Name: detection of alternative splicing during epithelial-mesenchymal transition
Uploaded: 2014-10-09T14:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

1 Cella Cultura della EMT induzione Nota: EMT può essere indotta da trattamenti di TGFβ o espressione ectopica di fattori di trascrizione Twist, Chiocciola, o Zeb1 / 2 in cellule epiteliali. Descritto in questo protocollo è un sistema EMT inducibile mediante espressione della proteina di fusione Twist-ER in cellule immortalizzate umane epiteliali mammarie (HMLE / Twist-ER, un dono di Dr. J Yang, UCSD) 9,11,29. Al 4-hydroxytamoxifen (TAM) il trattamento, la proteina di fusione Twist-ER trasloca nel nucleo di guidare la trascrizione e risultati in una transizione completa EMT in 12-14 giorni 9,11,29. Altri sistemi inducibili EMT, come HMLE / Snail-ER, HMLE / TGFβ, e MCF10A / TGFβ, si possono trovare nelle nostre precedenti pubblicazioni 11,30. <ol><li> Mantenere le cellule HMLE / Twist-ER in mammaria mezzo di crescita delle cellule epiteliali privo di siero (MEGM) in un incubatore a 37 ° C con il 5% di CO 2. Passage le cellule ogni 2-3 giorni quando raggiungono l&#39;80% di confluenza. Incubare le cellule con tripsina 0,15% a 37 ° C per 5-10 minuti, quindi inattivare la tripsina con siero di vitello al 5% / DMEM. Spin le cellule e risospendere in MEGM. Cellule portapiatti in un nuovo piatto di coltura tissutale con un rapporto di 1 a 4. </li><li> Sciogliere TAM in etanolo per ottenere una soluzione 200 mM. Proteggere TAM dalla luce e conservare a -20 ° C. Prima dell&#39;uso, appena aggiungere TAM in MEGM materie ad una diluizione 1: 10.000 per effettuare una concentrazione finale di 20 nM TAM. </li><li> Per l&#39;induzione di EMT, piastra cellule 1,6 x 10 6 HMLE / Twist-ER in un 10 centimetri piatto di coltura di tessuti in duplice copia. Celle di coltura con 20 supporti nM TAM-contenenti MEGM. </li><li> Piatto cellule 1,6 x 10 6 HMLE / Twist-ER in un piatto di 10 centimetri in duplice copia e il trattamento di cellule con 0,01% di etanolo come controllo non-TAM-trattati. </li><li> Due giorni dopo l&#39;incubazione, scattare foto sotto un microscopio ottico a 10X di ingrandimento per registrare cambiamenti morfologici delle cellule. </li><li> Aspirare il mezzo da un piatto di controllo o di TAM-trattaticellule. Lavare le cellule con PBS freddo e celle raccolto da demolizione metà della piastra in tampone di lisi RNA per l&#39;isolamento dell&#39;RNA e l&#39;altra metà in tampone RIPA per l&#39;analisi di proteine. </li><li> Cellule passaggio dal controllo o piatti TAM-trattati con 1,6 x 10 6 cellule ri-placcatura in un piatto di 10 centimetri in duplice copia. Continuamente trattare le cellule con 20 nM TAM o di un veicolo. </li><li> Ripetere i passaggi da 5 a 7 una volta ogni due giorni fino al giorno 14, in cui il tempo, le cellule Twist-ER TAM-trattati mostrano una mesenchimale morfologia fusiforme, mentre le cellule di controllo del veicolo trattate mantengono la morfologia epiteliale ciottoli-like. </li></ol> 2 Caratterizzazione di EMT Nota: A completamento della EMT è indicato da: (1) cambiamenti morfologici delle cellule da una morfologia epiteliale ciottoli come a un aspetto fibroblastica-come a forma di fuso; (2) Perdita di localizzazione E-caderina a giunzioni cellula-cellula; e (3) L&#39;espressione commutazione di marcatori EMT, definito dalla perditadi marcatori epiteliali e aumento dei marcatori mesenchimali. Cellulari cambiamenti morfologici vengono acquisite da microscopio ottico a 10X di ingrandimento nel corso del tempo di induzione EMT, descritto nella Sezione 1 Immuno-fluorescenza rilevamento di E-caderina in cellule-giunzioni è descritto in questa sezione. L&#39;espressione di markers di EMT viene rilevato mediante immunoblotting. Marcatori epiteliali comuni sono E-caderina, γ-catenina, e occludina, e marcatori mesenchimali includono fibronectina, N-caderina e vimentina. Procedure generali di immunoblotting sono descritti nella sezione 4. <ol><li> Al giorno 12 del trattamento TAM, semi di 1 x 10 5 cellule su un vetro coprioggetti circolare 12 millimetri disposte in un pozzetto di una piastra da 24 pozzetti. </li><li> 48 ore dopo che le cellule di semina, medie Aspirare e lavare con pre-riscaldato PBS. </li><li> Fissare le cellule con pre-riscaldato paraformaldeide 4% per 15 min. </li><li> Lavare le cellule tre volte con PBS. </li><li> Incubare le cellule con 0,2% Triton X-100 in PBS per 15minuti a temperatura ambiente. </li><li> Lavare le cellule tre volte con PBS. </li><li> Incubare le cellule a 5% BSA / PBS per 1 ora a temperatura ambiente per bloccare legame non specifico. </li><li> Incubare le cellule con l&#39;anticorpo E-caderina primario in una diluizione 1:50 in 1% BSA / PBS in una camera di umidificata O / N a 4 ° C. NOTA: L&#39;intervallo per la diluizione anticorpo primario è normalmente compreso tra 1:50 e 1: 200, e la diluizione ottimale per ogni anticorpo deve essere determinato sperimentalmente. </li><li> Decantare l&#39;anticorpo primario e lavare le cellule tre volte con PBS. </li><li> Incubare le cellule con l&#39;anticorpo secondario fluorescente marcato in una diluizione 1: 500 per 1 ora a temperatura ambiente. Da questo punto in poi, proteggere le cellule dalla luce. </li><li> Decantare l&#39;anticorpo secondario e lavare tre volte con PBS. </li><li> Cellule macchia con 0,1-1 g / ml DAPI o Hoechst per 10 min. </li><li> Lavare le cellule con PBS per tre volte. </li><li> Montare coprioggetto con una goccia di mezzo di montaggio, e coprioggetto tenuta con smalto. </li><li> Osservare le cellule e prendereimmagini sotto un microscopio a fluorescenza 63X. </li></ol> Colorazione con anticorpi supplementari può essere eseguita per confermare il fenotipo EMT. Ad esempio, N-caderina e α-actina del muscolo liscio possono essere colorati per un fenotipo mesenchimale 29,31,32. Riorganizzazione della struttura del citoscheletro può essere monitorato mediante colorazione delle cellule con falloidina per la F-actina 11. Inoltre, saggi di motilità cellulare e la resistenza delle cellule morte possono essere eseguite per esaminare le proprietà del EMT 11. 3 Rilevamento di Splice Isoforme Utilizzo qRT-PCR saggi <ol><li> Disegno Primer NOTA: Le coppie di primer deve essere accuratamente studiato per amplificare isoforme di splicing distinti. Exon skipping, l&#39;evento splicing alternativo più comunemente osservato, è illustrata qui come esempio per illustrare la strategia di progettazione di primer. Figura 1A mostra una quattro esone pre-mRNA, con il terzo esone come esone variabile. L&#39;inclusione dell&#39;esone 3 risultati inla produzione di un giunto isoforma più lunga, mentre skipping dell&#39;esone 3 genera una giuntura isoforma più corta. I primer devono essere progettati per distinguere l&#39;isoforma esone-3-incluso e esone-3-saltato isoforma, e per rilevare la trascrizione completa della mRNA. <ol><li> Per il rilevamento di inclusione dell&#39;esone, progettare con cura un primer in avanti all&#39;interno della variabile esone 3, e un primer reverse all&#39;interno della vicina esone costitutiva 4, permettendo l&#39;amplificazione specifica della variabile isoforma esone-incluso. Assicurarsi di non progettare sia in avanti e reverse primer all&#39;interno dello stesso esone variabile al fine di evitare i prodotti della PCR amplificati a partire dal DNA genomico o pre-mRNA. </li><li> Per amplificare specificamente esone eventi saltare, di design uno dei primer che abbracciano la giunzione dell&#39;esone costitutiva 2 e esone 4 che altrimenti sarebbe distrutto quando esone variabile 3 è incluso. Progettare l&#39;altro fondo all&#39;interno del esone costitutiva 4 Come tale, i prodotti di PCR vengono generati solo quando la variabile esone 3 è excluded e la costitutiva esone 2 e l&#39;esone 4 sono successivamente uniti. </li><li> Per rilevare le trascrizioni totali del gene, disegnare i primer entro due esoni costitutivi 1 e 2 Pertanto, i prodotti di PCR sono amplificati da tutte le isoforme. </li><li> Assicurarsi che la temperatura di fusione (Tm) per tutti i primer è di circa 55 ° C, la lunghezza di ogni primer è di circa 18-22 nucleotidi, e la dimensione dei prodotti di PCR è compresa tra 80 e 150 paia di basi. </li></ol></li><li> Estrazione di RNA: RNA estratto utilizzando un kit di totale isolamento di RNA (vedi tabella dei Materiali). <ol><li> Raccogliere le cellule in 350 microlitri di buffer di lisi RNA. </li><li> Aggiungere 350 ml di etanolo al 70% al lisato e mescolare accuratamente. </li><li> Trasferire il campione alla colonna di purificazione dell&#39;RNA. </li><li> Centrifugare a 10.000 xg per 1 min e scarto flow-through. </li><li> Lavare la colonna una volta con 500 ml di RNA di lavaggio buffer di I e due volte con RNA tampone di lavaggio II. </li><li> Rimuovere il tampone di lavaggio RNA residuoII dalla colonna mediante centrifugazione a velocità massima per 2 min. </li><li> Trasferire la colonna in una nuova provetta da 1,5 ml, aggiungere acqua priva di nucleasi 50 microlitri al centro della matrice colonna ed eluire l&#39;RNA per centrifugazione a velocità massima. </li></ol></li><li> Sintesi di cDNA First-strand <ol><li> Determinare la concentrazione e la qualità di RNA con la misura di assorbimento UV. NOTA: Buona RNA qualità dovrebbe avere un / 280 nm rapporto di assorbanza 260 nm di circa 2.0. </li><li> Impostare trascrittasi inversa (RT) reazioni che contengono 250 ng di RNA totale, 0,05 mg primer esameriche casuale, 50 pmol MgCl 2, 10 pmol dNTP, 2 microlitri di buffer 5 × GoScript, e 1 ml RT enzima in un volume finale di 20 ml. </li><li> Eseguire reazioni RT a 42 ° C per 1 ora, seguito da incubazione a 70 ° C per 5 minuti per inattivare l&#39;enzima RT. </li></ol></li><li> Real-time PCR <ol><li> Impostare 20 microlitri reazioni che consistono di 10 microlitri SYBR master mix verde,0.1-1.0 modello cDNA microlitri, e 5 pmol avanti e indietro primer. </li><li> Eseguire PCR in tempo reale con 40 cicli dei seguenti passi: 95 ° C denaturazione per 10 sec, 58 ° C ricottura per 10 sec e 60 ° C per 30 sec estensione. Eseguire reazioni PCR in triplice copia. </li><li> Controllare le curve di dissociazione e fare in modo che un singolo picco si osserva per ogni set di primer. </li><li> Ottenere i valori di ciclo di quantificazione (Cq) calcolando i secondi valori derivati ​​curve di amplificazione. </li><li> Calcolare la quantità relativa (RQ) del mRNA bersaglio in campioni indotto, rispetto al campione uninduced utilizzando il &#39;delta delta Cq (ΔΔCq)&#39; metodo come mostrato dalla seguente formula. Utilizzare geni housekeeping, come GAPDH o TBP, come riferimento. </li></ol></li></ol><img alt="Equazione 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> Per calcolare con maggiore precisione i valori relativi delle isoforme di splicing, tegli usa di più geni di riferimento dovrebbe essere considerato. I risultati possono essere analizzati utilizzando un metodo di quantificazione del valore assoluto modificato descritto da Pfaffl et al 33,34. 4 Esame di Splice Isoforme a livello proteico <ol><li> Raccogliere le cellule in 100 microlitri di buffer RIPA ghiacciato. </li><li> Impostare lisati su ghiaccio per circa 10 min. </li><li> Centrifugare a 14000 g per 10 min a 4 ° C, e raccogliere il surnatante. </li><li> Misurare la concentrazione di proteine ​​mediante saggi Bradford. </li><li> Diluire i campioni di proteine ​​in tampone di caricamento SDS e caricare 20-40 mg di proteine ​​in gel 10% SDS-PAGE. </li><li> Eseguire gel SDS-PAGE a 20 mA per 1.5 ore. </li><li> Trasferimento proteine ​​di membrane PVDF in un sistema di trasferimento umida a 4 ° C per 3 ore a 85 V. Regolare il tempo di trasferimento secondo il peso molecolare delle proteine ​​bersaglio. </li><li> Membrana blocco in 5% di latte senza grassi per 30 minuti a 1 ora a temperatura ambiente. </li><li> Incubare la membrana with un anticorpo primario a 4 ° CO / N. La gamma di diluizione anticorpo primario è normalmente compresa tra 1: 200 e 1: 5.000, determinare la diluizione ottimale per ciascun anticorpo sperimentalmente. La diluizione di anticorpi utilizzati in questo protocollo può essere trovato in &quot;Tabella di reagenti e attrezzature specifiche&quot;. <ol><li> Per rilevare isoforme di splicing, utilizzare anticorpi specifici che riconoscono una particolare isoforma. In alternativa, utilizzare un anticorpo che riconosce l&#39;epitopo nella costitutiva esone regione codificante e rilevare le isoforme di splicing simultaneamente in base alle loro dimensioni diverse proteine. </li><li> Allo stesso tempo, monitorare EMT mediante immunoblotting per i marcatori di EMT. Utilizzare E-caderina, γ-catenina, e occludina come marcatori epiteliali, e fibronectina, N-caderina e vimentina come marcatori mesenchimali. </li></ol></li><li> Membrana Lavare tre volte con TBS-T (50 mM Tris, 150 mM NaCl, 0,05% Tween 20, pH 7.4) in un intervallo di 5 min. </li><li> Incubare la membrana con una formica secondario HRP-coniugatoiBody in una diluizione 1: 10.000 per 1 ora a RT. </li><li> Membrana Lavare tre volte con TBS-T in un intervallo di 5 min. </li><li> Visualizzare proteina con un sistema di rilevazione chemiluminescenza ed esporre la membrana di un film autoradiografia. </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+mutations+in+the+U2AF1+splicing+factor+in+myelodysplastic+syndromes.">Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes.</a> Nat Genet. 44, 53-57 (2012).</li><li>Papaemmanuil, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+SF3B1+mutation+in+myelodysplasia+with+ring+sideroblasts.">Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.</a> N Engl J Med. 365, 1384-1395 (2011).</li><li>Yoshida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+pathway+mutations+of+splicing+machinery+in+myelodysplasia.">Frequent pathway mutations of splicing machinery in myelodysplasia.</a> Nature. 478, 64-69 (2011).</li><li>Mani, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial-mesenchymal+transition+generates+cells+with+properties+of+stem+cells.">The epithelial-mesenchymal transition generates cells with properties of stem cells.</a> Cell. 133, 704-715 (2008).</li><li>Reinke, L. M., Xu, Y., Cheng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snail+represses+the+splicing+regulator+epithelial+splicing+regulatory+protein+1+to+promote+epithelial-mesenchymal+transition.">Snail represses the splicing regulator epithelial splicing regulatory protein 1 to promote epithelial-mesenchymal transition.</a> J Biol Chem. 287, 36435-36442 (2012).</li><li>Serini, G., Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+myofibroblast+activity+and+phenotypic+modulation.">Mechanisms of myofibroblast activity and phenotypic modulation.</a> Exp Cell Res. 250, 273-283 (1999).</li><li>Zavadil, J., Bottinger, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+and+epithelial-to-mesenchymal+transitions.">TGF-beta and epithelial-to-mesenchymal transitions.</a> Oncogene. 24, 5764-5774 (2005).</li><li>Pfaffl, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nucleic Acids Res. 29, e45 (2001).</li><li>Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., Vandesompele, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=qBase+relative+quantification+framework+and+software+for+management+and+automated+analysis+of+real-time+quantitative+PCR+data.">qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data.</a> Genome Biol. 8, R19 (2007).</li><li>Boutz, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+post-transcriptional+regulatory+switch+in+polypyrimidine+tract-binding+proteins+reprograms+alternative+splicing+in+developing+neurons.">A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons.</a> Genes Dev. 21, 1636-1652 (2007).</li><li>Salomonis, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+regulates+mouse+embryonic+stem+cell+pluripotency+and+differentiation.">Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation.</a> Proc Natl Acad Sci USA. 107, 10514-10519 (2010).</li><li>Calarco, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vertebrate+nervous+system+alternative+splicing+and+development+by+an+SR-related+protein.">Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein.</a> Cell. 138, 898-910 (2009).</li><li>Grabowski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+takes+shape+during+neuronal+development.">Alternative splicing takes shape during neuronal development.</a> Curr Opin Genet Dev. 21, 388-394 (2011).</li><li>Seol, D. W., Billiar, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+caspase-9+variant+missing+the+catalytic+site+is+an+endogenous+inhibitor+of+apoptosis.">A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis.</a> J Biol Chem. 274, 2072-2076 (1999).</li><li>Srinivasula, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+endogenous+dominant-negative+short+isoform+of+caspase-9+that+can+regulate+apoptosis.">Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis.</a> Cancer Res. 59, 999-1002 (1999).</li><li>Akgul, C., Moulding, D. A., Edwards, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+Bcl-2-related+genes+functional+consequences+and+potential+therapeutic+applications.">Alternative splicing of Bcl-2-related genes functional consequences and potential therapeutic applications.</a> Cell Mol Life Sci. 61, 2189-2199 (2004).</li><li>Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+minigene+systems+to+dissect+alternative+splicing+elements.">Use of minigene systems to dissect alternative splicing elements.</a> Methods. 37, 331-340 (2005).</li><li>Cheng, C., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CD44+alternative+splicing+by+SRm160+and+its+potential+role+in+tumor+cell+invasion.">Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion.</a> Mol Cell Biol. 26, 362-370 (2006).</li></ol>

The authors declare that they have no competing financial interests.

La procedura qui descritta consente il rilevamento di splicing alternativo in un modello EMT inducibile. Come tali, le alterazioni dinamiche di espressione delle isoforme di splicing possono essere catturate nel corso tempo di EMT. Questo metodo ha vantaggi rispetto all&#39;uso di epithelial- diverso o linee di cellule mesenchimali-che rappresenta per il confronto di splicing alternativo, perché background genetici distinti da linee cellulari connessi alle Nazioni Unite potrebbero influenzare indebitamente splicing alt...

La transizione epitelio-mesenchimale (EMT) è un programma di sviluppo che guida la morfogenesi di organi e rimodellamento tissutale durante l&#39;embriogenesi. Quando anormalmente attivato, EMT promuove la metastasi del tumore e degli organi fibrosi 1,2. Studi convincenti hanno descritto l&#39;importanza della regolazione trascrizionale durante il processo di EMT, definito da diversi fattori di trascrizione, come il Twist, Lumaca, e ZEB, che reprimono l&#39;espressione della proteina di giunzione adherens E-caderina, con conseguente perdita di un cobble- pietra come morfologia epiteliale e il guadagno di un a forma di fuso fenotipo mesenchimale 3-8. Recenti studi attraverso l&#39;analisi dell&#39;intero genoma di RNA hanno rivelato che esiste un gruppo di geni i cui modelli di splicing sono associati sia con fenotipi epiteliali e mesenchimali 9,10. Il lavoro dal nostro laboratorio funzionalmente collegato splicing alternativo e EMT. Studiando la superficie cellulare molecola di adesione CD44, abbiamo dimostrato che il CD44 Alternative splicing è strettamente regolata durante EMT, e più importante, che CD44 giuntura isoforma passaggio causalmente contribuisce alla EMT 11. Splicing alternativo rappresenta un modello diffuso e conservato di regolazione genica, in quanto fino al 95% dei geni multi-esone umani sono splicing alternativo 12-14. Generando più prodotti proteici da un singolo gene, splicing alternativo costituisce un meccanismo essenziale per la diversità di proteine, aggiungendo un ulteriore livello di complessità al genoma umano. Come tale, disregolazione di splicing alternativo potrebbe potenzialmente portare a profondi effetti biologici che causano malattie umane. Infatti, aberrante splicing alternativo nelle malattie è stato documentato da oltre un decennio 15-25, compresi recenti scoperte che le mutazioni in geni che codificano per le macchine spliceosoma trovano comunemente nelle sindromi mielodisplastiche 26-28. Pertanto, lo sviluppo di metodi affidabili per la rilevazione di i splicing alternativosoforms è di grande importanza nello studio dei processi biologici diversi, tra cui EMT. Qui forniamo un protocollo per rilevare i cambiamenti nella splicing alternativo utilizzando un modello di EMT inducibile. I metodi per la progettazione di primer PCR e rilevazione isoforme di splicing sono adatte non solo per lo studio di splicing alternativo durante EMT, ma anche per lo studio di splicing alternativo in altri processi biologici. Indagare splicing alternativo durante EMT è indispensabile al fine di comprendere meglio i meccanismi di EMT e metastasi tumorali, facilitando così lo sviluppo di strategie efficaci per il trattamento di metastasi del cancro.

<table><tbody><tr><td>4-hydroxytamoxifen</td><td>Sigma</td><td>H7904</td><td>Make stock solution by dissolving 4-hydroxytamoxifen in ethanol to 200 &amp;#956;M. Keep the solution at -20 &amp;#176;C protected from light.</td></tr><tr><td>E.Z.N.A. Total RNA Isolation kit</td><td>Omega Bio-Tek</td><td>R6731</td><td>Total RNA isolation kit</td></tr><tr><td>GoScript Reverse Transcription System</td><td>Promega</td><td>A5001</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6002</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>LightCycler 480 Real-Time PCR System</td><td>Roche</td><td></td><td>Equipment for qRT-PCR assay</td></tr><tr><td>CD44 antibody</td><td>R&amp;amp;D Systems</td><td>BBA10</td><td>1:1,000 dilution</td></tr><tr><td>E-cadherin antibody</td><td>Cell Signaling Technology</td><td>4065</td><td>1:2,500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td></tr><tr><td>&amp;gamma;-Catenin antibody</td><td>Cell Signaling Technology</td><td>2309</td><td>1:1,000 dilution</td></tr><tr><td>Occludin antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-5562</td><td>1:500 dilution</td></tr><tr><td>Fibronectin antibody</td><td>BD Transduction Laboratories</td><td>610077</td><td>1:5,000 dilution</td></tr><tr><td>N-cadherin antibody</td><td>BD Transduction Laboratories</td><td>610920</td><td>1:2,000 dilution</td></tr><tr><td>Vimentin antibody</td><td>NeoMarkers</td><td>MS-129-p1</td><td>1:500 dilution</td></tr><tr><td>GAPDH antibody</td><td>Millipore Corporation</td><td>MAB374</td><td>1:10,000 dilution</td></tr><tr><td>Amasham ECL Western blotting detection reagent</td><td>GE Health Life Science</td><td>RPN2209</td><td>Chemiluminescence system</td></tr></tbody></table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Le procedure sopra descritte forniscono un metodo affidabile per rilevare splicing alternativo durante EMT. Risultati rappresentativi di CD44 giunzione commutazione isoforma durante EMT Twist-indotta sono riportati di seguito come esempio. EMT Twist-indotta nelle cellule HMLE / Twist-ER è stato caratterizzato da una transizione da un ciottolo-pietra come fenotipo epiteliale ad un fenotipo fibroblastica allungata (Figura 2A), l&#39;assenza di marcatori epiteliali E-caderina,...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Rilevamento di splicing alternativo Durante epiteliale-mesenchimale transizione

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

Research

JoVE Journal

Biology

12.9K Views.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Video: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Utilizzando RNA-sequenziamento per rilevare Novel Splice varianti correlati alla resistenza ai farmaci in<em&gt; In Vitro</em&gt; modelli di cancro

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Ricerca

Ricerca sul cancro

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Ingegneria fattori artificiali per manipolare in modo specifico l&#39;alternanza di splicing nelle cellule umane

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Genetica

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Analisi quantitativa di impionbatura alternativo Pre-mRNA nelle sezioni del cervello del Mouse usando analisi di RNA In Situ di ibridazione

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

Induzione di mesenchimali-epiteliale transizioni in cellule di sarcoma

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Biologia dello sviluppo

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Studio della segnalazione β TGF e della transizione epiteliale-mesenchimale indotta da TGF-β nel cancro al seno e nelle cellule normali

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

Utilizzo dello strumento Minigene E1A per studiare le modifiche allo splicing dell'mRNA

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

Un test cellulare basato su reporter per il monitoraggio dell'efficienza di giunzione

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Biochimica

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Biologia

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

Identificazione di splicing alternativo e poliadenilazione in dati di RNA-seq

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded together.
Splicing is Mediated by the Spliceosome
Splicing occurs in the nucleosome and is mediated by a complex of proteins and small RNAs called small nuclear ribonucleoproteins (snRNPs). snRNPs, together with other proteins, form the spliceosome, which recognizes specific nucleotide sequences at the ends of the exon and intron. First, it binds to a GU-containing sequence at the 5&#39; end of the intron and to a branch point sequence containing an A towards the 3&#39; end of the intron. In a number of carefully-orchestrated steps, other snRNPs then bring the branch point close to the 5&#39; splice site. Subsequently, a chemical reaction cleaves the 5&#39; end of the intron from its upstream exon and attaches it to the branch point, forming a loop called a lariat. To release the lariat, the AG-containing sequence of the intron near the 5&#39; end of the downstream exon reacts with the 3&#39; end of the upstream exon. This reaction patches the two exons together, concluding the splicing process.
Splicing Allows the Expression of Several Proteins from a Single Gene
Typically, exons are joined together in the order in which they appear in a gene. However, during alternative splicing, different combinations of exons in pre-mRNA are combined to form mature mRNA. This produces several different proteins from a single pre-mRNA transcript.
Different patterns of alternative splicing include exon skipping, alternative 5&#39; or 3&#39; splice sites, and intron retention. These patterns are guided by the length of exons or introns and the strength of the splicing signal at the splice sites. Because of this, exons that are shorter than other exons may be overlooked by the spliceosome and omitted from the mature mRNA. In contrast, introns that are significantly shorter than other introns may evade removal by the spliceosome and are retained in the mature mRNA. As a result, alternative splicing generates variants of mature mRNA that were copied from the same stretch of DNA. The RNA sequence variants produce different proteins with additional or fewer amino acids, shifts in the reading frame, or a premature stop codon. These protein isoforms have different biological properties, including function, cellular localization, and interaction with other proteins, thereby playing a vital role in tissue- and environment-specific gene expression.
Abnormal Splicing Can Cause Diseases
Errors in splicing can produce aberrant protein isoforms, which may contribute to diseases, including cancer. For instance, alternative splicing of the BCL2L1 gene generates a long and short protein isoform&#8212;BCL-XL and BCL-XS, respectively&#8212; through the use of alternative 5&#39; splice sites. The longer BCL-XL isoform promotes cell survival and is highly expressed in several types of cancers (e.g., blood, breast, and liver cancers). Expression of the short BCL-XS isoform that promotes cell death is suppressed in cancer.

RNA Splicing, Lariat and the Spliceososme

Splicing dell'RNA

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called ...

Rilevamento di splicing alternativo Durante epiteliale-mesenchimale transizione

Riepilogo

Esplora Altri Video

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Ingegneria fattori artificiali per manipolare in modo specifico l'alternanza di splicing nelle cellule umane