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.).

Die folgende Protokoll beschreibt die Verfahren zur Durchführung der fluoreszenzbasierten Assays RNA-Editierung. Der Assay kann in einem PCR-Röhrchen, 96-Well-oder 384-Well-Platten in Abhängigkeit von dem Umfang des Experiments durchgeführt werden. Anschließend wird das Fluoreszenzsignal kann auf einem geeigneten Echtzeit-PCR-Nachweissystem gelesen werden. Der Assay wird hier im Zusammenhang mit der 384-Well-Platten beschrieben. 1. Kultivierung T. brucei Cells <ol><li> Bereiten Sie ein Wachstumsmedium für T. brucei prozyklischen Form Zellen. Für 1 l Medium: <ol><li> Lösen 25,4 g SDM-79-Pulver in 800 ml MilliQ-Wasser. </li><li> 2 g NaHCO 3 und pH-Wert auf 7,3 mit 10 M NaOH. </li><li> In Nanopure-Wasser auf ein Volumen von 900 ml, Filter zu sterilisieren. </li><li> Hinzufügen Fetal Bovine Serum (FBS), Penicillin-Streptomycin-Lösung und Hämin (2,5 mg / ml) bis zu Endkonzentrationen von 10% (v / v), 100 U / ml und 7,5 mg / l auf. </li></ol></li><li> Wachsen 300 ml T. &lt;em&gt; brucei 1.7A Wildtyp (prozyklischen Form)-Zellen bei 28 ° C, Schütteln bei 70 Umdrehungen pro Minute bis zu einer Dichte von 1,5 x 10 7 Zellen / ml. HINWEIS: Dies sollte 3 ml der aktiven editosome produzieren mit ~ 0,5 mg Gesamtprotein, ausreichend für 600 Bearbeitung Reaktionen. </li><li> Ernte der Zellen durch Zentrifugation bei 6.000 × g für 10 min bei 4 ° C. </li><li> Mit 50 ml gekühltem PBSG-Puffer (10 mM Na 2 HPO 4, 10 mM NaH 2 PO 4, 145 mM NaCl und 6 mM Glucose) Waschen des Pellets und Spin-down Zellen werden erneut durch Zentrifugation bei 10.000 × g für 10 min bei 4 ° C. </li></ol> 2. Isolierung von Mitochondrien Crude HINWEIS: Alle Schritte sollten auf Eis oder bei 4 ° C durchgeführt, um editosome Aktivität zu bewahren. <ol><li> Resuspendieren der geernteten Zellen in 30 ml DEE-Puffer (1 mM Tris-HCl pH 8,0 und 1 mM EDTA). Verwenden Sie eine 40 ml sterile Dounce Homogenisator (vorgekühlte), um die Zellmembran durch Streicheln auf und ab mindestens 10-mal auf Eis zu stören. </li&gt; <li> 4,3 ml 60% Saccharose (w / v, dh 1,75 M) unmittelbar in den Homogenat zu einer Endkonzentration von 0,25 M Zentrifugieren bei 15.800 × g für 10 min bei 4 ° C, bevorzugt zu bringen Mitochondrien. </li><li> Resuspendieren des Mitochondrienpellet in 4,6 ml STM-Puffer (20 mM Tris-HCl pH 8,0, 250 mM Saccharose und 2 mM MgCl 2). Hinzufügen 13,8 ul 0,1 M CaCl 2 und 4 &amp;mgr; l RNase-freier DNase I, um Endkonzentrationen von 0,3 mM und 9 U / ml. Inkubieren der Mischung für 1 Stunde auf Eis. </li><li> Hinzufügen von 4,6 ml STE-Puffer (20 mM Tris-HCl pH 8,0, 250 mM Saccharose und 2 mM EDTA), um das DNase I. Zentrifugieren bei 15.800 × g für 10 min bei 4 ° C inaktiviert </li><li> Das Pellet in 400 ul Lysepuffer (10 mM Tris-HCl pH 7,2, 10 mM MgCl 2, 100 mM KCl, 1 ug / ml Pepstatin, 1 mM DTT und 1x komplett EDTA-freie Protease-Inhibitor) und Übertragung auf eine Mikrozentrifugenröhrchen. </li><li> Hinzufügen von 10% Triton X-100 auf eine Endkonzentration von 1%nd bebrüten das Lysat für 15 min bei 4 ° C auf einem Schwenker. </li><li> Deaktivieren des mitochondrialen Lysat durch Zentrifugieren zweimal bei 17.000 × g für 15 min bei 4 ° C; Beibehaltung der geklärte Überstand jeder Zeit. </li></ol> 3. Editosome Reinigung <ol><li> Pour à 10 ml 10% -30% (v / v) Glycerin linearen Gradienten (Tabelle 1) in ein Ultrazentrifugenröhrchen mit 2x HHE Gradienten-Puffer (40 mM HEPES pH 7,9, 20 mM Mg (OAc) 2, 100 mM KCl, und 2 mM EDTA) und ein Gradient Maker, indem Sie die Gebrauchsanleitung. </li><li> Entfernen Sie vorsichtig 500 ul Lösung von der Spitze der Glycerin-Gradienten und schonend geladen werden 500 ul der mitochondrialen Lysat gelöscht. Schleudern bei 178.000 × g für 6 Stunden bei 4 º C unter Verwendung einer Ultrazentrifuge. </li><li> Sequentielles Sammeln 500 ul-Fraktionen von der Oberseite zum Boden des Gradienten bei 4 ° C. Dann schnappen Einfrieren der Fraktionen mit flüssigem Stickstoff und bei -80 ° C bis zur Nutzung. </li></ol><ol><li> Annealing der jeweiligen DNA-Matrize, die komplementäre Sequenz T7-Promotorsequenz (Tabelle 2) mit einem T7-Promotor-Oligonukleotid (5&#39;-TAATACGACTCACTATAGGG-3 &#39;) in einem Molverhältnis von 1:1 durch Erhitzen bei 90 ° C für 3 Minuten und Abkühlen auf RT für mindestens 10 min. </li><li> Transkribieren RNA mit Hilfe eines in-vitro-Transkription-Kit, indem Sie die Gebrauchsanleitung. </li><li> Stoppen Sie die Transkription Reaktion durch Zugabe von gleichen Volumen von 7 M Harnstoff-Farbstoff (7 M Harnstoff, 0,05% Xylol Cynol und 0,05% Bromphenolblau). Führen Sie auf einem Filter sterilisiert 9% denaturierenden Polyacrylamid-Gel (9% Acrylamid, 7 M Harnstoff, 1x TBE). </li><li> Verwenden Sie die Ultraviolett (UV)-Shadowing mit einem kurzwelligen UV-Lampe zu finden und Verbrauch jeweiligen RNA. Platzieren der ausgeschnittenen Gelstück in ein Mikrozentrifugenröhrchen und füge 400 ul Gel Elutionspuffer (20 mM Tris-HCl pH 7,5, 250 mM NaOAc, 1 mM EDTA und 0,25% SDS). Eluiere über Nacht bei RT auf einem Schwenker. </li><li> PreciPitate der eluierten RNA durch Zugabe von 1 ml kaltem 100% Ethanol und Inkubation entweder bei -80 ° C für 30 min und 20 ° C über Nacht. </li><li> Zentrifuge bei 16.000 × g für 30 min bei 4 ° C, um die RNA zu pelletieren unten. </li><li> Mit 1 ml 75% Ethanol Waschen des Pellets. Zentrifugieren bei 16000 × g für 20 min bei 4 ° C </li><li> Die RNA-Pellet in geeigneter Weise in RNase-freiem Wasser auf die gewünschten Konzentrationen zu erreichen, wie in Tabelle 2 gezeigt. </li></ol> 5. Fluoreszenz-basierte RNA-Editing-Assay <ol><li> Für einen Reaktions kombinieren 1 pmol (1 ul) preA6Rbz und 2,5 pmol (1 ul) gA6Rbz (Molverhältnis 1:2,5) in einem Mikrozentrifugenröhrchen, Inkubation bei 70 º C für 3 min und lassen Sie es bei RT sitzen an mindestens 10 min. </li><li> Unterdessen bereiten einen Master-Mix unter Verwendung von Tabelle 3, ohne preA6Rbz und gA6Rbz, für die Bearbeitung aus 1x HHE Reaktionspuffer (25 mM HEPES pH 7,9, 10 mM Mg (OAc) 2, 50 mM KCl und 10 mM EDTA), 1 mMATP, 5 mM CaCl 2, 16 ng / ul Torula-Hefe-RNA, 0,1% Triton X-100, und das gereinigte editosome. </li><li> In geglüht preA6Rbz und gA6Rbz, um den Master-Mix zu vervollständigen. </li><li> Verzichten 18 &amp;mgr; l der Mastermischung (Tabelle 3) in die Vertiefungen, die entweder 2 &amp;mgr; l RNase-freies Wasser (Vertiefungen ohne Verbindungen) oder 2 &amp;mgr; l 200 &amp;mgr; M chemischen Verbindungen und umfassen Kontrollproben in der Platte nach Fig. 5. </li><li> Verschließen Sie die Platte mit einem Plattenabdeckung und drehen Sie die Platte nach unten, um die Luftblasen zu entfernen. Inkubieren bei 28 ° C für 4 Stunden. </li><li> In 25 pmol (2 ul) gA6Rbz Wettbewerber in jede Vertiefung. Legen Sie eine frische Versiegelung, drehen Sie die Platte um und legen Sie es auf einer Echtzeit-PCR-Maschine. Programmieren Sie das folgende Experiment: Schritt 1: 85 ° C für 5 min; Schritt 2: 24 ° C für 10 min; Schritt 3: Stop. </li><li> Werden 15 pmol (1 ul) des FRET-Substrat zu jeder Vertiefung bis zu einem Endvolumen von 23 ul. Verschließen Sie die Platte mit einem frischen Versiegelung.Schnell drehen die Platte und legen Sie sie wieder auf die Echtzeit-PCR-Maschine. </li><li> Programmieren Sie einen neuen Versuch mit den folgenden Schritten: Schritt 1: 37 ° C für 1 min; Schritt 2: Lesen; Schritt 3: Gehen Sie auf 1, 40 mal Schritt; Schritt 4: Stop. </li><li> Setup die Platte, indem Sie alle Brunnen, die Lektüre erfordern, und wählen Sie den FAM-Filter. Eingangsvolumen 23 ul und den Lauf starten. </li><li> Berechnen Sie die Steigung der von jedem gut / Probe erhalten, um eine kinetische Messung durch Auftragen der Pisten auf einem Balkendiagramm für die Analyse zu erhalten Werte. HINWEIS: Eine kinetische Lese verbessert das Signal-Rausch-Verhältnis zwischen der Probe und dem Hintergrund; als der Hintergrund Probe würde eine Steigung nahe Null haben. Ein Endpunkt Lesen hat eine höhere Hintergrundgeräusche. </li></ol>

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The authors have nothing to disclose.

Ein neuartiges Hochdurchsatz-Screening-Verfahren, um Inhibitoren gegen die RNA-Editing-Komplex von Trypanosomen identifizieren vorgestellt wurde, bietet ein neues Werkzeug für die Wirkstoffforschung an Krankheiten, die durch Trypanosomatiden verursacht zu begegnen. FRET-basierten Ribozym Assay wurde ausgiebig für verschiedene Zwecke verwendet, 20-22; Jedoch haben wir die Fähigkeit von FRET-Ribozym-Assay für die in vitro Überwachung der RNA-Editing Aktivität 19 verwendet. Dieser Test ...

Der Prozess der RNA-Editing, ein Post-Transkriptions mRNA Änderung, wurde zuerst in Trypanosomatiden 1 entdeckt. Seitdem hat umfangreiche Arbeit bei der Untersuchung der Mechanismus hinter RNA-Editing in Trypanosoma brucei 2,3 durchgeführt. In einer Reihe von enzymatischen Reaktionen, die editosome ein Kernkomplex von etwa 20 Proteinen, erstellt reifen mitochondrialen mRNAs für mehrere Komponenten der oxidativen Phosphorylierung Energieerzeugungssystems. Die Reihenfolge der Ereignisse ist endonukleolytische katalytische Spaltung Uridylat (U) Hinzufügen oder Löschen, und Ligation, wie durch Führungs RNAs (gRNAs) diktiert 4. Neben den Kern editosome komplexe Proteine ​​haben eine Reihe von Hilfsfaktoren auch identifiziert 5-7. Diese Proteine ​​sind meist gesehen in unabhängigen Komplexen gruppiert. Jedoch ist die Reihenfolge der Proteinanordnung in den Kern editosome komplex und die Interaktionsmuster des Kernkomplex mit dem ZubehörKomplexe noch bestimmt werden. Targeting der RNA-Editing-Prozess in Trypanosomatiden können chemische Dissectoren sorgen, dass die Hilfe bei der Untersuchung der Montage und Funktion der editosome komplex. Darüber hinaus haben funktionelle Untersuchungen auf mehreren editosome Proteine ​​Wesentlichkeit in verschiedenen Lebensphasen dargestellt, was auf ihr Potenzial als Medikament zielt 12.08. Daher kann die gefundenen Inhibitoren der editosome auch als Bleiverbindungen gegen Trypanosomatiden handeln. Dies ist an der Zeit, wie sie derzeit gegen Krankheiten, die durch Trypanosomatida verursacht verfügbaren Medikamente sind giftig, ineffizient und teuer 13,14. Eine effiziente und bequeme in-vitro-Test ist notwendig, um die chemische Universum für spezifische Inhibitoren, RNA-Editing blockieren erkunden. Drei Tests wurden entwickelt und verwendet werden, um Aktivitäten zu überwachen editosome: (a) volle Runde in vitro RNA-Editing-Test 15, (b) in vitro RNA-Editing-Assay 16,17, vorge gespalten einnd (c) Hammerhead-Ribozym (HHR) basierenden Assay 18. Die ersten beiden Tests beruhen auf direkte Visualisierung des bearbeiteten Produktes (6 ATPase mRNA) mit Hilfe von Radioaktivität. Der HHR-basierten Assay verwendet eine modifizierte Version der ATPase 6 mRNA, die modelliert wird als ein Ribozym nach Bearbeitung verhalten. Die funktionelle Ribozym dann spaltet spezifisch eine radioaktiv markierte RNA-Substrat, als Reporter dient. Kürzlich Moshiri et al. Entwickelt ein &quot;mischen und zu messen&quot; In-vitro-Reporter-Assay HHR-Basis von RNA-Editing überwachen, wo das radioaktiv markierte RNA-Substrat mit einem Fluoreszenz-Resonanz-Energie-Transfer (FRET) Substrat 19 ersetzt. Die prinzipiellen Vorteile dieses Tests sind: (a) es ist eine schnelle und bequeme Mischen und Messen Assaytyp, die Herstellung von aktiven Ribozym und Substratspaltung gleichzeitig erfolgen im gleichen Röhrchen in geringen Mengen (z. B. 20 ul), (b ) es die Verwendung von radioaktiv markierten Materialien vermeidet, (c) die Empfindlichkeit dass Adurch Fluoreszenz Instrumentierung in einem Mikrotiter-Plattenformat, und (d) ein hohes Signal-Rausch-Verhältnis fforded. Unter Verwendung dieses Tests wurde die Wirkung von bekannten RNA-Editing-Ligase Inhibitoren gegen gereinigtes editosome 19 bestätigt. Dieses Experiment bestätigt der Test für die schnelle Identifizierung von RNA-Editing-Hemmer, in erster Linie gegen ganze editosomes von T. brucei. Fig. 1 ist eine detaillierte Schritt-für-Schritt-Schema des fluoreszenzbasierten In-vitro-RNA-Editing-Assay. Dieses Protokoll kann entweder für die Überwachung der RNA-Editing in vitro oder einfach für das Screening von Substanzbibliotheken von verschiedenen Maßstäben angepasst werden verwendet werden.

<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

Erhebliche Fortschritte bei der Bestimmung des Mechanismus der mitochondrialen RNA-Editing in Trypanosomen gemacht worden. Ebenso hat erhebliche Fortschritte bei der Identifizierung der Komponenten des editosome Komplex, der RNA-Editing katalysieren gemacht worden. Es ist jedoch noch nicht klar, wie diese Proteine ​​zusammen zu arbeiten. Von einem Hochdurchsatz-Screening gegen die editosome erhalten Chemische Verbindungen können blockieren oder beeinträchtigen einen oder mehrere Schritte im Bearbeitungszyklus. Daher wird die Identifizierung von neuen chemischen Verbindungen wertvolle molekulare Sonden zur Zerlegung der editosome Funktion und Anordnung zu erzeugen. In früheren Studien wurden in vitro-Assays unter Verwendung von Bearbeitungsradioaktiv markierten RNA durchgeführt. Diese Assays sind zeitaufwendig, ineffizient und für Hochdurchsatz-Zwecke ungeeignet. Hier eine homogene Fluoreszenz-basierten &quot;zu mischen und zu messen&quot; Hammerhead-Ribozym in-vitro-Reporter-Assay von RNA-Editing zu überwachen, wird vorgestellt. Nur als Folge von RNA-Editingdas Hammerhead-Ribozym ein Fluoreszenz-Resonanz-Energie-Transfer (FRET)-Substrat Oligoribonukleotid Spaltung erfährt. Dies führt wiederum zu einer Trennung des Fluorophors vom Quencher, wodurch ein Signal erzeugt wird. Im Gegensatz dazu, wenn die editosome Funktion gesperrt ist, wird das Fluoreszenzsignal gelöscht werden. Dies ist eine sehr empfindliche und einfache Tests, der allgemein für in vitro-RNA-Editing oder Hochdurchsatz-Screening von Chemikalien, die die editosome Funktion hemmen können überwacht werden sollte.

Um die notwendigen Schritte für den Aufbau einer großen Leinwand zeigen, erforderlich, Fig. 2-5 sind repräsentative Kontrollexperimenten auf die Qualität des Tests zusammen. Dies sind wesentliche Kontrollversuche für eine konsistente Assay über mehrere Tage zum Screenen oder zum Vergleich von verschiedenen Bildschirmen. Die Bewertung der Fluoreszenz-Signal-zu-Rausch-Verhältnis Um die Stabilität und die Qualität der Fluor...

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-Katalysator als Reporter für Drogen-Screening gegen RNA-Editing in Trypanosomen

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 Aufrufe.  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.

Serie: 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.

Visualizing RNA Localization in Xenopus Oozyten

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

Forschung

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.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

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.

Chromatin Isolierung von RNA-Reinigung (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.

Substrat Generation für Endonukleasen der CRISPR / Cas-Systeme

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.

Depletion der ribosomalen RNA für Mosquito Gut metagenomische 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; In situ</em&gt; Hybridisierung in whole mount Embryonen und Histologie für Marine Elasmobranchs Angepasst

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.

Eine Stoff Screening Verfahren für die Glucocorticoid-Signalisierung mit einem Zebrafisch-Larve Luciferase-Reportersystems

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.

Nachweis von RNA-bindenden Proteinen durch<em&gt; In Vitro</em&gt; RNA Pull-down in Adipocyte Kultur

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.

Praktische Anwendung der RNA-Interferenz: mündliche Lieferung von Double-Stranded RNA in Liposomen Träger für Schaben

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.

Imaging-FITC-Dextran als Reporter für regulierte Exozytose

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