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.).

Le protocole ci-dessous décrit la procédure pour effectuer l&#39;ARN essai de montage à base de fluorescence. Le dosage peut être effectué dans un seul tube de PCR, 96 puits ou 384 puits en fonction de la portée de l&#39;expérience. Ensuite, le signal de fluorescence peut être lu sur un système de détection de PCR en temps réel approprié. Le dosage est décrit ici dans le contexte de plaques à 384 puits. 1. T. La culture Les cellules brucei <ol><li> Préparer un milieu de croissance de T. brucei procyclique forment des cellules. Pour 1 litre de milieu: <ol><li> Dissoudre SDM-79 poudre de 25,4 g dans l&#39;eau de 800 ml. </li><li> Ajouter 2 g de NaHCO 3 et pH à 7,3 avec du NaOH 10. </li><li> Ajouter de l&#39;eau nanopure jusqu&#39;à un volume final de 900 ml, stériliser par filtration. </li><li> Ajouter de sérum bovin fœtal (FBS), de la pénicilline-streptomycine et de la solution d&#39;hémine (2,5 mg / ml) à des concentrations finales de 10% (v / v), 100 U / ml et 7,5 mg / L respectivement. </li></ol></li><li> Cultivez 300 ml de T. &lt;em&gt; brucei 1.7A type sauvage (de forme procyclique) cellules à 28 ° C, en agitant à 70 tours par minute à une densité de 1,5 x 10 7 cellules / ml. NOTE: Ceci devrait produire 3 ml de éditosome actif avec ~ 0,5 mg de protéine totale, suffisante pour 600 réactions de modification. </li><li> Récolter les cellules par centrifugation à 6000 xg pendant 10 min à 4 ° C. </li><li> Laver le culot avec 50 ml de tampon de PBSG refroidi (10 mM de Na 2 HPO 4, 10 mM de NaH 2 PO 4, NaCl 145 mM et glucose 6), et centrifuger les cellules à nouveau par centrifugation à 10 000 xg pendant 10 min à 4 ° C. </li></ol> 2. Isolement des mitochondries brut NOTE: Toutes les étapes doivent être effectuées sur la glace ou à 4 ° C pour préserver éditosome activité. <ol><li> Remettre en suspension les cellules récoltées dans 30 ml de tampon DTE (1 mM Tris-HCl pH 8,0 et 1 mM EDTA). Utilisez un 40 ml stérile homogénéisateur Dounce (pré-réfrigérées) pour perturber la membrane cellulaire en caressant de haut en bas au moins 10 fois sur la glace. </li&gt; <li> Immédiatement ajouter 4,3 ml de saccharose à 60% (poids / volume, c&#39;est à dire 1,75 M) à l&#39;homogénat à une concentration finale de 0,25 M. Centrifuger à 15 800 g pendant 10 min à 4 ° C, de préférence vers le bas pour amener les mitochondries. </li><li> Reprendre le culot mitochondrial dans 4,6 ml de tampon STM (20 mM Tris-HCl pH 8,0, 250 mM de saccharose et 2 mM de MgCl2). Ajouter 13,8 ul de 0,1 M de CaCl2 et 4 ul de DNase sans RNase I à des concentrations finales de 0,3 mM et de 9 U / ml, respectivement. Faire incuber le mélange pendant 1 heure sur de la glace. </li><li> Ajouter 4,6 ml de tampon STE (20 mM Tris-HCl pH 8,0, 250 mM de saccharose et de l&#39;EDTA 2 mM) pour inactiver la DNase I. Centrifuger à 15 800 g pendant 10 min à 4 ° C. </li><li> Remettre en suspension le culot dans 400 pl de tampon de lyse (10 mM Tris-HCl pH 7,2, 10 mM MgCl 2, 100 mM de KCl, 1 ug / ml de pepstatine, 1 mM de DTT, et l&#39;inhibiteur de protéase de 1x complet sans EDTA) et le transfert à un tube de centrifugeuse. </li><li> Ajouter 10% de Triton X-100 à une concentration finale de 1% parnd incuber le lysat pendant 15 minutes à 4 ° C sur un agitateur de tubes. </li><li> Désactivez le lysat mitochondrial par centrifugation à deux reprises à 17 000 g pendant 15 min à 4 ° C; en conservant le surnageant clarifié chaque fois. </li></ol> 3. Éditosome Purification <ol><li> Verser 10 ml de 10% -30% (v / v) gradient linéaire de glycérol (tableau 1) dans un tube d&#39;ultracentrifugation en utilisant un tampon à gradient 2x HHE (40 mM HEPES pH 7,9, mM de Mg 20 (OAc) 2, 100 mM de KCl, et 2 mM EDTA) et une machine à gradient en suivant le manuel d&#39;instruction. </li><li> Retirez délicatement 500 pi de solution à partir du haut de la pente de glycérol et charger doucement 500 pi du lysat mitochondrial dégagé. Spin à 178 000 g pendant 6 heures à 4 ° C en utilisant une ultracentrifugation. </li><li> Collecter des fractions de 500 ul de manière séquentielle à partir du haut vers le bas de la pente à 4 ° C. Puis enclenchez geler les fractions en utilisant de l&#39;azote liquide et conserver à -80 ° C jusqu&#39;à utilisation. </li></ol><ol><li> Recuire la matrice d&#39;ADN respective contenant une séquence complémentaire de T7 séquence promotrice (tableau 2) avec un oligonucléotide promoteur T7 (5&#39;-TAATACGACTCACTATAGGG-3 &#39;) dans un rapport molaire de 1:1 par chauffage à 90 ° C pendant 3 min et le refroidissement à la température ambiante pendant au moins 10 min. </li><li> Transcrire l&#39;ARN en utilisant un kit de transcription in vitro en suivant le manuel d&#39;instruction. </li><li> Arrêter la réaction de transcription en ajoutant un volume égal d&#39;urée 7 M colorant (7 M d&#39;urée, 0,05% Xylène Cynol, et 0,05% de bleu de bromophénol). Fonctionner sur un filtre stérilisé à 9% gel de polyacrylamide dénaturant (9% d&#39;acrylamide, urée 7 M, TBE 1x). </li><li> Utilisez l&#39;observation ultraviolet (UV) avec une lampe UV à ondes courtes pour localiser et accise ARN respectif. Placer le morceau de gel excisée dans un tube de centrifugeuse et ajouter 400 ul de tampon d&#39;élution de gel (20 mM Tris-HCl pH 7,5, 250 mM de NaOAc, EDTA 1 mM et SDS 0,25%). Éluer nuit à température ambiante sur un agitateur de tube. </li><li> Precipitate élue l&#39;ARN en ajoutant 1 ml d&#39;éthanol à 100% froid et incubation soit à -80 ° C pendant 30 min ou à -20 ° C pendant une nuit. </li><li> Centrifuger à 16 000 g pendant 30 min à 4 ° C, pour former un culot vers le bas de l&#39;ARN. </li><li> Laver le culot avec 1 ml d&#39;éthanol à 75%. Centrifuger à 16 000 g pendant 20 min à 4 ° C. </li><li> Remettre en suspension le culot d&#39;ARN de manière appropriée dans l&#39;eau exempte de RNase pour atteindre les concentrations désirées, comme indiqué dans le tableau 2. </li></ol> 5. Base de fluorescence test ARN édition <ol><li> Pour une seule réaction, combiner 1 pmol (1 pi) de preA6Rbz et 2,5 pmol (1 pi) de gA6Rbz (rapport molaire 1:2,5) dans un tube de centrifugeuse, incuber à 70 º C pendant 3 minutes et laisser reposer à température ambiante pendant au moins de 10 min. </li><li> Pendant ce temps préparer un mélange maître en utilisant le tableau 3, sans le preA6Rbz et gA6Rbz, pour la réaction de l&#39;édition contenant un tampon 1x HHE (25 mM HEPES pH 7,9 mM Mg 10 (OAc) 2, KCl 50 mM et 10 mM d&#39;EDTA), 1 mMATP, 5 mM de CaCl2, 16 ng / ul d&#39;ARN de levure Torula, 0,1% de Triton X-100, et le éditosome purifiée. </li><li> Ajouter recuit preA6Rbz et gA6Rbz pour compléter le mélange maître. </li><li> Distribuer 18 ul de la solution mère (Tableau 3) dans les puits contenant soit 2 ul d&#39;eau exempte de RNase (puits sans composés) ou 2 pi de 200 composés uM chimiques et comprennent des échantillons de contrôle de la plaque selon la Figure 5. </li><li> Sceller la plaque avec un scellant pour plaques et tourner la plaque vers le bas, pour éliminer les bulles d&#39;air. Incuber à 28 ° C pendant 4 heures. </li><li> Ajouter 25 pmol (2 pi) de gA6Rbz concurrent à chaque puits. Placez un nouveau scellant, tourner la plaque et placez-le sur une machine de PCR en temps réel. Programmer l&#39;expérience suivante: Etape 1: 85 ° C pendant 5 min; Etape 2: 24 ° C pendant 10 min; Étape 3: Arrêtez. </li><li> Ajouter 15 pmol (1 pi) de substrat FRET à chaque puits pour un volume final de 23 ul. Sceller la plaque avec un nouveau scellant.Tourner rapidement la plaque et le replacer sur la machine PCR en temps réel. </li><li> Programmer une nouvelle expérience avec les étapes suivantes: Etape 1: 37 ° C pendant 1 min; Étape 2: Lire; Étape 3: Passez à l&#39;étape 1, 40 fois; Étape 4: Stop. </li><li> Configuration de la plaque en sélectionnant tous les puits qui nécessitent la lecture et de choisir le filtre de FAM. Le volume d&#39;entrée que 23 pi et commencer la course. </li><li> Calculer la pente des valeurs obtenues à partir de chaque / puits d&#39;échantillon pour obtenir une mesure cinétique en traçant des pistes sur un graphique à barres pour l&#39;analyse. REMARQUE: La lecture cinétique améliore le rapport signal-sur-bruit entre l&#39;échantillon et l&#39;arrière-plan; que l&#39;échantillon de fond aurait une pente proche de zéro. Un point de lecture de fin a plus de bruit de fond. </li></ol>

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The authors have nothing to disclose.

Une méthode de criblage à haut débit roman pour identifier des inhibiteurs contre le complexe d&#39;édition de l&#39;ARN de trypanosomes a été présenté, en fournissant un nouvel outil pour la découverte de médicaments pour lutter contre les maladies causées par trypanosomatidés. Dosage de ribozyme à base de FRET a été largement utilisé à des fins différentes de 20 à 22; Cependant, nous avons utilisé le test de capacité de ribozyme à base de FRET pour la surveillance in vitro de ...

Le processus d&#39;édition de l&#39;ARN, une modification de l&#39;ARNm post-transcriptionnelle, a été découvert en trypanosomatidés 1. Depuis lors, d&#39;importants travaux ont été réalisés en étudier le mécanisme derrière édition de l&#39;ARN chez T. brucei 2,3. Dans une série de réactions enzymatiques, la éditosome, un complexe de base de l&#39;ordre de 20 protéines, crée des ARNm matures mitochondriales pour plusieurs composants du système de la phosphorylation oxydative de la production d&#39;énergie. L&#39;ordre des événements est catalytiques clivage endonucléolytique, uridylate (U) ajout ni suppression, et la ligature, comme dicté par ARN guides (ARNg) 4. En plus des protéines essentielles éditosome complexes, un certain nombre de facteurs accessoires ont également été identifiés 5-7. Ces protéines sont observés essentiellement regroupés dans les complexes indépendants. Cependant, l&#39;ordre d&#39;assemblage des protéines dans le complexe de éditosome de base et les modèles d&#39;interaction du complexe de base de l&#39;accessoirecomplexes sont encore à déterminer. Cibler le processus d&#39;édition de l&#39;ARN dans trypanosomatidés peut fournir aux ciseaux chimiques que l&#39;aide à l&#39;étude de l&#39;assemblage et de la fonction du complexe éditosome. En outre, des études fonctionnelles sur plusieurs protéines éditosome ont montré essentialité dans les différents stades de la vie, en indiquant leur potentiel en tant que médicament cible 8-12. Par conséquent, les inhibiteurs de la éditosome trouvés peuvent également agir en tant que composés de plomb contre trypanosomatidés. C&#39;est rapide, comme les médicaments actuellement disponibles contre les maladies causées par trypanosomatide sont toxiques, inefficace et coûteux 13,14. Un essai in vitro efficace et pratique est nécessaire d&#39;explorer l&#39;univers chimique d&#39;inhibiteurs spécifiques qui bloquent édition de l&#39;ARN. Trois tests ont été mis au point et utilisé pour surveiller éditosome activités: (a) tour complet test in vitro de l&#39;édition de l&#39;ARN 15, (b) pré-coupés test in vitro de l&#39;édition de l&#39;ARN 16,17, une (c) ribozyme en tête de marteau (RHS) à base de dosage 18. Les deux premiers essais reposent sur la visualisation directe du produit modifié (ATPase six ARNm) avec l&#39;aide de la radioactivité. L&#39;analyse basée sur la HHR utilise une version modifiée de l&#39;ATPase 6 ARNm qui est modélisée à se comporter comme un ribozyme à l&#39;édition. Le ribozyme fonctionnelle clive ensuite spécifiquement un substrat d&#39;ARN radiomarqué, servant en tant que journaliste. Récemment, Moshiri et al. Développé un &quot;mélange et mesure&quot; HHR-fondé en essai rapporteur in vitro pour surveiller édition de l&#39;ARN, où le substrat de l&#39;ARN radiomarqué est remplacé par un transfert d&#39;énergie par résonance de fluorescence (FRET) du substrat 19. Les principaux avantages de ce dosage sont les suivants: (a) il s&#39;agit d&#39;un mélange rapide et commode et le type de dosage de mesure, comme la production de ribozyme actif et clivage du substrat se produisent simultanément dans le même tube à faible volume (20 ul), (b ) elle permet d&#39;éviter l&#39;utilisation de matières radiomarquées, (c) la sensibilité qui est unfforded par fluorescence instrumentation dans un format de plaque de micro-titrage, et (d) un signal de niveau haut par rapport au bruit. En utilisant ce dosage, l&#39;effet des inhibiteurs connus de l&#39;ARN ligase de montage de contre éditosome purifié a été confirmée 19. Cette expérience a validé le test pour l&#39;identification rapide des inhibiteurs de l&#39;édition de l&#39;ARN, principalement contre editosomes entiers de T. brucei. La figure 1 est un schéma détaillé étape par étape de l&#39;essai in vitro de l&#39;édition de l&#39;ARN à base de fluorescence. Ce protocole peut être utilisé soit pour la surveillance de l&#39;édition de l&#39;ARN in vitro ou facilement être adapté pour le criblage de banques de composés de diverses échelles.

<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

Des progrès substantiels ont été accomplis dans la détermination du mécanisme de l&#39;édition de l&#39;ARN mitochondrial chez les trypanosomes. De même, des progrès considérables ont été réalisés dans l&#39;identification des composants du complexe de éditosome qui catalysent édition de l&#39;ARN. Cependant, il n&#39;est pas encore clair comment ces protéines fonctionnent ensemble. Les composés chimiques obtenus à partir d&#39;un écran à haut débit contre la éditosome peuvent bloquer ou affecter une ou plusieurs étapes du cycle d&#39;édition. Par conséquent, l&#39;identification de nouveaux composés chimiques va générer des sondes moléculaires utiles pour disséquer la fonction de éditosome et l&#39;assemblage. Dans des études antérieures, des essais in vitro de modification ont été réalisées en utilisant l&#39;ARN radiomarqué. Ces tests prennent du temps, inefficace et inadapté à des fins à haut débit. Ici, une base de fluorescence homogène &quot;mélanger et mesurer&quot; marteau ribozyme test in vitro de reporter à surveiller édition de l&#39;ARN, est présenté. Qu&#39;à la suite de l&#39;édition d&#39;ARN d&#39;le ribozyme en tête de marteau d&#39;un substrat de oligoribonucléotide transfert d&#39;énergie de résonance de fluorescence (FRET) subit un clivage. Ceci à son tour conduit à la séparation du fluorophore de l&#39;agent d&#39;extinction en produisant ainsi un signal. En revanche, lorsque la fonction de éditosome est inhibé, le signal de fluorescence est éteinte. Il s&#39;agit d&#39;un test très sensible et simple qui devrait être généralement applicables à surveiller dans l&#39;édition de l&#39;ARN in vitro ou criblage à haut débit de produits chimiques qui peuvent inhiber la fonction de éditosome.

Pour démontrer les étapes nécessaires pour la mise en place d&#39;un écran de grande envergure, les figures 2-5 sont des expériences de contrôle représentatifs liés à la qualité de l&#39;analyse. Ce sont des expériences de contrôle essentielles pour une analyse cohérente sur plusieurs jours de dépistage ou de comparaison des différents écrans. Évaluer le ratio fluorescence signal-bruit Pour assurer la stabilité ...

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.

ARN catalyseur en tant que reporter pour le criblage de médicaments contre l&#39;ARN Édition dans trypanosomes

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 vues.  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.

Vidéo: 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.

Visualisation de localisation de l&#39;ARN Xenopus Les ovocytes

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

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Biologie

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.

Transcription in vitro et plafonnement de l&#39;ARNm de luciférase Gaussia Suivie par la transfection de cellules 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.

Isolement de la chromatine par purification d&#39;ARN (CHIRP)

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

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.

Génération substrat pour des endonucléases de CRISPR / Cas Systems

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.

L&#39;appauvrissement de l&#39;ARN ribosomique pour métagénomique Gut Mosquito ARN-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</emHybridation&gt; en embryons entiers de montage et histologie Adapté pour les élasmobranches marins

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.

Une procédure de dépistage chimique pour les glucocorticoïdes de signalisation avec un système de Reporter poisson zèbre Larve luciférase

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

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 détection des protéines liant l&#39;ARN par<em&gt; In vitro</em&gt; ARN Pull-down en adipocytes 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 ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

Utilisation pratique de l’interférence d’ARN : l’administration orale d’ARN Double brin dans Liposome transporteurs pour les blattes

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

L’imagerie FITC-dextran comme Reporter pour l’exocytose régulée

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...