We are grateful to the past and present members of the laboratory, in particular Urszula Liwak, for critical discussion regarding polysome profiling protocols and sharing of data. This work was supported by operating grants from the Canadian institutes of Health Research, Cancer Research Society, and the National Science and Engineering Research Council of Canada to MH. MDF was supported by the Vanier Canada Graduate Scholarship Doctoral Award and the Ontario Graduate Scholarship, and this work forms part of her Ph.D. dissertation.

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The authors have nothing to disclose.

Perfilado polysomal es una técnica poderosa que permite la cuantificación del estado de la traducción de las transcripciones específicas bajo diferentes condiciones de tratamiento. Es una técnica muy sencilla, en principio, sin embargo, requiere varios pasos de ejecución, algunos de los cuales son críticos para la producción de buenos perfiles de polisomas. Estos pasos clave son: 1) el uso de productos químicos de RNasa libre y utensilios de plástico, 2) la preparación de buenos gradientes de sacarosa (en nuestra experiencia de un 10-50% gradientes de sacarosa funcionan mejor para resolver de manera eficiente 40 / 60S subunidades y los ARNm con los ribosomas unidos), y 3 ) utilizando células que están creciendo de manera exponencial y no más del 80% de confluencia con el fin de capturar las tasas más altas de traducción. Este último paso debe ser tenido en cuenta a la hora de hacer los tratamientos con células largas, como la caída de genes o sobreexpresión, por lo que la cantidad de células a la placa estará en función de cuánto serán las células confluentes en el punto final. En los resultados presented aquí, polisomas análisis del perfil era un instrumento importante para evaluar el papel PDCD4 en la regulación de la traducción de los IRES-albergan los ARNm de XIAP y Bcl-XL 12. El hecho de que no se observó ningún cambio en los perfiles de polisomas generales sobre PDCD4 knock-down (Figura 1B) permitió concluir que PDCD4 no perjudicó la traducción celular global bajo las condiciones del experimento. A continuación utiliza el análisis de RT-qPCR para determinar la distribución de la XIAP y Bcl-xL mRNAs en células tratadas con siRNAs PDCD4 o de control. qPCR es un método de elección para el análisis de perfiles de polisomas, en contraposición a la norma RT-PCR o transferencia de Northern, ya que permite una cuantitativa en lugar de análisis semi-cuantitativo de la distribución de ARNm y para la detección de cambios sutiles en la eficiencia de traducción entre los tratamientos. Usando esta técnica, hemos sido capaces de demostrar que la distribución de los XIAP y Bcl-XL ARNm (expresado como un porcentaje del total de ARNm diana a través de la gradient) se cambió significativamente en polirribosomas pesados ​​(mRNAs con un gran número de ribosomas asociados con ellos) después PDCD4 ronda hacia abajo (Figura 1D, E), lo que indica un aumento en la traducción de estos mRNAs. Esto se confirmó además por un incremento en XIAP y Bcl-XL niveles de proteína, pero no en sus niveles de ARN de estado estable (Figura 1F, G). Es importante destacar que la distribución de polisomas de la variante no IRES de XIAP permaneció sin cambios entre las células tratadas y no tratadas (Figura 1C). Alternativamente, la eficiencia de traducción podría ser presentado como una relación de contenido de ARNm en polisomas (normalmente fracciones 5 a 10) versus monosomas (normalmente fracciones 2 a 4) 14. El uso de los controles en el protocolo es importante en la validación de perfiles de polisomas cualquier resultado, como picos observados a partir de las lecturas de absorbancia a 254 nm no pueden por sí mismos ser atribuidos a ARN ribosomal (detergentes, tales como Triton X-100 absorben fuertemente en esta región del espectro y puede oscurecer 40 / 60S picos en la parte superior del gradiente). Gradientes blanco sin lisado y / o con tampón de lisado deben ejecutarse para determinar la contribución relativa de los tampones para la absorbancia a 254 nm. Artefactual estructuras de orden superior también pueden dar lugar a interpretaciones erróneas de polisomas en los que hay, de hecho, ninguno (por ejemplo, &quot;pseudo-polisomas&quot; 20). Secuestrantes de magnesio (utilizado durante todo el procedimiento para estabilizar los ribosomas 80S) con el EDTA quelante de cationes divalentes añadidos a los lisados ​​y al tampón de gradiente (20 mM durante 15 min) hará que la separación del ribosoma en sus pequeños componentes (40S) y grandes (60S subunidades). Por lo tanto, si picos de absorbancia registrados a 260 nm son de hecho polisomas, el tratamiento EDTA colapsará el perfil tal que se observó un único máximo cerca de la parte superior del gradiente que corresponde a liberar el ARNm y las unidades ribosómicas (datos no presentados). Coincidentecon el colapso inducido por EDTA-del perfil, todos los objetivos específicos analizados por qPCR ahora debería hallarse en la parte superior del gradiente. El número de ribosomas asociados con un mRNA no siempre es un indicador de la eficiencia con la que en particular mRNA se está traduciendo. De hecho, hay contextos específicos en los que la traducción activa en polisomas convierte estancó 21,22 que el lector debe tener en cuenta. La determinación de si o no las transcripciones se dedican activamente con la maquinaria de traducción se puede hacer por a) tratar la muestra con puromicina, que a diferencia de EDTA, provoca &#39;run-off&#39; de los ribosomas que se translocación activamente a través de un ARNm, o b) tratar la muestra con homoharringtonina que inhibe la translocación de sólo el primer ribosoma al codón de inicio, de nuevo causando &#39;escorrentía&#39; de cualquier ribosomas de translocación aguas abajo. El uso de las transcripciones de control para el análisis de qPCR también es crítica. La selección de las transcripciones que no se ve afectada por diferentes condiciones de tratamiento tales como algunos genes de limpieza (GAPDH, actina, tubulina, nucleolina), ARNm ribosómicos (18S, Rpl13a) o en este caso la variante no IRES de la XIAP IRES, es importante en verificar si el efecto del tratamiento sobre la traducción es específica o generalizada. Estas transcripciones de control pueden utilizarse para normalizar la distribución de los mRNAs analizados como se ha hecho aquí (XIAP y Bcl-XL ARNm se normalizaron a GAPDH mRNA y se expresó como un porcentaje del contenido total de ARNm representado por la suma de contenido de mRNA en cada fracción ) o, alternativamente, objetivo y de control mRNAs pueden expresarse como valores absolutos y se muestran por separado. análisis qPCR de los lisados ​​de entrada (por lo general el 10% del volumen total) es otro paso que va a controlar la distribución de los ARNm específicos. Idealmente, el contenido de mRNA de una transcripción específica en el lisado de entrada debe ser comparable a la suma de contenido de mRNA en todas las 10 fracciones analizadas. Finalmente, Un paso opcional para controlar el fenol: cloroformo etapa de extracción es a pico cada fracción polisomas con un ARNm reportero transcrito in vitro (tal como el CAT, cloranfenicol acetil transferasa) que será posteriormente cuantificada por qPCR, e incluso se puede utilizar para normalizar la datos 14. Como con cualquier técnica, la elaboración de perfiles de polisomas presenta algunas limitaciones. El hecho de que por lo general un mínimo de 10 fracciones (cambios más sutiles en la eficiencia de la traducción puede requerir 20 o más) necesitan ser recogidos con el fin de tener distribuciones de polisomas agradables hace que este paso limitativo, en el sentido de que sólo un máximo de 4 muestras puede ser analizada en un momento en que sea capaz de manejar cómodamente la extracción de RNA y análisis qPCR de 40 muestras y controles de entrada 4 a la vez. El tamaño de la muestra se puede añadir fácilmente hasta con el número de transcripciones deben ser analizados y cuántos qPCR replica que hacer, y esto puede ser costoso. Otra de las limitaciones de un polysomal perfilado un qPCR basado ennálisis es que sólo se puede hacer en un enfoque basado en la candidato (donde las transcripciones a ser analizados son conocidos de antemano) y no se pueden aplicar para el análisis de todo el genoma de la traducción. Sin embargo, a principios del siglo 21, los investigadores comenzaron a interrogar a su ARN polysomal a nivel genómico utilizando microarrays de ADN 15 y, más recientemente, con la secuenciación de próxima generación 16,17. En este enfoque de gran alcance, se realiza perfiles polysomal como se describe en este protocolo, salvo que en lugar de realizar un análisis qPCR de fracciones individuales, monosomas fracciones (por lo general de fracciones 2-4) y polisomas fracciones (normalmente fracciones) están agrupados juntos y variaciones en la traducción mundial analizados por microarrays de ADN o secuenciación de todo el genoma de ARN. De ahí que con este enfoque, es posible evaluar todos los ARNm cuya distribución turnos de polisomas a monosomas (disminución de la traducción) o viceversa (aumento en la traducción) a un tratamiento específicoción. Validación y cuantificación de la distribución específica de ARNm de los polisomas se pueden entonces ser realizadas por qPCR. Un uso aún más poderosa del principio de perfiles polysomal es perfilado ribosoma. Además extensión de alto rendimiento de esta técnica se informó recientemente de que permite la monitorización simultánea de cientos de polisomas fracciones 23. Esto proporciona una clara ventaja cuando sondeo eficiencias traduccionales de las colecciones de mutantes o en un RNAi pantallas de gran escala. Perfilado ribosoma es una técnica que se aprovecha de la secuenciación de próxima generación para mapear fragmentos de ARN protegidos por los ribosomas (huellas ribosoma) que participan en la síntesis de proteínas 24,25. En esta técnica, la translocación del ribosoma puede ser inhibida con cicloheximida (reversible) o emetina (irreversible) seguido por la lisis celular y la digestión de RNasa para producir una población de huellas ribosoma. Los ribosomas se enriquecen, el ARN total se aísla, y se elimina la contaminación ribosomal y el ARN ribosomal mitocondrial.Huellas de ARN de aproximadamente 26-28 nucleótidos son aislados, transcripción inversa, convertida en una biblioteca de cDNA y se secuenciaron 26. A diferencia de perfiles de polisomas estándar, este enfoque no sólo identifica mRNAs específicos que son regulados en traslación, sino también produce información ARNm específico a una resolución de codón tales como la ocupación exacta y la densidad de los ribosomas a lo largo de una transcripción específica. Esto es particularmente de interés para los ARNm con modos específicos de traducción, tales como ARNm IRES-albergan, ya que podría dar más información sobre el lugar en la transcripción del ribosoma-reclutamiento que está ocurriendo.

Regulación de la síntesis de proteínas (traducción) es un proceso celular clave que está íntimamente ligada a la supervivencia celular. Dado que la traducción consume más del 50% de la energía de la célula, no es sorprendente que la traducción está estrechamente regulada y que las perturbaciones en la traducción no se toleran bien. Por el contrario, muchos procesos celulares aberrantes que requieren maquinaria de traducción y la salida de la traducción (es decir proteoma) tienen que ser modificados. Esto es a menudo el caso en varios estados de respuesta al estrés y la enfermedad, y es, probablemente, el mejor ejemplo en el cáncer. Una ventaja clave de control de la traducción es la capacidad de las células para reprogramar rápidamente la salida de proteínas en respuesta a diversos disparadores externos e internos, por lo tanto mecanismo de ajuste fino que inclina la balanza entre la supervivencia celular y la muerte 1. Dos aspectos de la traducción de control son particularmente interesantes; comprensión de la naturaleza de la mec reguladorahanism (s) y elementos de control que permiten la traducción específica, y la identificación de mRNAs específicos y sus productos de proteínas que participan en la respuesta celular al gatillo particular que provocó traducción selectiva en el primer lugar. Perfiles de polisomas es una técnica que permite el estudio eficaz de ambos procesos. El objetivo general de la técnica de perfiles de polisomas es estudiar y cuantificar el estado de la traducción de ARNm específicos bajo diferentes condiciones celulares. El principio de la técnica es captar la traducción del ARNm por &#39;&#39; congelación &#39;&#39; Traducción activamente ribosomas en diferentes transcripciones y separar los polirribosomas resultantes por ultracentrifugación en un gradiente de sacarosa, permitiendo así una distinción entre las transcripciones altamente traducidas (obligado por varios ribosomas) y los mal traducido (unida por uno o dos ribosomas). En este protocolo particular, el antibiótico cicloheximida que se une a la60S subunidad ribosomal y bloquea la liberación de desacetilada tRNA del sitio E ribosoma 2, se utiliza para inhibir la translocación del ribosoma y estancar los ribosomas ARNm de traducción. Sin embargo, otros agentes de bloqueo tales como emetina que también bloquea la traducción de elongación 3, puede ser utilizado. A diferencia de la transferencia de western y 35 S-metionina técnicas de etiquetado que miden la salida final del proceso de traducción, polisomas perfilado tiene la ventaja de medir los ribosomas asociación con mRNAs activamente traducidas, permitiendo así un estudio más detallado de los mecanismos de traducción en condiciones diferentes. Por lo tanto, la técnica se puede adaptar fácilmente para estudiar específicamente los diferentes modos de traducción 4-6, proteínas factores involucrados en la regulación de la traducción 7-9, condiciones de estrés que afectan a la síntesis de proteínas 1,10,11 o los efectos de las estructuras de ARNm tales como IRES o aguas arriba marcos de lectura abiertos en sintetizador de proteínasESIS 12-14. Hoy en día, polisoma aplicaciones de perfiles son aún más amplia con el uso de microarrays de DNA 15 o secuenciación de próxima generación 16,17 analizar polisomas asociados-ARNm.

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			<td height="21" style="height:21px;width:351px;">Name of Material/ Equipment</td>
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			<td style="width:119px;">Catalog Number</td>
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			<td height="21" style="height:21px;width:351px;">Gradient Master 108 automated gradient maker</td>
			<td style="width:135px;">BIOCOMP</td>
			<td style="width:119px;">107-201M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Auto Densi-Flow two-chamber gradient maker</td>
			<td style="width:135px;">LABCONCO</td>
			<td align="right" style="width:119px;">4517000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">Beckman Ultracentrifuge&#160;</td>
			<td style="width:135px;">Beckman Coulter</td>
			<td style="width:119px;">Model # L-100-XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Density gradient fractionator</td>
			<td style="width:135px;">TELEDYNE ISCO</td>
			<td style="width:119px;">69-3873-246</td>
			<td>Composed of: tube piercing system,&#160; peristaltic pump, UA-6 Detector with 254 and 280nm filters and Foxy R1 Fraction Collector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">WinDaq data acquisition software</td>
			<td style="width:135px;">DATAQ INSTRUMENTS</td>
			<td style="width:119px;">WINDAQ/LITE</td>
			<td>http://www.dataq.com/products/software/acquisition.htm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">Pollyallomer ultracentrifuge tubes (for SW41, 14x89 mm)</td>
			<td style="width:135px;">Beckman Coulter</td>
			<td style="width:119px;">331372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Nuclease-free 2-ml centrifuge tubes (dolphin nose)</td>
			<td style="width:135px;">DiaMed</td>
			<td style="width:119px;">PRE1900-N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Nuclease-free 2-ml centrifuge tubes (flat bottom)</td>
			<td style="width:135px;">SafeSeal</td>
			<td style="width:119px;">12000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sucrose (nucleases-free)</td>
			<td style="width:135px;">Calbiochem</td>
			<td style="width:119px;">573113</td>
			<td>MW = 342.3 g/mol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Cycloheximide</td>
			<td style="width:135px;">SIGMA</td>
			<td style="width:119px;">C7698</td>
			<td>MW = 281.4 g/mol. Resuspend in DMSO and keep at -80C for long term sotrage. Caution: TOXIC! Can cause repiratory tracts and skin irritation. Wear mask when weighing.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sodium chloride (nucleases-free)</td>
			<td style="width:135px;">OmniPur</td>
			<td style="width:119px;">7710</td>
			<td>MW = 58.44 g/mol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">MgCl2.6H2O (nucleases-free)</td>
			<td style="width:135px;">OmniPur</td>
			<td style="width:119px;">5980</td>
			<td>MW = 203.3 g/mol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tris Base (nucleases-free)</td>
			<td style="width:135px;">J.T. Baker</td>
			<td style="width:119px;">4109-01</td>
			<td>MW = 121.14 g/mol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Triton X-100</td>
			<td style="width:135px;">OmniPur</td>
			<td align="right" style="width:119px;">9400</td>
			<td>Caution: TOXIC! Can cause repiratory tracts and skin irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Recombinant RNasin Ribonuclease Inhibitor</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">N2515</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">RNaseZap Rnase inactivation solution</td>
			<td style="width:135px;">Ambion, Life Technologies</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">GlycoBlue Coprecipitant (15 mg/mL)</td>
			<td style="width:135px;">Invitrogen, Life Technologies</td>
			<td style="width:119px;">AM9516</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td style="width:135px;">Ambion, Life Technologies</td>
			<td style="width:119px;">AM9722</td>
			<td>Caution: TOXIC ! Can cause repiratory tracts irritation as well as skin irritation and corrosion</td>
		</tr>
	</tbody>
</table>

assessment of selective mrna translation in mammalian cells by polysome profiling

Regulación de la síntesis de proteínas representa un punto de control clave en la respuesta celular al estrés. En particular, los elementos reguladores de ARN discreto se muestran para permitir a la traducción selectiva de mRNAs específicos, que típicamente codifican para las proteínas requeridas para una respuesta de estrés particular. La identificación de estos mRNAs, así como la caracterización de los mecanismos reguladores responsables de la traducción selectiva ha estado en la vanguardia de la biología molecular durante algún tiempo. Perfiles de polisomas es un método piedra angular en estos estudios. El objetivo de los perfiles de polisomas es capturar la traducción del ARNm mediante la inmovilización de los ribosomas traducen activamente en diferentes transcripciones y separar los polirribosomas resultantes por ultracentrifugación en un gradiente de sacarosa, lo que permite una distinción entre las transcripciones altamente traducidos y mal traducido queridos. Estos pueden ser caracterizados adicionalmente por métodos tradicionales bioquímicos y de biología molecular. Es importante destacar que la combinación de polysome perfiles con enfoques genómicos de alto rendimiento permite un análisis a gran escala de la regulación de traducción.

Recientemente hemos descrito un nuevo papel para el supresor de tumor PDCD4 como un inhibidor de la traducción selectiva de mRNAs que codifican inhibidores apoptóticos XIAP y Bcl-xL IRES-12 que alberga. Perfiles de polisomas era una técnica clave para demostrar la participación específica de PDCD4 en la traducción mediada por IRES-. Células HEK293 se transfectaron transitoriamente para agotar los niveles endógenos de PDCD4 (Figura 1A) y después se sometieron a análisis del perfil de polisomas. Hemos observado que la reducción de los niveles de PDCD4 no afectó la traducción celular global, a juzgar por la falta de cambios en el perfil de polisomas cuando se comparan siCTRL y siPDCD4 células tratadas (Figura 1B). Del mismo modo, polisomas distribución de variante no IRES de XIAP 19 se mantuvo sin cambios entre las células tratadas y no tratadas (Figura 1C). En contraste, polisoma distribución de los ARNm dos IRES-albergan, XIAP y Bcl-XL, se modificó significativamente. En ausencia de PDCD4 la distribución de estas dos mRNAs se desplaza en ribopolysomes pesados ​​(Figura 1D, E). Como esto no se acompaña de cambios en los niveles de estado estacionario de XIAP y Bcl-XL de mRNA (Figura 1F) Estos resultados demuestran una mayor traducción de XIAP y Bcl-XL en células con niveles reducidos PDCD4, que fue confirmado por Western Blot (Figura 1G). <img alt="Figura 1" src="/files/ftp_upload/52295/52295fig1highres.jpg" /> Figura 1:. Perfiles de polisomas PDCD4 identifica como inhibidor selectivo de la traducción XIAP y Bcl-xL (A) HEK293 células fueron tratadas con PDCD4 siRNA o control (CTRL), no la orientación siRNA, se lisaron y se sometieron a análisis de transferencia de Western con anti-PDCD4 y anticuerpos antinucleolina para verificar el grado de PDCD4 knock-down. (B) PDCD4 fue derribado como en (A) ylisados ​​celulares se sometieron a polisomas perfilado. Polisomas perfil representativo se muestra en el panel superior; distribución de la XIAP no IRES (C), Bcl-XL (D), y XIAP IRES (E) mRNAs relativos a la de GAPDH se muestra como el porcentaje de mRNA total (% ARNm) en cada fracción. (F) Steady los niveles de mRNA -Estado se midieron por RT-qPCR en PDCD4 siRNA o control (CTRL) siRNA células tratadas. (G) Los lisados ​​de (A) también se sometieron a análisis de Western blot con anti-XIAP, anti-Bcl-xL, y anticuerpos anti-nucleolina. (NB. Los datos presentados en la Figura 1 se publicaron originalmente en 12) <a href="https://www.jove.com/files/ftp_upload/52295/52295fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta cifra.</a>

Watch this Scientific Journal Video about Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling at JoVE.com

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

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 ...

assessment-selective-mrna-translation-mammalian-cells-polysome

Research

JoVE Journal

Biology

27.9K vistas.  University of Ottawa. 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. 

Video: Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Análisis de la cinética de exportación nuclear de ARNm en células de mamífero por microinyección

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Investigación

Biología

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análisis de la posición del ciclo celular en células de mamífero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Supervisión de la replicación del plásmido en células de mamíferos en vivo durante varias generaciones por microscopía de fluorescencia

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

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. ...

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. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medición de niveles hemo síntesis en células de mamífero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Sondeo de mRNA cinética en el espacio y el tiempo en Escherichia coli usando hibridación de fluorescencia de una sola molécula de dos colores

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...