Najmeh Nikpour and Fiona Alum provided suggestions and edited this manuscript. Department of Biochemistry at McGill University supported HM and VM with the CIHR Training Initiative in Chemical Biology. This work was supported by Canadian Institute of Health Research (CIHR) grant 119464 (to R. S.).

El protocolo siguiente se describe el procedimiento para realizar el ensayo de edición de ARN basado en la fluorescencia. El ensayo se puede realizar en un único tubo de PCR, 96 así-, o placas de 384 pocillos dependiendo del alcance del experimento. Posteriormente, la señal de fluorescencia se puede leer en un tiempo real, sistema de detección de PCR adecuados. El ensayo que aquí se describe en el contexto de las placas de 384 pocillos. 1. El cultivo de T. Las células brucei <ol><li> Preparar un medio de crecimiento para T. brucei células procíclico de formulario. Para 1 l de medio: <ol><li> Disolver 25,4 g de polvo SDM-79 en agua 800 ml MiliQ. </li><li> Añadir 2 g de NaHCO3 y pH a 7,3 con 10 M de NaOH. </li><li> Añadir agua nanopura a un volumen final de 900 ml, esterilizar filtro. </li><li> Añadir suero fetal bovino (FBS), penicilina-estreptomicina y solución de hemina (2,5 mg / ml) a concentraciones finales de 10% (v / v), 100 U / ml y 7,5 mg / L, respectivamente. </li></ol></li><li> Grow 300 ml de T. &lt;em&gt; tipo salvaje 1.7A brucei (forma procíclico) las células a 28 º C, agitando a 70 rpm a una densidad de 1,5 x 10 7 células / ml. NOTA: Esto debería producir 3 ml de editosome activa con ~ 0,5 mg de proteína total, suficiente para 600 reacciones de edición. </li><li> Recoger las células por centrifugación a 6000 xg durante 10 min a 4 ° C. </li><li> Lavar el precipitado con 50 ml de tampón de PBSG enfriada (10 mM de Na 2 HPO 4, 10 mM NaH 2 PO 4, NaCl 145 mM, y glucosa 6 mM), y girar hacia abajo las células de nuevo por centrifugación a 10.000 xg durante 10 min a 4 ° C. </li></ol> 2. Aislamiento de crudo mitocondrias NOTA: Todos los pasos deben realizarse en hielo oa 4 ° C para preservar editosome actividad. <ol><li> Resuspender las células recolectadas en 30 ml de tampón de DTE (1 mM de Tris-HCl pH 8,0 y EDTA 1 mM). Utilice un homogeneizador Dounce de 40 ml estéril (previamente enfriado) para interrumpir la membrana celular por acariciar arriba y hacia abajo al menos 10 veces en el hielo. </li&gt; <li> Inmediatamente después, añadir 4,3 ml de 60% ​​de sacarosa (w / v, es decir, 1,75 M) al homogenado hasta una concentración final de 0,25 M. Se centrifuga a 15.800 xg durante 10 min a 4 ° C, para llevar preferentemente hacia abajo mitocondrias. </li><li> Resuspender el sedimento mitocondrial en 4,6 ml de tampón STM (20 mM de Tris-HCl, pH 8,0, sacarosa 250 mM y 2 mM de MgCl 2). Añadir 13,8 l de 0,1 M de CaCl 2 y 4 l de RNasa libre de DNasa I a concentraciones finales de 0,3 mM y 9 U / ml, respectivamente. Incubar la mezcla durante 1 hora en hielo. </li><li> Añadir 4,6 ml de tampón STE (20 mM de Tris-HCl, pH 8,0, sacarosa 250 mM y EDTA 2 mM) para inactivar la DNasa I. Se centrifuga a 15.800 xg durante 10 min a 4 ° C. </li><li> Resuspender el sedimento en 400 l de tampón de lisis (10 mM Tris-HCl pH 7,2, 10 mM MgCl2, KCl 100 mM, 1 mg / ml de pepstatina, DTT 1 mM, y 1x de inhibidor de proteasa completo sin EDTA) y la transferencia a un tubo de microcentrífuga. </li><li> Añadir 10% de Triton X-100 a una concentración final de 1% de unND incubar el lisado durante 15 min a 4 ° C en un rotador tubo. </li><li> Desactive la lisado mitocondrial por centrifugación dos veces a 17.000 xg durante 15 min a 4 ° C; conservando el sobrenadante aclarado cada vez. </li></ol> 3. Editosome Purificación <ol><li> Verter 10 ml de un 10% -30% (v / v) de glicerol gradiente lineal (Tabla 1) en un tubo de ultracentrífuga usando tampón de gradiente de 2x HHE (40 mM de HEPES pH 7,9, 20 mM de Mg (OAc) 2, 100 mM KCl, y 2 mM EDTA) y una máquina de gradiente, siguiendo el manual de instrucciones. </li><li> Retire cuidadosamente 500 l de solución de la parte superior de la gradiente de glicerol y cargar suavemente 500 l de lisado mitocondrial despejado. Giran a 178.000 xg durante 6 horas a 4 ° C utilizando una ultracentrífuga. </li><li> Recoger fracciones de 500 l secuencialmente desde la parte superior a la parte inferior del gradiente a 4 ° C. Entonces complemento congelar las fracciones usando nitrógeno líquido y se almacenan a -80 ° C hasta su uso. </li></ol><ol><li> Recocer el molde de ADN correspondiente que contiene la secuencia complementaria a la secuencia del promotor de T7 (Tabla 2) con un oligonucleótido promotor de T7 (5&#39;-TAATACGACTCACTATAGGG-3 &#39;) en una relación molar 1:1 por calentamiento a 90 ° C durante 3 min y enfriamiento a TA durante al menos 10 min. </li><li> Transcribir ARN usando un kit de transcripción in vitro siguiendo el manual de instrucciones. </li><li> Detenga la reacción de transcripción mediante la adición de un volumen igual de 7 M tinte urea (urea 7 M, 0,05% Xileno Cynol, y 0,05% Azul de bromofenol). Ejecutar en un filtro esterilizado 9% de desnaturalización en gel de poliacrilamida (9% de acrilamida, 7 M urea, 1x TBE). </li><li> Utilice la sombra la luz ultravioleta (UV) con una lámpara UV de onda corta para localizar e impuestos especiales correspondiente ARN. Coloque la pieza de gel escindido en un tubo de microcentrífuga y añadir 400 l de tampón de elución en gel (20 mM de Tris-HCl pH 7,5, mM de NaOAc 250, EDTA 1 mM y 0,25% de SDS). Eluir noche a temperatura ambiente en un mezclador de tubo. </li><li> PreciPitate el ARN se eluyó mediante la adición de 1 ml de etanol frío al 100% e incubando ya sea a -80 ° C durante 30 min o -20 ° C durante la noche. </li><li> Centrifugar a 16.000 xg durante 30 min a 4 ° C, para sedimentar por la ARN. </li><li> Lavar el pellet con 1 ml de etanol al 75%. Centrifugar a 16.000 xg durante 20 min a 4 ° C. </li><li> Resuspender el sedimento de ARN apropiadamente en RNasa libre de agua para alcanzar las concentraciones deseadas, como se muestra en la Tabla 2. </li></ol> 5. Ensayo Edición de ARN basados ​​en fluorescencia <ol><li> Para una sola reacción, combine 1 pmol (1 l) de preA6Rbz y 2,5 pmol (1 l) de gA6Rbz (relación molar 1:2,5) en un tubo de microcentrífuga, se incuba a 70 º C durante 3 minutos y dejar reposar a temperatura ambiente durante al menos 10 min. </li><li> Mientras tanto preparar una mezcla maestra usando la Tabla 3, sin la preA6Rbz y gA6Rbz, para la reacción de edición que contiene tampón 1x HHE (25 mM de HEPES pH 7,9, 10 mM de Mg (OAc) 2, KCl 50 mM y EDTA 10 mM), 1 mMATP, 5 mM de CaCl2, 16 ng / l de ARN de levadura Torula, 0,1% de Triton X-100, y la editosome purificada. </li><li> Añadir recocido preA6Rbz y gA6Rbz para completar la mezcla maestra. </li><li> Dispensar 18 l de la mezcla maestra (Tabla 3) en los pocillos que contenían ya sea 2 l de agua libre de RNasa (pocillos sin compuestos) o 2 l de 200 compuestos químicos y M incluir muestras de control en la placa de acuerdo con la Figura 5. </li><li> Sellar la placa con un sellador de placas y hacer girar el plato, para eliminar cualquier burbuja de aire. Se incuba a 28 ° C durante 4 horas. </li><li> Añadir 25 pmol (2 l) de gA6Rbz competidor a cada pocillo. Colocar un sellador fresco, girar la placa de abajo y colocarlo en una máquina de PCR en tiempo real. Programar el siguiente experimento: Paso 1: 85 º C durante 5 min; Paso 2: 24 ° C durante 10 min; Paso 3: Detener. </li><li> Añadir 15 pmol (1 mu l) de sustrato de FRET a cada pocillo para un volumen final de 23 l. Sellar la placa con un sellador fresco.Girar rápidamente la placa y vuelva a colocarlo en la máquina en tiempo real PCR. </li><li> Programe un nuevo experimento con los siguientes pasos: Paso 1: 37 º C durante 1 min; Paso 2: Lee; Paso 3: Vaya al Paso 1, 40 veces; Paso 4: Detener. </li><li> Configuración de la placa mediante la selección de todos los pozos que requieren la lectura y eligen el filtro FAM. El volumen de entrada de hasta 23 l y empezar la carrera. </li><li> Calcular la pendiente de los valores obtenidos a partir de cada pocillo / muestra para obtener una medición cinética por el trazado de las pistas en un gráfico de barras para el análisis. NOTA: Una lectura cinética mejora la relación señal a ruido entre la muestra y el fondo; como el fondo de la muestra tendría una pendiente cercana a cero. Una lectura de punto final tiene un mayor ruido de fondo. </li></ol>

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The authors have nothing to disclose.

Un método de cribado de alto rendimiento novedoso para identificar inhibidores contra el complejo de la edición de ARN de tripanosomas se presentó, proporcionando una nueva herramienta para el descubrimiento de fármacos para combatir enfermedades causadas por los tripanosomátidos. Ensayo FRET basado ribozima-ha sido ampliamente utilizado para diferentes propósitos 20-22; Sin embargo, hemos utilizado la capacidad de ensayo de ribozima basada en FRET para el control in vitro de la activi...

El proceso de edición de ARN, una modificación post-transcripcional del mRNA, fue descubierto por primera vez en tripanosomátidos 1. Desde entonces, el trabajo sustancial se ha llevado a cabo en el estudio del mecanismo detrás de la edición de ARN en Trypanosoma brucei 2,3. En una serie de reacciones enzimáticas, la editosome, un complejo de núcleo de aproximadamente 20 proteínas, crea ARNm mitocondriales maduros para múltiples componentes del sistema de la fosforilación oxidativa de generación de energía. El orden de los eventos catalíticos es la escisión endonucleolítica, uridilato (U) adición o supresión, y la ligadura, según lo dictado por los ARN guía (gRNAs) 4. Además de las principales proteínas editosome complejos, un número de factores accesorios también se han identificado 5-7. Estas proteínas se consideran sobre todo agrupadas en complejos independientes. Sin embargo, el orden de ensamblaje de proteínas en el complejo editosome núcleo y los patrones de interacción del complejo central con el accesoriocomplejos están aún por determinar. Orientación del proceso de edición del ARN en tripanosomátidos puede proporcionar disectores químicas que ayudan en el estudio de la asamblea y función del complejo editosome. Además, los estudios funcionales en varias proteínas editosome han demostrado esencialidad a través de diferentes etapas de la vida, lo que indica su potencial como medicamento actúa 8-12. Por lo tanto, los inhibidores de la editosome han encontrado también pueden actuar como compuestos de plomo contra los tripanosomátidos. Esto es oportuna, ya que los fármacos actualmente disponibles contra las enfermedades causadas por trypanosomatid son tóxicos, ineficiente y costoso 13,14. Un ensayo eficiente y conveniente in vitro es necesario explorar el universo químico de inhibidores específicos que bloquean la edición del ARN. Tres ensayos se han desarrollado y utilizado para monitorear editosome actividades: (a) ronda completa en el ensayo de la edición de ARN in vitro 15, (b) pre-troceados en el ensayo de la edición de ARN in vitro 16,17, unnd ensayo 18 (c) de martillo ribozimas (HHR)-basado. Los dos primeros ensayos se basan en la visualización directa del producto editado (ATPasa 6 ARNm) con la ayuda de la radiactividad. El ensayo basado en HHR utiliza una versión modificada de la ATPasa 6 ARNm que se modela a comportarse como una ribozima en la edición. La ribozima funcional entonces hiende específicamente a un sustrato de ARN radiomarcado, que sirve como un reportero. Recientemente, Moshiri et al. Desarrolló una &#39;mezcla y medir&#39; HHR-basado en reportero de ensayo in vitro para monitorizar la edición del ARN en el que el sustrato de ARN radiomarcado se sustituye con una transferencia de energía de resonancia de fluorescencia (FRET) sustrato 19. Las ventajas principales de este ensayo son: (a) es una mezcla rápida y conveniente y el tipo de medida de ensayo, como la producción de ribozima activa y de escisión de sustrato se producen simultáneamente en el mismo tubo en bajo volumen (es decir, 20 l), (b ) que evita el uso de materiales marcados radiactivamente, (c) la sensibilidad que es unfforded por la instrumentación de fluorescencia en un formato de placa de microtitulación, y (d) una elevada relación señal-ruido. Utilizando este ensayo, el efecto de los inhibidores de la edición de ARN ligasa conocidos contra editosome purificado se confirmó 19. Este experimento valida el ensayo para la identificación rápida de los inhibidores de la edición de ARN, principalmente contra editosomes enteros de T. brucei. La figura 1 es un esquema detallado paso a paso del ensayo en la edición de ARN in vitro basado en la fluorescencia. Este protocolo puede ser utilizado para el seguimiento de la edición de ARN in vitro o ser fácilmente adaptado para el cribado de bibliotecas de compuestos de diversas escalas.

<table border="0" cellpadding="0" cellspacing="0" width="979">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:248px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDM-79 Medium</td>
			<td>Gibco by life technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fetal Bovine Serum</td>
			<td>life technologies</td>
			<td style="width:139px;">12483-020</td>
			<td>heat inactivation at 550C for 1 h</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Hemin, minimum 80%</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:139px;">H5533-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Penicillin-Streptomycin Sollution</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:139px;">MT-30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Dnase 1 recombinant, Rnase Free&#160;</td>
			<td style="width:203px;">Roche</td>
			<td style="width:139px;">4716728001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">T7 RiboMax Express Large&#160; scale RNA&#160; production system</td>
			<td style="width:203px;">Promega</td>
			<td style="width:139px;">P1320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Kimble Kontes Dounce Tissue Grinders&#160;&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:139px;">K885300-0040&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gradient Master, ver 5.25&#160;</td>
			<td style="width:203px;">Biocomp</td>
			<td style="width:139px;">107-201M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Ultra Clear Tube, 13.2ML</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Optima L-100XP&#160; Ultracentrifuge&#160;</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">392052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">SW 41 Ti ROTOR</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">MicroSeal &#39;B&#39; Seal, Seals</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">MSB1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">CFX 384 Touch Real-Time PCR Detection System</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">185-5484</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Acryl/Bis solution (19: 1), 40% (w/v)</td>
			<td style="width:203px;">Bio Basic</td>
			<td style="width:139px;">A0006-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Urea, Molecular biology grade, 1Kg</td>
			<td style="width:203px;">life technologies</td>
			<td style="width:139px;">AM9902</td>
			<td style="width:248px;"></td>
		</tr>
	</tbody>
</table>

rna catalyst as a reporter for screening drugs against rna editing in trypanosomes

Se ha avanzado considerablemente en la determinación del mecanismo de edición del ARN mitocondrial en tripanosomas. Del mismo modo, se ha avanzado considerablemente en la identificación de los componentes del complejo editosome que catalizan la edición del ARN. Sin embargo, todavía no está claro cómo estas proteínas trabajan juntos. Los compuestos químicos obtenidos a partir de una pantalla de alto rendimiento contra el editosome pueden bloquear o afectar a una o más etapas en el ciclo de edición. Por lo tanto, la identificación de nuevos compuestos químicos generará valiosos sondas moleculares para disecar la función editosome y montaje. En estudios anteriores, los ensayos in vitro de edición en se llevaron a cabo utilizando ARN radiomarcado. Estos ensayos consumen mucho tiempo, ineficiente e inadecuada para fines de alto rendimiento. Aquí, una basada en la fluorescencia homogénea &quot;de mezcla y medida&quot; reportero de ensayo martillo ribozima in vitro para monitorear la edición de ARN, se presenta. Sólo como consecuencia de la edición de ARN dela ribozima cabeza de martillo un sustrato oligorribonucleótido transferencia de energía de resonancia de fluorescencia (FRET) se somete a la escisión. Esto a su vez resulta en la separación del fluoróforo de la inhibidor de la fluorescencia produciendo de este modo una señal. En contraste, cuando la función de editosome se inhibe, se apagará la señal de fluorescencia. Se trata de un ensayo altamente sensible y simple que debe ser de aplicación general para vigilar en la edición de ARN in vitro o cribado de alto rendimiento de los productos químicos que pueden inhibir la función editosome.

Para demostrar los pasos necesarios para la creación de una pantalla de gran escala, las figuras 2-5 son experimentos de control representativos relacionados con la calidad del ensayo. Estos son experimentos de control esenciales para un ensayo consistente a lo largo de varios días de cribado o para la comparación de diferentes pantallas. La evaluación de la relación señal-ruido de fluorescencia Para garantizar la estabilid...

Watch this Scientific Journal Video about RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes at JoVE.com

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA catalizador como reportero para rastrear fármacos contra ARN de edición en tripanosomas

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

rna-catalyst-as-reporter-for-screening-drugs-against-rna-editing

Research

JoVE Journal

Biology

8.4K vistas.  McGill University. A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

Video: RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizar la localización del ARN en Xenopus Los ovocitos

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Investigación

Biología

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

En la transcripción in vitro y la nivelación de mRNA de luciferasa Gaussia Seguido por transfección de células HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Aislamiento de la cromatina mediante la purificación de ARN (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Bioensayo electroantenográfico como herramienta de detección de compuestos volátiles de las plantas hospederas

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Sustrato Generación para endonucleasas de CRISPR / Cas Sistemas

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

El agotamiento de ARN ribosomal de metagenómica intestino del mosquito RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

ARN<em&gt; In situ</emHibridación&gt; Los embriones en todo el montaje y la histología de células Adaptado para elasmobranquios marinos

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Un procedimiento de detección química de glucocorticoides Señalización con un sistema indicador de pez cebra Larva luciferasa

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Un método para la detección y validación de mutaciones de resistencia contra la quinasa Inhibidores

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

La detección de las proteínas de unión a ARN por<em&gt; In Vitro</em&gt; ARN desplegable de adipocitos Cultura

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...