Ringraziamo i membri del laboratorio Tuschl per le discussioni utili. MH è supportato dal Deutscher Akademischer Austauschdienst (DAAD). Questo lavoro è stato sostenuto dal Fondo Nazionale Svizzero di Grant # 3100A0-114001 a MZ; TT è un investigatore HHMI, e il lavoro nel suo laboratorio è stato sostenuto da NIH GM073047 e MH08442 e la Fondazione Starr.

Il protocollo seguente descrive il PAR-Clip procedura per cellule HEK293 che esprimono FLAG / HA-tag IGF2BP1 su induzione con doxiciclina. Useremo un anti-FLAG anticorpo per immunoprecipitazione. PAR-Clip funziona con qualsiasi linea di cellule che esprimono livelli rilevabili di endogeno, senza tag legame con le proteine ​​RNA (RBP) di interessi se un anticorpo efficace per immunoprecipitazione è disponibile. Le cellule in espansione <ol><li> Espandere FlpIn-HEK293/TO/FLAG/HA-IGF2BP1 cellule in terreno di crescita. Si consiglia di utilizzare tra 100-400 x 10 6 cellule (circa 15 centimetri 10-40 piastre di coltura cellulare) come punto di partenza. Crescere a circa l&#39;80% confluenza. </li><li> 14 h prima di aggiungere un reticolazione) 4-thiouridine ad una concentrazione finale di 100 mM (1:1000 v / v di un 1 M 4-thiouridine soluzione madre) direttamente al mezzo di coltura cellulare e b) inducono l&#39;espressione del FLAG / HA tagged IGF2BP1 mediante aggiunta di 1 mg / ml di doxiciclina (1:10.000 v / v di 10 mg / ml soluzione madre doxiciclina). NOTA: al posto di 4-thiouridine è anche possibile utilizzare il 100 mM di 6-thioguanosine. </li></ol> UV-reticolazione <ol><li> Lavare le cellule una volta con 10 ml ghiacciata PBS per piastra e rimuovere completamente PBS. </li><li> Piastre posto su un vassoio con ghiaccio e irradiare scoperto con 0,15 J / cm 2 di 365 nm luce UV in un Stratalinker 2400 (Stratagene) o un dispositivo simile. </li><li> Raschiare le cellule con un poliziotto in gomma in 1 ml di PBS per piastra, trasferimento in tubi da 50 ml centrifugazione e raccogliere per centrifugazione a 500 xg per 5 minuti a 4 ° C e scartare il surnatante. 100 x 10 6 cellule HEK293 (10 15 lastre cm) produrrà circa. 1 ml di pellet umido. </li><li> (Opzionale) A meno che non si desidera continuare direttamente con la lisi cellulare, shock congelare il pellet di cellule in azoto liquido e conservare a -80 ° C. Pellet cellulare può essere conservato per almeno 12 mesi. </li></ol> Lisi cellulare e RNaseT1 digerire <ol><li> Raccogliere pellet cellulare delle cellule reticolato in 3 volumi di tampone di lisi 1x NP40 e incubare in ghiaccio per 10 minuti. </li><li> Lisato di cellule chiare per centrifugazione a 13.000 xg per 15 minuti a 4 ° C. </li><li> Cancella il lisato ulteriormente filtrandola attraverso una siringa da 0,2 micron filtro a membrana (Acrodisc Pall o equivalente). </li><li> Aggiungi RNasi T1 (Fermentas, 10.000 U / mL) a una concentrazione finale di 1 U / mL e incubare a bagnomaria per 15 minuti a 22 ° C. Reazione fredda successivamente per 5 minuti in ghiaccio prima di procedere. </li></ol> Immunoprecipitazione ed il recupero di frammenti di RNA bersaglio reticolato Utilizzando il separatore magnetico Seguire queste linee guida per tutta la preparazione del campione per evitare che il sfere magnetiche si secchi. <ol><li> Collocare la provetta contenente le perline sul supporto magnetico per 1 2 minuti. </li><li> Aggiungere la soluzione tampone al tubo mentre il tubo si trova sul separatore magnetico. </li><li> Chiudere la provetta, rimuoverlo dal separatore magnetico, e risospendere le sfere. Si può risospendere le sfere muovendo il tubo con le dita o utilizzare un vortex fissato a 5 6. </li><li> Centrifugare brevemente per raccogliere le sfere che possono rimanere nel tappo del tubo. </li><li> Ripetere i passaggi da 1 a 4 come richiesto. </li></ol> Preparazione di perline magnetiche <ol><li> Trasferire 10 ml di particelle di proteina G Dynabeads magnetico (Invitrogen) per cella ml di lisato (per un tipico esperimento che dovrebbe essere di ca. 40 50 ml di perline) in una provetta da microcentrifuga 1,5 ml. Lavare perle due volte con 1 ml di tampone citrato-fosfato. </li><li> Risospendere in doppio del volume di tampone citrato-fosfato rispetto al volume originario di sospensione tallone. </li><li> Aggiungi 0,25 mg di anti-FLAG anticorpi monoclonali M2 (Sigma) per ogni ml di sospensione e incubare su una ruota girevole per 40 minuti a temperatura ambiente. </li><li> Lavare perle due volte in 1 ml di tampone citrato-fosfato per rimuovere l&#39;anticorpo non legato. </li><li> Risospendere le sfere in doppio del volume di tampone citrato-fosfato rispetto al volume originario di sospensione tallone. </li></ol> Immunoprecipitazione (IP), secondo la digestione RNasi T1, e defosforilazione <ol><li> Aggiungere 20 ml di preparati al momento sfere magnetiche anticorpi coniugati per ml di lisato cellulare parziale RNasi T1 trattati e incubare in provette da 15 ml centrifugazione su una ruota girevole per 1 ora a 4 ° C. </li><li> Raccogliere sfere magnetiche su un collettore di particelle magnetiche per 15 e 50 tubi di centrifugazione ml (Invitrogen) e trasferimento in tubi da 1,5 ml microcentrifuga. </li><li> Lavare perle 3 volte in 1 ml di tampone di lavaggio IP. </li><li> Aggiungi RNaseT1 (Fermentas, 10.000 U / mL) a una concentrazione finale di 100 U / mL e incubare la sospensione tallone a bagnomaria per 15 minuti a 22 ° C. Raffreddare successivamente sul ghiaccioper 5 min. </li><li> Lavare perle 3 volte in 1 ml di alto sale tampone di lavaggio. </li><li> Perline risospendere in 1 volume di tampone defosforilazione </li><li> Aggiungi vitello fosfatasi alcalina intestinale (CIAP, NEB) ad una concentrazione finale di 0,5 U / mL, e incubare la sospensione per 10 minuti a 37 ° C. </li><li> Lavare perle due volte in 1 ml di tampone di lavaggio fosfatasi </li><li> Lavare perle due volte in polinucleotide chinasi (PNK) buffer senza digitale terrestre (DTT la concentrazione necessaria per la reazione enzimatica è abbastanza alta da danneggiare le sfere magnetiche). </li><li> Perline risospendere in un unico volume originale cordolo di PNK tampone </li></ol> Radiomarcatura reticolato di segmenti di RNA a proteine ​​immunoprecipitato <ol><li> Per la sospensione tallone descritto sopra, aggiungere Υ-32 P-ATP ad una concentrazione finale di 0,5 μCi / mL e T4 PNK (ONA) a 1 U / mL in un unico volume originale tallone. Incubare la sospensione per 30 minuti a 37 ° C. </li><li> Aggiungi non radioattivo ATP per ottenere una concentrazione finale di 100 mM e incubare per altri 5 minuti a 37 ° C. </li><li> Lavare le sfere magnetiche 5 volte con 800 ml di PNK buffer senza DTT. </li><li> Risospendere le sfere in 70 ml di SDS-PAGE tampone di caricamento. </li></ol> SDS-PAGE e electroelution reticolato di RNA-proteina complessi da fette di gel <ol><li> Incubare la sospensione radiomarcato per 5 minuti in un blocco di calore a 95 ° C per denaturare e rilasciare il RBP immunoprecipitati con RNA reticolato e vortice. </li><li> Rimuovere le sfere magnetiche sul separatore e trasferire il supernatante in una provetta da microcentrifuga pulita 1,5 ml. </li><li> Carico massimo di 40 ml di supernatante per pozzetto di una Novex Bis-Tris 4-12% (Invitrogen) gel di poliacrilammide prefabbricati ed eseguire il gel per 55 min a 200 V. </li><li> Smontare la camera di gel e smantellare il gel, lasciandolo montato su una piastra. Per facilitare l&#39;allineamento del gel alla lista su carta phosphorimager, si consiglia di impiantare tre pezzi minuscoli gel radioattivi asimmetrico a tre dei quattro angoli del gel. Pezzi gel radioattivi possono essere raccolti dal gel che in precedenza erano utilizzate per purificare radiomarcati oligonucleotidi sintetici. Avvolgere il gel in film plastico (ad esempio pellicola trasparente) per evitare contaminazioni. </li><li> Esporre il gel per uno schermo oscurato phosphorimager per 1 ora e visualizzare su un phosphorimager. </li><li> Allineare il gel sulla parte superiore della stampa phosphorimager utilizzando i pezzi di gel impiantati per l&#39;orientamento. Tagliare le bande che corrispondono alle dimensioni previste di RBP (IGF2BP1, ca. 75 kDa) e trasferirla in un D-Tube Tubo dializzatore Midi e aggiungere 800 microlitri 1x tampone SDS esecuzione. </li><li> Electroelute il reticolato RNA-RBP complesso in 1x SDS gestione del buffer a 100 V per 2 ore escisse dal gel e electroeluted in un D-Tube dializzatore Midi (Novagen) in 800 microlitri di buffer SDS esecuzione secondo le istruzioni del produttore. </li></ol> Proteinasi K digestione <ol><li> Aggiungere un volume equivalente di tampone 2x proteinasi K rispetto alla electroeluate, seguito con l&#39;aggiunta di proteinasi K (Roche) ad una concentrazione finale di 1,2 mg / ml. Incubare per 30 minuti a 55 ° C. </li><li> Recuperare l&#39;RNA da acido fenolo / cloroformio / IAA di estrazione (25:24:1, pH 4,0) seguita da un&#39;estrazione con cloroformio. Aggiungere 1 ml di glicogeno (10 mg / ml di brodo) precipitare l&#39;RNA con l&#39;aggiunta di 3 volumi di etanolo. Sciogliere il precipitato in acqua 10,5 microlitri. </li></ol> biblioteca preparazione cDNA e sequenziamento profondo Portare l&#39;RNA recuperato attraverso un protocollo standard cDNA preparazione biblioteca originariamente descritta per la clonazione dei piccoli RNA normativo 19. Il primo passo, la legatura adattatore 3 &#39;, è stata effettuata come descritto in una scala 20 l con 10,5 ml di RNA recuperato. Utilizzare il Solexa sequenziamento adattatore set descritto. A seconda della quantità di RNA recuperato, 5&#39;-adattatore-3&#39;-adattatore prodotti senza inserti possono essere rilevati dopo l&#39;amplificazione del cDNA come ulteriori bande PCR. In tali casi, il prodotto più accise PCR delle dimensioni previste dal NuSieve 3% a basso punto di fusione su gel di agarosio, eluire il prodotto di PCR dai pezzi gel con il kit GelElute (Qiagen) e sequenza utilizzando la tecnologia Solexa. Un Solexa sequenziamento eseguire offre di solito tra i 6 ei 10 milioni di sequenza si legge che sono sufficienti per una copertura trascrittoma gamma di siti di legame delle proteine ​​RNA vincolanti. Analisi bioinformatica Un&#39;attenta analisi bioinformatica della sequenza di legge deve essere fatto per ottenere la intuizioni significative nei siti RNA vincolante per la RBP esame, come l&#39;elemento di riconoscimento di RNA, le regioni preferito vincolante la RBP ha (exonic vs intronico, codifica sequenza vs non tradotte sequenza). La sequenza si legge devono essere allineati contro le banche dati del genoma e EST. Noi di solito utilizzare legge MAPPIng esclusivamente al genoma con fino a un disadattamento, inserimento o l&#39;eliminazione di costruire gruppi di sequenza si legge che possono poi essere ulteriormente analizzato. La frequenza di mutazioni caratteristiche del cluster sequenziato legge, T alle transizioni C quando si usa 4-SU e G ad A transizioni quando si utilizzano 6-SG, sono indicativi di sequenze con successo reticolato. Nella nostra esperienza RNA uncrosslinked etichettati con 4-SU mostrano un tasso di mutazione sfondo di circa il 20%. Questo tasso è aumenta di ca. 50-80% su reticolazione. Una descrizione dettagliata delle analisi bioinformatica può essere trovato nel materiale supplementare della pubblicazione da parte Hafner et al 18. Passi opzionali Determinazione dei livelli di incorporazione di 4-thiouridine in RNA totale Isolare RNA totale dalla linea cellulare che esprimono stabilmente la RBP di interesse dopo essere cresciuto in media integrato con 100 mM 4SU 16 ore prima del raccolto. Come controllo, le cellule raccolta coltivati ​​senza aggiunta 4SU. Isolare RNA totale con l&#39;aggiunta di 3 volumi di reagente Trizol (Sigma) al pellet cellulari lavato seguendo le istruzioni del produttore s. è stata ulteriormente purificare l&#39;RNA totale usando Qiagen RNeasy secondo il protocollo del produttore. Per prevenire l&#39;ossidazione del 4SU durante l&#39;isolamento di RNA e analisi, aggiungere 0,1 mM ditiotreitolo (DTT) a tamponi di lavaggio e successive reazioni enzimatiche. Digerire e defosforilata RNA totale di nucleosidi singolo per l&#39;analisi HPLC come descritto in precedenza 20. In breve, in un volume di 30 microlitri, incubare a 40 mcg di RNA totale sono stati purificati per 16 ore a 37 ° C con 0,4 U fosfatasi alcalina batterica (Biochemical Worthington) e 0,09 veleno di serpente U fosfodiesterasi (Worthington Biochemical). Come standard di riferimento, utilizzare una sintesi di RNA 4SU marcato, (siamo in standard uso CGUACGCGGAAUACUUCGA (4SU) U) e anche soggetti per completare la digestione enzimatica. Separare le miscele risultante di ribonucleosides mediante HPLC su Discovery Supelco C18 (particelle di silice legata fase mM 5, 250 x 4,6 mm) colonna a fase inversa (Bellefonte PA, USA). Buffer HPLC sono 0,1 M TEAA nel 3% acetonitrile (A) e il 90% acetonitrile in acqua (B). Utilizzare un gradiente isocratica: 0% B per 15 minuti, da 0 a 10 B% per 20 minuti, dal 10 al 100% B per 30 min. Applicare un min 5 100% B lavare applicata tra le esecuzioni per pulire la colonna HPLC. Rappresentante Risultati <img src="/files/ftp_upload/2034/2034fig1.jpg" alt="Figura 1" /> Figura 1 (pannello di destra) mostra un risultato rappresentativo di un PAR-Clip eseguita con linee cellulari che esprimono FLAG / HA-tag IGF2BP1 con 4-SU e 6-SG. Si noti che l&#39;efficienza reticolazione di 6-SG per IGF2BP1 è inferiore al rendimento reticolazione per 4-SU. L&#39;efficienza reticolazione inferiore si tradurrà in un contesto più elevato di sequenze derivate da abbondanti frammenti di RNA cellulare e quindi si dovrebbe considerare di scala l&#39;esperimento quando si utilizzano nucleosidi fotoreattivo meno efficienti. Il pannello di sinistra della figura 1 mostra un confronto di utilizzare diversi analoghi uridina fotoreattivo che potrebbero essere potenzialmente utilizzate per PAR-Clip rispetto ai tradizionali reticolazione UV 254 nm. L&#39;intensità della banda radioattiva della lunghezza corretta che dà una buona idea se il PAR-Clip esperimento ha funzionato e che è stato isolato l&#39;RNA sufficiente per portare a termine una piccola sequenza di RNA protocollo (step-by-step descrizione per la preparazione di piccole librerie di cDNA sequenziamento RNA può essere trovato in 19). La frequenza di mutazioni caratteristiche della legge in sequenza, T alle transizioni C quando si usa 4-SU e G ad A transizioni quando si utilizzano 6-SG, sono indicativi di sequenze con successo reticolato. Nella nostra esperienza RNA uncrosslinked etichettati con 4-SU mostrano un tasso di mutazione sfondo di circa il 20%. Questo tasso è aumenta di ca. 50-80% su reticolazione.

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base+composition+analysis+of+nucleosides+using+HPLC.">Base composition analysis of nucleosides using HPLC.</a> Current Protocols in Nucleic Acid Chemistry. Chapter 10 (Unit 10.6), (2001).</li></ol>

TT è una co-fondatore e consulente scientifico per Alnylam Pharmaceuticals e consulente di Regulus Therapeutics.

Tamponi e reagenti Crescita media cellule HEK293 <ul><li> DMEM </li><li> 10% FBS </li><li> 2 mM L-Glutammina </li><li> 100 U / ml di penicillina </li><li> 100 U / ml streptomicina </li><li> 100 mg / ml Hygromycin </li><li> 15 mg / ml blasticidin </li></ul> 4-thiouridine soluzione madre (1 M) <ul><li> 260,27 mg 4-thiouridine </li><li> 1 ml di DMSO </li></ul> Doxycyclin magazzino (10 mg / ml) <ul><li> 10 mg doxycyclin </li><li> 1 ml di DMSO </li></ul> 1x NP40 buffer di lisi Preparare uno stock di tampone 5x senza DTT e inibitori della proteasi. Aggiungi DTT e inibitore della proteasi direttamente prima dell&#39;esperimento. <ul><li> HEPES 50 mM, pH 7,5 </li><li> 150 mM KCl </li><li> 2 mM EDTA </li><li> 1 mM NaF </li><li> 0,5% (v / v) NP40 </li><li> 0,5 mM DTT </li><li> completa EDTA senza inibitore della proteasi cocktail (Roche) </li></ul> Tampone citrato-fosfato <ul><li> 4,7 g / l di acido citrico </li><li> 9,2 g / l Na 2 HPO 4 </li><li> pH 5,0 </li></ul> IP-tampone di lavaggio <ul><li> 50 mM HEPES-KOH, pH 7,5 </li><li> 300 mM KCl </li><li> 0,05% (v / v) NP40 </li><li> 0,5 mM DTT (aggiungere direttamente prima dell&#39;esperimento) </li><li> completa EDTA senza inibitore della proteasi cocktail (Roche) (aggiungere direttamente prima dell&#39;esperimento) </li></ul> High-sale tampone di lavaggio <ul><li> 50 mM HEPES-KOH, pH 7,5 </li><li> 500 mM KCl </li><li> 0,05% (v / v) NP40 </li><li> 0,5 mM DTT (aggiungere direttamente prima dell&#39;esperimento) </li><li> completa EDTA senza inibitore della proteasi cocktail (Roche) (aggiungere direttamente prima dell&#39;esperimento) </li></ul> Defosforilazione tampone <ul><li> 50 mM Tris-HCl, pH 7,9 </li><li> 100 mM NaCl </li><li> 10 mM MgCl 2 </li><li> 1 mM DTT </li></ul> Fosfatasi tampone di lavaggio <ul><li> 50 mM Tris-HCl, pH 7,5 </li><li> 20 mM EGTA </li><li> 0,5% (v / v) NP40 </li></ul> Polinucleotide chinasi (PNK) buffer senza DTT <ul><li> 50 mM Tris-HCl, pH 7,5 </li><li> 50 mM NaCl </li><li> 10 mM MgCl 2 </li></ul> PNK tampone <ul><li> 50 mM Tris-HCl, pH 7,5 </li><li> 50 mM NaCl </li><li> 10 mM MgCl 2 </li><li> 5 mM DTT </li></ul> SDS PAGE tampone di caricamento <ul><li> 10% glicerolo (v / v) </li><li> 50 mM Tris-HCl, pH 6.8 </li><li> 2 mM EDTA </li><li> 2% SDS (w / v) </li><li> 100 mM DTT </li><li> 0,1% bromofenolo blu </li></ul> 2x proteinasi K tampone <ul><li> 100 mM Tris-HCl, pH 7,5 </li><li> 150 mM NaCl </li><li> 12,5 mm EDTA </li><li> 2% (w / v) di SDS </li></ul>

par-clip - a method to identify transcriptome-wide the binding sites of rna binding proteins

Trascrizioni di RNA sono sottoposti a regolazione post-trascrizionale genica interagendo con centinaia di RNA-binding proteins (RBPs) e microRNA contenenti complessi ribonucleoproteina (miRNPs) che sono spesso espressi in una cellula di tipo dipendente. Per capire come l&#39;interazione di questi RNA-binding fattori influisce sulla regolazione della trascrizione individuale, mappe ad alta risoluzione in vivo le interazioni proteina-RNA sono necessari 1. Una combinazione di fattori genetici, biochimici e approcci computazionali sono generalmente applicati per individuare l&#39;RNA-RBP o RNA-RNP interazioni. Profiling microarray di RNA associate a RBPs immunopurified (RIP-Chip) 2 definisce gli obiettivi a livello del trascrittoma, ma la sua applicazione è limitata alla caratterizzazione delle interazioni cineticamente stabile e solo in rari casi 3,4 permette di identificare l&#39;elemento RBP riconoscimento (RRE ) all&#39;interno del RNA bersaglio lungo. Più diretto obiettivo informazioni RBP sito è ottenuto dalla combinazione di reticolazione UV 5,6 vivo con immunoprecipitazione 7-9 seguito da l&#39;isolamento reticolato di segmenti di RNA e sequenziamento del DNA (CLIP) 10. CLIP è stato utilizzato per identificare gli obiettivi di un certo numero di RBPs 11-17. Tuttavia, CLIP è limitata dalla scarsa efficienza dei raggi UV 254 nm RNA-proteina reticolazione, e la posizione della reticolazione non è facilmente individuabile all&#39;interno dei frammenti sequenziati reticolato, il che rende difficile separare reticolati UV bersaglio segmenti di RNA da sfondo non reticolati anche frammenti di RNA presenti nel campione. Abbiamo sviluppato una potente cellula approccio basato reticolazione a determinare in alta risoluzione e trascrittoma livello i siti di legame di RBPs cellulare e miRNPs che chiamiamo PAR-Clip (Photoactivatable-ribonucleosidi-Enhanced reticolazione e immunoprecipitazione) (vedi fig. 1A per uno schema del metodo). Il metodo si basa sulla costituzione di analoghi ribonucleosidi fotoreattivo, come thiouridine 4-(4-SU) e 6-thioguanosine (6-SG) nella nascente trascrizioni di RNA da cellule viventi. L&#39;irradiazione delle cellule dalla luce UV di 365 nm induce la reticolazione efficiente delle fotoreattivo nucleosidi marcati RNA cellulare RBPs interagenti. Immunoprecipitazione della RBP di interesse è seguita da isolamento del RNA reticolato e coimmunoprecipitated. L&#39;RNA isolato è trasformata in una libreria di cDNA e profondo in sequenza usando la tecnologia Solexa. Una caratteristica di librerie cDNA preparati da PAR-Clip è che la posizione precisa di reticolazione può essere identificata da mutazioni che risiedono nella sequenza del DNA. Quando si utilizza 4-SU, sequenze reticolato timidina di transizione citidina, mentre l&#39;uso di 6-SG risultati in guanosina di adenosina mutazioni. La presenza di mutazioni in sequenze reticolato rende possibile la loro separazione dallo sfondo di sequenze derivate da abbondanti RNA cellulare. Applicazione del metodo ad una serie di diverse proteine ​​RNA binding è stata riportata in Hafner et al 18.

Watch this Scientific Journal Video about PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins at JoVE.com

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

Trascrizioni di RNA sono soggetti ad ampie regolamentazione posttranscriptional che è mediato da una moltitudine di trans-acting RNA-binding proteins (RBPs). Qui vi presentiamo un metodo generalizzabile per identificare con precisione e trascrittoma su scala i siti di legame di RNA RBPs.

PAR-Clip - un metodo per identificare trascrittoma livello i siti di legame delle proteine ​​RNA binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

par-clip-method-to-identify-transcriptome-wide-binding-sites-rna

Research

JoVE Journal

Biology

53.2K Views.  Rockefeller University. Trascrizioni di RNA sono soggetti ad ampie regolamentazione posttranscriptional che è mediato da una moltitudine di trans-acting RNA-binding proteins (RBPs). Qui vi presentiamo un metodo generalizzabile per identificare con precisione e trascrittoma su scala i siti di legame di RNA RBPs.

Video: PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentazione di analisi, per l&#39;Analisi del Binding Protein a F-Actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Ricerca

Biologia

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteomica per identificare proteine ​​che interagiscono con P2X2 Ligand-Gated canali di comunicazione

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profilatura di metiltransferasi e altri S-Adenosil- L-Omocisteina-binding proteine ​​mediante spettrometria di massa Compound Capture (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Raccolta variabile concentrazione dataset Calorimetria isotermica di titolazione per determinare meccanismi vincolanti

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iClip - trascrittoma livello Mappatura delle interazioni proteina-RNA con risoluzione individuale Nucleotide

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Pull-down di Calmodulina proteine ​​leganti

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificato lievito-sistema del doppio ibrido per identificare proteine ​​che interagiscono con il fattore di crescita progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Protein Purification-libera Metodo di Binding Affinity Determinazione Thermophoresis microscala

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Un metodo di quantificazione sensibile e specifico per la determinazione del siero miosina cardiaca Binding Protein-C da elettrochemiluminescenza immunoenzimatico

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

PAR-Clip - un metodo per identificare trascrittoma livello i siti di legame delle proteine ​​RNA binding

PAR-Clip - un metodo per identificare trascrittoma livello i siti di legame delle proteine RNA binding