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.).

O protocolo que se segue descreve o processo para a realização do ensaio de edição de ARN baseada em fluorescência. O ensaio pode ser realizado num tubo de PCR, 96 bem-, ou placas de 384 poços, consoante o âmbito da experiência. Subsequentemente, o sinal de fluorescência pode ser lida em tempo real, um sistema de detecção de PCR adequados. O ensaio aqui descrito no contexto de placas de 384 poços. 1. T. A cultura brucei Células <ol><li> Prepare um meio de crescimento para T. brucei procíclicos formam células. Para 1 litro de meio: <ol><li> Dissolver 25,4 g SDM-79 em pó em 800 ml de água MiliQ. </li><li> Adicionar 2 g de NaHCO3 e o pH para 7,3 com NaOH 10 M. </li><li> Adicionar água nanopura para um volume final de 900 ml, filtro de esterilizar. </li><li> Adicionar Soro Fetal Bovino (FBS), penicilina-estreptomicina a solução e hemina (2,5 mg / ml) para uma concentração final de 10% (v / v), 100 U / ml e 7,5 mg / L, respectivamente. </li></ol></li><li> Crescer 300 ml de T. &lt;in&gt; brucei 1.7A tipo selvagem (forma procíclicos) as células a 28 ° C, agitando a 70 rpm para uma densidade de 1,5 x 10 7 células / ml. NOTA: Este deve produzir 3 ml de editosome activo com ~ 0,5 mg de proteína total, suficiente para 600 reacções de edição. </li><li> Colher as células por centrifugação a 6000 xg durante 10 min a 4 ° C. </li><li> Lavar o sedimento com 50 ml de tampão de PBSG gelado (10 mM de Na 2 HPO 4, 10 mM de NaH 2 PO 4, NaCl 145 mM, e glucose 6 mM), e girar as células novamente por centrifugação a 10.000 xg durante 10 min a 4 ° C. </li></ol> 2. Isolamento de mitocôndrias Crude NOTA: Todos os passos devem ser realizados em gelo ou a 4 ° C para preservar editosome actividade. <ol><li> Ressuspender as células colhidas em 30 ml de tampão de DTE (1 mM Tris-HCl pH 8,0 e EDTA 1 mM). Use um 40 ml homogeneizador Dounce estéril (pré-refrigerados) para interromper a membrana celular acariciando cima e para baixo pelo menos 10 vezes no gelo. </li&gt; <li> Imediatamente adicionar 4,3 ml de 60% ​​de sacarose (w / v, ou seja 1,75 M) ao homogenato para uma concentração final de 0,25 M. Centrifuga-se a 15.800 xg durante 10 min a 4 ° C, para trazer para baixo, preferencialmente mitocôndrias. </li><li> Ressuspender o sedimento mitocondrial em 4,6 ml de tampão STM (20 mM Tris-HCl pH 8,0, 250 mM de sacarose e 2 mM de MgCl 2). Adicionar 13.8 ul de 0,1 M de CaCl2 e 4 ul de ADNase I isenta de RNase para concentrações finais de 0,3 mM e 9 U / ml, respectivamente. Incubar a mistura durante 1 hora em gelo. </li><li> Adicionar 4,6 ml de tampão STE (20 mM Tris-HCl pH 8,0, sacarose 250 mM e EDTA 2 mM), para inactivar a DNase I. Centrifugar a 15.800 xg durante 10 min a 4 ° C. </li><li> Ressuspender o sedimento em 400 ul de tampão de lise (10 mM Tris-HCl pH 7,2, 10 mM MgCl 2, KCl 100 mM, 1 ug / ml de pepstatina, 1 mM de DTT, e 1 x completo de inibidor da protease livre de EDTA) e transferência para uma tubo de microcentrífuga. </li><li> Adicionar 10% de Triton X-100 para uma concentração final de 1% de umnd incubar o lisado durante 15 minutos a 4 ° C num rotor de tubo. </li><li> Limpar o lisado mitocondrial por centrifugação duas vezes a 17,000 xg durante 15 min a 4 ° C; mantendo o sobrenadante clarificado de cada vez. </li></ol> 3. Editosome Purificação <ol><li> Despeje a 10 ml de 10% -30% (v / v), gradiente linear de glicerol (Tabela 1) em um tubo de ultracentrifugação usando tampão de gradiente de HHE 2x (40 mM de HEPES pH 7,9, 20 mM de Mg (OAc) 2, KCl 100 mM, e EDTA 2 mM) e uma máquina de gradiente, seguindo o manual de instruções. </li><li> Remover cuidadosamente 500 mL de solução a partir do topo do gradiente de glicerol e carregar suavemente 500 ul de lisado mitocondrial apagada. Girar em 178.000 xg durante 6 horas a 4 ° C usando uma ultracentrífuga. </li><li> Recolha fracções de 500 ul sequencialmente a partir do topo para o fundo do gradiente, a 4 ° C. Em seguida, encaixe congelar as frações, utilizando nitrogênio líquido e armazenar a -80 ° C até o uso. </li></ol><ol><li> Recozer o molde de ADN contendo respectiva sequência complementar à sequência do promotor de T7 (Tabela 2) com um oligonucleótido do promotor de T7 (5&#39;-TAATACGACTCACTATAGGG-3 &#39;) numa razão molar de 1:1 por aquecimento a 90 ° C durante 3 min e arrefecer à TA durante pelo menos 10 min. </li><li> Transcrever RNA usando um kit de transcrição in vitro, seguindo o manual de instruções. </li><li> Parar a reacção de transcrição por adição de um volume igual de 7 M corante ureia (ureia 7 M, 0,05% de xileno Cynol, e 0,05% de azul de bromofenol). Correr num filtro esterilizado 9% desnaturante em gel de poliacrilamida (9% de acrilamida, 7 M de ureia, 1 x TBE). </li><li> Use o sombreamento ultravioleta (UV) com uma lâmpada UV de ondas curtas para localizar e respectivo RNA especial de consumo. Coloque o pedaço de gel excisado num tubo de microcentrífuga e adicionar 400 ul de tampão de eluição em gel (20 mM Tris-HCl pH 7,5, NaOAc 250 mM, EDTA 1 mM e 0,25% de SDS). Eluir a noite à temperatura ambiente num rotor tubo. </li><li> Precipitate o ARN eluído por adição de 1 ml de etanol frio a 100% e incubação, quer a -80 ° C durante 30 min ou a -20 ° C durante a noite. </li><li> Centrifugar a 16.000 xg durante 30 min a 4 ° C, para precipitar o ARN para baixo. </li><li> Lava-se a pelete com 1 ml de etanol a 75%. Centrifugar a 16000 xg durante 20 min a 4 ° C. </li><li> Ressuspender o sedimento de ARN apropriada em RNase isenta de água para atingir as concentrações desejadas, como se mostra na Tabela 2. </li></ol> 5. Baseado em fluorescência Edição RNA Assay <ol><li> Para uma única reacção, combinar 1 pmol (1 ul) de preA6Rbz e 2,5 pmol (1 ul) de gA6Rbz (proporção molar 1:2,5), num tubo de microcentrífuga, incubar a 70 º C durante 3 min e deixar repousar à TA durante a pelo menos, 10 min. </li><li> Enquanto isso preparar uma mistura principal utilizando a Tabela 3, sem o preA6Rbz e gA6Rbz, para a reacção de edição contendo tampão 1x EHH (25 mM de HEPES pH 7,9, 10 mM de Mg (OAc) 2, 50 mM de KCl e 10 mM de EDTA), 1 mMATP, 5 mM de CaCl2, 16 ng / mL de ARN de levedura Torula, 0,1% de Triton X-100, e o editosome purificada. </li><li> Adicionar recozido preA6Rbz e gA6Rbz para completar a mistura principal. </li><li> Dispensar 18 ul da mistura principal (Tabela 3) em poços contendo 2 ul de água isenta de RNase (poços sem compostos) ou 2 ul de 200 uM e compostos químicos incluem amostras de controlo na placa de acordo com a Figura 5. </li><li> Selar a placa com o selador de placa e girar a placa para baixo, para remover as bolhas de ar. Incubar a 28 ° C durante 4 h. </li><li> Adicionar 25 pmol (2 ul) de competidor gA6Rbz a cada poço. Coloque um novo aferidor, girar a placa para baixo e coloque-o em uma máquina de PCR em tempo real. Programe o seguinte experimento: Passo 1: 85 ° C durante 5 min; Passo 2: 24 ° C durante 10 min; Passo 3: Pare. </li><li> Adicionam-se 15 pmol (1 ul) de substrato de FRET para cada poço a um volume final de 23 ul. Selar a placa com um novo aferidor.Girar rapidamente a placa e coloque-a de volta na máquina de PCR em tempo real. </li><li> Programe um novo experimento com os seguintes passos: Passo 1: 37 ° C durante 1 min; Passo 2: Leia; Passo 3: Vá para a Etapa 1, 40 vezes; Passo 4: Pare. </li><li> Instalação da placa, selecionando todos os poços que exigem leitura e escolher o filtro FAM. Volume de entrada de 23 mL e começar a correr. </li><li> Calcular o declive dos valores obtidos a partir de cada poço / amostra para obter uma medição de cinética traçando as pistas sobre um gráfico de barras para análise. NOTA: A leitura cinética melhora a razão sinal-para-ruído entre a amostra e o fundo; como a amostra de fundo teria uma inclinação perto de zero. Uma leitura ponto final tem maior ruído de fundo. </li></ol>

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The authors have nothing to disclose.

Um método de triagem romance de alto rendimento para identificar inibidores contra o complexo RNA edição de Tripanossomas foi apresentado, fornecendo uma nova ferramenta para a descoberta de medicamentos para combater as doenças causadas por tripanosomatídeos. Ensaio ribozyme baseado trastes tem sido amplamente utilizado para diferentes fins 20-22; no entanto, nós utilizamos a capacidade de ensaio ribozyme baseado em FRET para o monitoramento in vitro de edição de RNA atividade 19....

O processo de edição de RNA, uma modificação mRNA pós-transcricional, foi descoberto pela primeira vez em tripanosomatídeos 1. Desde então, o trabalho substancial foi realizada em estudar o mecanismo por trás edição de RNA em Trypanosoma brucei 2,3. Em uma série de reacções enzimáticas, a editosome, um complexo de núcleo de cerca de 20 proteínas, cria mRNAs mitocondriais maduros para vários componentes do sistema de geração de energia a fosforilação oxidativa. A ordem dos eventos catalíticos é clivagem endonucleolítica, uridylate (U) adição ou exclusão, e ligadura, como ditado por RNAs guia (gRNAs) 4. Além das proteínas complexas editosome núcleo, um certo número de factores acessórios também foram identificadas 5-7. Estas proteínas são vistas principalmente agrupadas em complexos independentes. No entanto, a ordem de montagem de proteínas no complexo editosome núcleo e os padrões de interacção do complexo de núcleo com o acessóriocomplexos estão ainda a ser determinado. Visando o processo de edição de RNA em tripanosomatídeos pode fornecer dissectores químicas que ajudam no estudo do conjunto e de função do complexo editosome. Além disso, estudos funcionais em várias proteínas editosome mostraram essencialidade em diferentes fases da vida, indicando o seu potencial como droga atinge 8-12. Portanto, os inibidores da editosome encontrados também podem actuar como compostos de chumbo contra tripanosomatideos. Este é oportuna, como drogas atualmente disponíveis contra doenças causadas por tripanossomatídeo são tóxicos, ineficiente e caro 13,14. Um ensaio eficiente e conveniente in vitro é necessário explorar o universo químico para inibidores específicos que bloqueiam edição RNA. Três ensaios foram desenvolvidos e utilizados para monitorar editosome atividades: (a) volta completa em ensaio edição de RNA in vitro 15, (b) pré-clivada em ensaio edição de RNA in vitro 16,17, umª ensaio 18 (c) martelo ribozyme (HHR)-based. Os dois primeiros ensaios de contar com visualização direta do produto editado (ATPase 6 mRNA) com a ajuda de radioatividade. O ensaio baseia-HHR utiliza uma versão modificada da ATPase 6 ARNm que é modelada para se comportar como um ribozima mediante edição. A ribozima funcional então cliva especificamente um substracto de ARN marcado radioactivamente, que serve como um repórter. Recentemente, Moshiri et al. Desenvolveu um &quot;misturar e medir &#39;HHR-base no ensaio in vitro repórter para monitorizar a edição de ARN em que o substrato de RNA marcado radioactivamente é substituído por uma transferência de energia de ressonância de fluorescência (FRET) do substrato 19. As principais vantagens deste ensaio são os seguintes: (a) é uma mistura rápida e conveniente e medida do tipo de ensaio, como a produção de ribozima activa e substrato de clivagem ocorrer simultaneamente no mesmo tubo em baixo volume (isto é, 20 mL), (b ) que evita a utilização de materiais marcados radioactivamente, (c), que é uma sensibilidadefforded por instrumentação de fluorescência em um formato de placa de micro-titulação, e (d) um sinal de alta-ruído. Utilizando este ensaio, o efeito de inibidores conhecidos de ligase de RNA de edição contra editosome purificada foi confirmada 19. Este experimento validou o ensaio para a rápida identificação de inibidores de edição de RNA, principalmente contra editosomes inteiros de T. brucei. A Figura 1 é um esquema detalhado passo-a-passo do ensaio na edição de ARN in vitro baseados em fluorescência. Este protocolo pode ser utilizado para monitorizar a edição de ARN in vitro ou ser facilmente adaptado para o rastreio de bibliotecas de compostos 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>
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		<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>
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		<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>
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		<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

Um progresso substancial foi feito para determinar o mecanismo de edição de RNA mitocondrial em tripanossomas. Da mesma forma, um progresso considerável foi feito na identificação dos componentes do complexo editosome que catalisam edição RNA. No entanto, ainda não está claro como as proteínas funcionam juntos. Os compostos químicos obtidos a partir de uma tela de alto rendimento contra o editosome pode bloquear ou afectar um ou mais passos no ciclo de edição. Portanto, a identificação de novos compostos químicos que geram sondas moleculares valiosos para dissecar a função editosome e montagem. Em estudos anteriores, os ensaios in vitro de edição foram realizadas utilizando ARN marcado com rádio. Estes ensaios são demorados, ineficiente e inadequado para fins de alto rendimento. Aqui, um baseado em fluorescência homogênea &quot;misturar e medir&quot; ensaio repórter martelo ribozyme in vitro para monitorar a edição de RNA, é apresentado. Apenas como conseqüência da edição de RNAa ribozima uma transferência de energia de ressonância de fluorescência (FRET) do substrato oligoribonucleotide sofre clivagem. Isto por sua vez resulta na separação do fluoróforo do extintor, produzindo assim um sinal. Em contraste, quando a função editosome é inibida, o sinal de fluorescência será extinta. Este é um método altamente sensível e simples, que devem ser aplicadas para controlar a edição de ARN in vitro ou elevado rendimento de rastreio de produtos químicos que podem inibir a função editosome.

Para demonstrar os passos necessários para a criação de uma tela em grande escala, as Figuras 2-5 são experiências representativas de controlo relacionados com a qualidade do ensaio. Estas são experiências de controlo essenciais para um ensaio consistente ao longo de vários dias ou de rastreio para a comparação de diferentes telas. Avaliação da fluorescência Relação sinal-ruído Para assegurar a estabilidade e a qu...

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 Catalisador como repórter para Triagem Drogas contra edição de RNA em Tripanossomas

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 visualizações.  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.

Vídeo: 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.

Visualizando Localização RNA em Xenopus Oócitos

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

Biologia

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.

Transcrição in vitro e nivelamento de Gaussia mRNA luciferase Seguido por HeLa transfecção celular

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.

Isolamento de cromatina por purificação de RNA (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.

Bioensaio eletroantenográfica como uma ferramenta de triagem para voláteis da planta hospedeira

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.

Geração de substrato para Endonucleases 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.

Esgotamento de RNA ribossômico para metagenomico Gut 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.

RNA<em&gt; Em situ</em&gt; Hibridação em embriões de Mount inteiros e histologia de células Adaptado para Elasmobrânquios Marinhos

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.

A Triagem procedimento químico para Glucocorticoid Sinalização com um Sistema Reporter Zebrafish Zergling luciferase

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.

Um método de triagem e Validação de mutações resistentes aos inibidores da quinase

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.

Detecção de proteínas de ligação a ARN pela<em&gt; In Vitro</em&gt; RNA Pull-down em Cultura adipócito

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 ...