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.).

Il protocollo che segue descrive la procedura per eseguire il test RNA editing basato sulla fluorescenza. L&#39;analisi può essere eseguita in un unico tubo PCR, 96 ben-, o 384 pozzetti a seconda del campo di applicazione dell&#39;esperimento. Successivamente il segnale di fluorescenza può essere visualizzato su un sistema di rilevazione adeguato tempo reale PCR. Il saggio qui è descritta nel contesto di piastre a 384 pozzetti. 1. Culturing T. Le cellule brucei <ol><li> Preparare un terreno di coltura per T. brucei prociclico cellule formano. Per 1 L di terreno: <ol><li> Sciogliere 25.4 g SDM-79 polvere in acqua 800 ml miliQ. </li><li> Aggiungere 2 g di NaHCO3 e il pH a 7,3 con NaOH 10 M. </li><li> Aggiungere acqua Nanopure ad un volume finale di 900 ml, filtro sterilizzare. </li><li> Aggiungere siero fetale bovino (FBS), penicillina-streptomicina soluzione e emina (2,5 mg / ml) a concentrazioni finali di 10% (v / v), 100 U / ml e 7,5 mg / L rispettivamente. </li></ol></li><li> Grow 300 ml di T. &lt;em&gt; brucei 1.7A wild type (forma prociclico) le cellule a 28 ° C, si stringono a 70 giri per una densità di 1,5 x 10 7 cellule / ml. NOTA: Questo dovrebbe produrre 3 ml di editosome attivo con ~ 0,5 mg di proteine ​​totali, sufficienti per 600 reazioni di editing. </li><li> Raccogliere le cellule per centrifugazione a 6.000 xg per 10 min a 4 ° C. </li><li> Lavare il pellet con 50 ml di tampone PBSG refrigerati (10 mM Na 2 HPO 4, 10 mM NaH 2 PO 4, 145 mM NaCl e 6 mM di glucosio), e centrifugare le cellule di nuovo per centrifugazione a 10.000 xg per 10 min a 4 ° C. </li></ol> 2. Isolamento di greggio mitocondri NOTA: Tutte le procedure devono essere eseguite su ghiaccio oppure a 4 ° C per preservare editosome attività. <ol><li> Risospendere le cellule raccolte in 30 ml di tampone DTE (1 mM Tris-HCl pH 8,0 e EDTA 1 mM). Usare un 40 ml sterile omogeneizzatore Dounce (pre-refrigerata) per distruggere la membrana cellulare da accarezzare su e giù almeno 10 volte su ghiaccio. </li&gt; <li> Immediatamente aggiungere 4,3 ml di 60% di saccarosio (w / v; cioè 1,75 M) al omogenato ad una concentrazione finale di 0,25 M. Centrifugare a 15.800 xg per 10 min a 4 ° C, per portare preferenzialmente giù mitocondri. </li><li> Risospendere il pellet mitocondriale in 4,6 ml di tampone STM (20 mM Tris-HCl pH 8,0, 250 mM saccarosio e 2 mM MgCl 2). Aggiungere 13,8 ml di 0,1 M CaCl 2 e 4 ml di DNasi I RNasi-free a concentrazioni finali di 0,3 mm e 9 U / ml, rispettivamente. Incubare la miscela per 1 ora su ghiaccio. </li><li> Aggiungere 4,6 ml di tampone STE (20 mM Tris-HCl pH 8,0, saccarosio 250 mM e EDTA 2 mM) per inattivare la DNasi I. Centrifugare a 15.800 xg per 10 min a 4 ° C. </li><li> Risospendere il pellet in 400 ml di tampone di lisi (10 mM Tris-HCl pH 7.2, 10 mM MgCl 2, KCl 100 mM, 1 mg / ml pepstatina, 1 mM DTT, e 1x completo inibitori della proteasi libero-EDTA) e trasferimento ad un provetta per microcentrifuga. </li><li> Aggiungere 10% Triton X-100 ad una concentrazione finale di 1% and incubare il lisato per 15 min a 4 ° C su un rotatore tubo. </li><li> Cancellare il lisato mitocondriale mediante centrifugazione due volte a 17.000 xg per 15 min a 4 ° C; mantenendo il surnatante eliminato ogni volta. </li></ol> 3. Editosome Purificazione <ol><li> Versare 10 ml 10% -30% (v / v) di glicerolo gradiente lineare (Tabella 1) in un tubo un&#39;ultracentrifuga utilizzando tampone gradiente HHE 2x (40 mM HEPES pH 7.9, 20 mM Mg (OAc) 2, KCl 100 mM, e 2 mM EDTA) e una macchinetta del gradiente seguendo il manuale di istruzioni. </li><li> Rimuovere con cautela 500 ml di soluzione dalla parte superiore del gradiente di glicerolo e caricare delicatamente 500 ml di lisato mitocondriale eliminato. Spin a 178.000 xg per 6 ore a 4 ° C utilizzando un&#39;ultracentrifuga. </li><li> Raccogliere 500 microlitri frazioni sequenzialmente dall&#39;alto verso il basso del gradiente a 4 ° C. Quindi far scattare congelare le frazioni utilizzando azoto liquido e conservare a -80 ° C fino al loro utilizzo. </li></ol><ol><li> Ricuocere stampo di DNA contenente rispettiva sequenza complementare alla sequenza del promotore T7 (Tabella 2) con un oligonucleotide promotore T7 (5&#39;-TAATACGACTCACTATAGGG-3 &#39;) in rapporto molare 1:1 in riscaldamento a 90 ° C per 3 min e raffreddamento a temperatura ambiente per almeno 10 min. </li><li> Trascrivere RNA utilizzando un kit di trascrizione in vitro seguendo il manuale di istruzioni. </li><li> Fermare la reazione di trascrizione con l&#39;aggiunta di volume uguale di 7 M urea colorante (7 M urea, 0.05% xilene Cynol, e lo 0,05% blu di bromofenolo). Eseguito su un filtro sterilizzato 9% gel di poliacrilammide denaturante (9% acrilammide, 7 M urea, 1x TBE). </li><li> Utilizzare il pedinamento raggi ultravioletti (UV) con una lampada UV ad onde corte per individuare e accise rispettivi RNA. Posizionare il pezzo gel asportata in una provetta per microcentrifuga e aggiungere 400 ml di tampone di eluizione gel (20 mM Tris-HCl pH 7.5, 250 NaOAc mM, EDTA 1 mM e 0,25% SDS). Eluire notte a RT su un rotatore tubo. </li><li> Precipitate l&#39;RNA eluito aggiungendo 1 ml di etanolo 100% freddo e incubando sia a -80 ° C per 30 minuti oa -20 ° C per una notte. </li><li> Centrifugare a 16.000 xg per 30 min a 4 ° C, per far sedimentare per RNA. </li><li> Lavare il pellet con 1 ml di etanolo al 75%. Centrifugare a 16.000 xg per 20 min a 4 ° C. </li><li> Risospendere il pellet di RNA opportunamente in acqua RNasi libero per ottenere le concentrazioni desiderate, come illustrato nella Tabella 2. </li></ol> 5. Sede a fluorescenza RNA Editing Assay <ol><li> Per una singola reazione, unire 1 pmol (1 ml) di preA6Rbz e 2,5 pmol (1 ml) di gA6Rbz (rapporto molare 1:2.5) in una provetta per microcentrifuga, incubare a 70 º C per 3 minuti e lasciate riposare a temperatura ambiente per almeno 10 min. </li><li> Nel frattempo preparare un master mix utilizzando la Tabella 3, senza preA6Rbz e gA6Rbz, per la reazione di modifica contenente tampone 1x HHE (25 mM HEPES pH 7,9, 10 mM Mg (OAc) 2, 50 mM KCl e EDTA 10 mM), 1 mMATP, 5 mM CaCl 2, 16 ng / ml di RNA Torula lievito, 0,1% Triton X-100, e il editosome purificata. </li><li> Aggiungi ricotto preA6Rbz e gA6Rbz per completare il master mix. </li><li> Pipettare 18 microlitri della miscela master (Tabella 3) in pozzetti contenente 2 ml di acqua priva di RNasi (pozzetti che non composti) o 2 ml di 200 mM composti chimici e comprendono campioni di controllo nella piastra di figura 5. </li><li> Sigillare la piastra con un sigillante piastra e girare il piatto, per eliminare eventuali bolle d&#39;aria. Incubare a 28 ° C per 4 ore. </li><li> Aggiungere 25 pmol (2 microlitri) di gA6Rbz concorrente di ogni bene. Posizionare un sigillante fresco, girare il piatto e posizionarlo su una macchina PCR real-time. Programmare il seguente esperimento: Passo 1: 85 ° C per 5 min; Passo 2: 24 ° C per 10 min; Passo 3: Stop. </li><li> Aggiungere 15 pmol (1 mL) di substrato FRET a ciascun pozzetto per un volume finale di 23 microlitri. Sigillare la piastra con un nuovo sigillante.Girare velocemente la piastra e rimetterlo sulla macchina PCR real-time. </li><li> Programmare un nuovo esperimento con le seguenti operazioni: Passo 1: 37 ° C per 1 min; Fase 2: Leggi; Fase 3: Andare al passo 1, 40 volte; Fase 4: Stop. </li><li> Installazione della piastra selezionando tutti i pozzi che richiedono la lettura e scegliere il filtro FAM. Volume d&#39;ingresso come 23 microlitri e iniziare la corsa. </li><li> Calcolare la pendenza dei valori ottenuti da ciascun / pozzetto per ottenere una misurazione cinetica riportando le piste su un grafico a barre per l&#39;analisi. NOTA: Una lettura cinetica di migliorare il rapporto segnale-rumore tra il campione e lo sfondo; come il campione sfondo avrebbe una pendenza prossima allo zero. Un punto di lettura finale ha una maggiore rumore di fondo. </li></ol>

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The authors have nothing to disclose.

Un metodo di screening high-throughput romanzo per identificare inibitori contro il complesso RNA editing dei tripanosomi è stato presentato, fornendo un nuovo strumento per la scoperta di farmaci per combattere malattie causate da trypanosomatids. Test ribozyme-based FRET è stato ampiamente utilizzato per diversi scopi 20-22; Tuttavia, abbiamo utilizzato la capacità di dosaggio ribozima basato FRET per il monitoraggio di attività in vitro editing dell&#39;RNA 19. Questa analisi potreb...

Il processo di editing dell&#39;RNA, una modifica mRNA post-trascrizionale, è stato scoperto nel trypanosomatids 1. Da allora, il lavoro sostanziale è stato condotto nello studio del meccanismo che sta dietro RNA editing in Trypanosoma brucei 2,3. In una serie di reazioni enzimatiche, la editosome, un complesso nucleo di circa 20 proteine, crea mRNA mitocondriali maturi per più componenti del sistema di fosforilazione ossidativa di generazione di energia. L&#39;ordine degli eventi catalitici è scissione endonucleolitico, uridylate (U), aggiunta o cancellazione, e legatura, come dettato dalla RNA guida (gRNAs) 4. Oltre al nucleo proteine ​​editosome complessi, un numero di fattori accessori sono stati anche identificati 5-7. Queste proteine ​​si osservano principalmente raggruppati in complessi indipendenti. Tuttavia, l&#39;ordine di montaggio proteina nel complesso nucleo editosome e gli schemi di interazione del complesso nucleo con l&#39;accessoriocomplessi sono ancora da determinare. Targeting il processo di editing RNA in trypanosomatids può prevedere dissettori chimici che aiuti a studiare l&#39;assemblaggio e la funzione del complesso editosome. Inoltre, studi funzionali in diverse proteine ​​editosome hanno dimostrato l&#39;essenzialità attraverso diverse fasi della vita, indicando la loro potenziale come bersagli farmacologici 8-12. Pertanto, gli inibitori trovate del editosome possono anche agire come composti di piombo contro trypanosomatids. Questo è tempestivo, in quanto i farmaci attualmente disponibili contro le malattie causate da trypanosomatid sono tossici, inefficiente e costoso 13,14. Un test efficiente e conveniente in vitro è necessario per esplorare l&#39;universo chimica per inibitori specifici che bloccano RNA editing. Tre saggi sono stati sviluppati e utilizzati per monitorare editosome attività: (a) ciclo completo in vitro RNA editing saggio di 15, (b) pre-spaccati in vitro RNA editing saggio di 16,17, unnd dosaggio 18 (c) martello ribozyme (HHR)-based. I primi due saggi si basano sulla visualizzazione diretta del prodotto modificato (ATPasi 6 mRNA) con l&#39;aiuto di radioattività. Il saggio basato HHR utilizza una versione modificata del ATPasi 6 mRNA che viene modellato a comportarsi come un ribozima su editing. Il ribozima funzionale poi specificamente ad un substrato RNA radiomarcato, che serve come reporter. Recentemente, Moshiri et al. Sviluppato un &#39;mescolare e misurare&#39; HHR-basato in vitro reporter per monitorare RNA editing dove il substrato RNA radiomarcato viene sostituito con un trasferimento di energia di risonanza di fluorescenza (FRET) substrato 19. I vantaggi principali di questo saggio sono: (a) è un mix rapido e conveniente e misura tipo di dosaggio, come la produzione di ribozima attivo e substrato fenditura si verificano simultaneamente nella stessa provetta a basso volume (cioè 20 microlitri), (b ) evita l&#39;uso di materiali marcati radioattivamente, (c) sensibilità che è unfforded da fluorescenza strumentazione in un formato di piastra di micro-titolazione, e (d) un alto rapporto segnale-rumore. Usando questo test, l&#39;effetto di inibitori noti RNA editing ligasi contro editosome purificato è stato confermato 19. Questo esperimento convalidato il test per la rapida identificazione di inibitori di RNA editing, soprattutto contro editosomes interi da T. brucei. La figura 1 è uno schema dettagliato passo-passo del saggio in vitro RNA editing basato sulla fluorescenza. Questo protocollo può essere utilizzato sia per il monitoraggio RNA editing in vitro o facilmente essere adattato per lo screening di librerie di composti di varie scale.

<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

Notevoli progressi sono stati compiuti nel determinare il meccanismo mitocondriale RNA editing in trypanosomes. Allo stesso modo, sono stati compiuti notevoli progressi nell&#39;identificazione dei componenti del complesso editosome che catalizzano RNA editing. Tuttavia, non è ancora chiaro come queste proteine ​​lavorino insieme. Composti chimici ottenuti da uno schermo ad alta velocità contro il editosome possono bloccare o influenzare una o più fasi del ciclo di editing. Pertanto, l&#39;identificazione di nuovi composti chimici genererà sonde molecolari importanti per sezionare la funzione editosome e assemblaggio. In studi precedenti, in vitro saggi di modifica in sono stati effettuati utilizzando RNA radio-marcato. Questi test sono molto tempo, inefficace e inadatto per scopi di high-throughput. Qui, una con sede a fluorescenza omogenea &quot;mix e misurare&quot; martello ribozyme in vitro giornalista per monitorare RNA editing, viene presentato. Solo come conseguenza di RNA editing diil ribozyme martello una risonanza di fluorescenza trasferimento di energia (FRET) substrato oligoribonucleotide subisce scissione. Questo a sua volta provoca la separazione del fluoroforo dal quencher producendo così un segnale. Al contrario, quando la funzione editosome è inibita, si spegnerà il segnale di fluorescenza. Questo è un test altamente sensibile e semplice che dovrebbe essere generalmente applicabile per monitorare in vitro RNA editing o high throughput screening delle sostanze chimiche che possono inibire la funzione editosome.

Per dimostrare i passi necessari per la creazione di uno schermo di grandi dimensioni, le figure 2-5 sono gli esperimenti rappresentativi di controllo relative alla qualità del test. Questi sono esperimenti di controllo essenziali per un dosaggio costante per diversi giorni di screening o per il confronto di diversi schermi. Valutare il segnale-rumore rapporto fluorescenza Per garantire la stabilità e la qualità del substrato ...

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 Catalyst come reporter per lo screening dei farmaci contro l&#39;RNA Editing in tripanosomi

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

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

Visualizing RNA Localization in Xenopus Oocytes

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

Visualizing RNA Localization in Xenopus Oocytes

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

Visualizzazione RNA localizzazione in Xenopus Gli oociti

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

Ricerca

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.

Trascrizione in vitro e tappatura mRNA Luciferase Gaussia Seguito da cellule HeLa Transfection

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 di cromatina Purificazione RNA (disturbo)

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.

Bioassay Electroantennographic come strumento di screening per Volatili piante ospiti

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.

Generazione Substrato per endonucleasi di CRISPR / Cas Sistemi

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;esaurimento delle RNA ribosomiale per metagenomiche 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; In situ</emIbridazione&gt; in embrioni Mount interi e istologia Adattato per Elasmobranchi Marine

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.

Una procedura di screening chimico per glucocorticoidi segnalazione con un reporter del sistema Zebrafish Larva 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.

Un metodo per lo screening e validazione di mutazioni resistenti contro 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 ...

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.

Rilevamento di RNA-binding proteins by<em&gt; In Vitro</em&gt; RNA a tendina nella cultura degli adipociti

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 ...