciación de la traducción es un proceso altamente ordenado que comienza con la formación de la eIF2a.GTP.Met-tRNA I Met complejo ternario, y su posterior unión a la subunidad 40S del ribosoma, lo que lleva a la formación del complejo de pre-iniciación. El siguiente paso es el reclutamiento del complejo pre-iniciación a ARNm, que implica la actividad de los factores de iniciación de la traducción como eIF4F y eIF3. El complejo de preiniciación 48S así formado sufre cambios conformacionales específicos que permiten esta maquinaria para iniciar la exploración de la región 5&#39;-no traducida del ARNm hasta que se reconoce el codón de iniciación agosto La mayoría de los factores de iniciación de la traducción se liberan y 60S subunidades son reclutados para formar un ribosoma 80S complejo competente para su traducción, unt que se inicia la síntesis de proteínas punto (Figura 1). Más de un 80S monosome se puede traducir la misma mRNA a la vez la producción de los llamados polisomas (o polirribosomas). La densidad de polisomas en un ARNm refleja la iniciación, elongación y las tasas de terminación y por lo tanto es una medida de la traducibilidad de una transcripción en particular. Sin embargo, el perfil de polisomas se utiliza principalmente para evaluar los cambios en la traducción del ARNm en la etapa de iniciación. Aquí hemos utilizado un inhibidor del proteasoma como inhibidor de la iniciación de la traducción. El tratamiento de las células cancerosas con este fármaco induce una respuesta de estrés que se caracteriza por la activación de la quinasa de estrés llamado HRI que fosforila el factor de iniciación de la traducción eIF2a 3. La fosforilación de la eIF2a es uno de los eventos más importantes que conducen a la inhibición de la iniciación de la traducción en células de mamífero 4.

<table border="0" cellpadding="0" cellspacing="0" width="875">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
			<td height="21" style="height:21px;width:279px;">Cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">HeLa cervical cancer cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Schneider Drosophila embryonic cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CRL-1963</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Culture medium and Supplements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Schneider&#8217;s Drosophila Medium</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>SO146-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM</td>
			<td>Life technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">FBS</td>
			<td>Fisher Scientist Scientist</td>
			<td>SH30396-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">penicillin/streptomycin</td>
			<td>Life technologies</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Sucrose solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">D-Sucrose</td>
			<td>Fisher Scientist</td>
			<td>BP220-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycerol</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>49767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Blue Bromophenol</td>
			<td>Fisher Scientist</td>
			<td>B3925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tris Hydrochloride</td>
			<td>Fisher Scientist</td>
			<td>BP153-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:279px;">MgCl2 </td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NaCl</td>
			<td>Tekniscience</td>
			<td>3624-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DTT</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D 9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Nonidet P40 (Igepal CA-630 )</td>
			<td>MJS Biolynx</td>
			<td>19628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">SDS</td>
			<td>Tekniscience</td>
			<td>4095-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor)</td>
			<td>Life technologies</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Antiproteases (complete, mini, EDTA free)</td>
			<td>Roche</td>
			<td>11,836,170,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">RNA Extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteinase K</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM2542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Phenol: Chloroforme</td>
			<td>Fisher Scientist</td>
			<td style="width:119px;">BP1754I-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Chloroforme</td>
			<td style="width:311px;">Fisher Scientist</td>
			<td style="width:119px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycogen</td>
			<td>Life technologies</td>
			<td>10814-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isopropanol</td>
			<td style="width:311px;">Acros organics</td>
			<td>327270010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-FMRP antibody</td>
			<td colspan="2" style="width:429px;">Fournier et al., Cancer Cell International, 2010</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-Ribosomal Protein L28 antibody</td>
			<td style="width:311px;">Santa Cruz Biotechnology, Inc.</td>
			<td style="width:119px;">SC-50362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Others</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteasome inhibitor : Bortezomib</td>
			<td style="width:311px;">LC Laboratories</td>
			<td style="width:119px;">B-1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DEPC (Diethylpyrocarbonate)</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D5758-25ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">RNaseZAP Solution</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">T25 cell culture flask</td>
			<td style="width:311px;">Corning</td>
			<td style="width:119px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">1cc U100 Insulin Syringe 28 G1/2</td>
			<td>Fisher Scientist</td>
			<td>148291B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tube ultra-centrifugation, PA, 12ml</td>
			<td>Fisher Scientist</td>
			<td>FSSP9763205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco Model 160 gradient former</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Sorvall OTD Combi</td>
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Thermo Scientific Sorvall Rotor TH-641</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;">54295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Automated Density Fractionation System</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;">67-9000-177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco UA-6 UV-vis detector</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ultracentrifuge C 5415</td>
			<td style="width:311px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Optical Microscope</td>
			<td style="width:311px;">Olympus CK2</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of translation initiation during stress conditions by polysome profiling

El control preciso de la traducción del ARNm es fundamental para la homeostasis de células eucariotas, particularmente en respuesta a estrés fisiológico y patológico. Las alteraciones de este programa pueden dar lugar al crecimiento de las células dañadas, un sello distintivo del desarrollo del cáncer, o a la muerte celular prematura tal como se ve en las enfermedades neurodegenerativas. Gran parte de lo que se conoce acerca de la base molecular para la traducción de control se ha obtenido a partir del análisis de polisomas utilizando un sistema de fraccionamiento en gradiente de densidad. Esta técnica se basa en la ultracentrifugación de extractos citoplásmicos en un gradiente lineal de sacarosa. Una vez que se ha completado el giro, el sistema permite el fraccionamiento y la cuantificación de las zonas centrifugadas correspondientes a diferentes poblaciones de traslación ribosomas, lo que resulta en un perfil de polisomas. Cambios en el perfil de polisomas son indicativos de cambios o defectos de iniciación de la traducción que se producen en respuesta a diversos tipos de estrés. Esta técnica también permite evaluar ªe papel de las proteínas específicas en la iniciación de la traducción, y para medir la actividad de traducción de los ARNm específicos. Aquí describimos nuestro protocolo para realizar perfiles de polisomas con el fin de evaluar iniciación de la traducción de las células y los tejidos eucariotas, ya sea en condiciones normales o de estrés de crecimiento.

Como se ha mencionado, polysome perfil permite el análisis de los cambios de iniciación de la traducción bajo condiciones de estrés. Figura 1 es una vista simplificada de iniciación de la traducción que, como se ha descrito anteriormente es un proceso de múltiples etapas que implica un conjunto ordenado de complejos de iniciación de la traducción. En condiciones normales de crecimiento, los complejos de iniciación de traducción se convierten en polirribosomas cuya detección por perfil polyso...

Watch this Scientific Journal Video about Analysis of Translation Initiation During Stress Conditions by Polysome Profiling at JoVE.com

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Aquí, se describe un método para analizar los cambios en la iniciación de la traducción de ARNm de células eucariotas en respuesta a condiciones de estrés. Este método se basa en la separación de velocidad en gradientes de sacarosa de la traducción de los ribosomas de los ribosomas no traducir.

Análisis de la Traducción Iniciación Durante condiciones de estrés por polisoma Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

analysis-translation-initiation-during-stress-conditions-polysome

Research

JoVE Journal

Biology

18.2K vistas.  Laval University.  Aquí, se describe un método para analizar los cambios en la iniciación de la traducción de ARNm de células eucariotas en respuesta a condiciones de estrés. Este método se basa en la separación de velocidad en gradientes de sacarosa de la traducción de los ribosomas de los ribosomas no traducir. 

Video: Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Monitoreo hormonas vegetales en las respuestas de estrés

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Investigación

Biología

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Perfiles de anticuerpos por los sistemas de inmunoprecipitación luciferasa (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Análisis de estrés oxidativo en embriones de pez cebra

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Perfiles de Células Madre Embrionarias Humanas individual por RT-PCR cuantitativa

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Polisoma Fraccionamiento y análisis de los mamíferos Translatomes en un genoma de gran escala

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Evaluación del selectivo ARNm traducción en células de mamífero por polisoma perfiles

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Un método fácil para la planta de perfiles de polisomas

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Análisis de la transferencia dependiente de la unión Gap de miARN con 3D-FRAP Microscopía

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Análisis de la función E3 Ubiquitina Ligasa

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...