Agradecemos Prof. Jeff Gerst e Boris Slobodin para os seus conselhos úteis na criação do protocolo rápida e fornecendo os plasmídeos necessários. Agradecemos também a Dr. Abigail Atir-Lande por sua ajuda no estabelecimento deste protocolo e Dr. Tamar Ziv do Smoler Proteomics Center for sua ajuda com a análise LC-MS / MS. Agradecemos Prof. TG Kinzy (Rutgers) para o anticorpo YEF3. Este trabalho foi apoiado pela concessão 2011013 do Binacional Science Foundation.

Nota: Inserir uma sequência que consiste em 12 locais de ligação MS2 (MS2 malhas; MS2L) no locus genómico desejado, geralmente entre a grelha de leitura aberta (ORF) e a 3 &#39;UTR. Um protocolo detalhado para essa integração é fornecido em outros lugares 22. Verifique a inserção correta e expressão por PCR, análise de Northern ou RT-PCR 20,23. É importante verificar que a integração não interferiu com a síntese do 3 &#39;UTR. Além disso, um plasmídeo que expressa MS2 CP-fundido com PAS sob a expressão de um promotor indutível (depleção de metionina) também deve ser introduzido nas células 24. Uma estirpe idêntica, excluindo os loops MS12 introduzidas, deve ser utilizado como um controlo para a detecção de sinais não específicos. 1. purificação rápida <ol><li> Cresça 500 ml de células de levedura (250-1,000 usar ml de células de levedura de acordo com o nível da expressão do gene de interesse) que expressam ARNm de MS2-tagged 20 eProteína de fusão MS2-CP-GFP-PAS 24 a DO600 0,8-1,0 a 30 ° C no meio de crescimento apropriado (por exemplo, dextrose sintética (SD) meio selectivo). </li><li> células Centrifugar a 3000 x g durante 4 min a RT e desprezar o sobrenadante. </li><li> Lavar células em 1x tampão de fosfatos salino (PBS), de centrifugação, como anteriormente (passo 1.2), ressuspender em um volume igual (passo 1.1) de SD sem metionina, e incubar durante 45 - 60 min a 30 ° C para induzir a expressão da MS2 -CP-GFP-PAS proteína de fusão. Nota: tempo de indução mais longos podem causar agregação de GFP. </li><li> Retiradas 10 ml de células e extrair RNA a partir desta amostra usando o método simples &quot;fenol quente&quot; 25. Atenção: O fenol é altamente tóxico e deve ser manuseado de acordo com as suas instruções de segurança. Nota: Este ARN é uma boa referência para a qualidade do ARN isolado (Figura 1A). </li><li> Por 500 ml células, adicionar 1,35 ml de 37% de formaldeído para a final concentration de 0,1% para reticular os complexos de ARN-proteína. Continue incubação a 30 o C por 10 min. Atenção: O formaldeído é altamente tóxico e deve ser manuseado de acordo com as suas instruções de segurança. </li><li> Adicionar 26,5 ml de 2,5 M de glicina para uma concentração final de 0,125 M e incuba-se durante 3 minutos para parar a reticulação. A partir deste passo, colocar tudo em gelo para minimizar a degradação do RNA. </li><li> Células centrifugar a 3000 xg durante 4 min a 4 ° C e lavar com 45 ml frio 1x PBS. </li><li> Ressuspender as células em 1 ml de tampão de lise rápida por 100 ml de cultura de células inicial. </li><li> Dividido em alíquotas de 500 mL em tubos de microcentrífuga com tampa de rosca e adicione ~ 400 mg de contas de vidro refrigerada a cada alíquota (cerca de um total de 0,2 ml tubo de PCR). </li><li> Lisar as células em um grânulo batedor durante 3 min. imediatamente colocar o lisado em gelo. Nota: lise celular lançamentos RNases celulares, que são a principal causa de degradação do RNA. tampão de lise rápida contém uma elevada concentração de RNase inhibitors, tais como a heparina não específica inibidor de RNase e inibidores específicos. Trabalhando rapidamente e manter tudo frio também é recomendado. </li><li> Transferir o lisado para novos tubos. Pierce um pequeno orifício na parte inferior de cada tubo de microcentrífuga com uma agulha quente (0,8 mm x 40 mm) e colocar os tubos de microcentrífuga de topo dos tubos de 15 ml. Utilizar a cabeça de uma seringa de 5 ml, como um adaptador entre os tubos de microcentrífuga e os tubos de 15 ml. Centrifugar a montagem de tubos a 3000 xg durante 1 min a 4 o C. </li><li> Transferir o fluxo através do tubo de 15 mL para um novo tubo de 1,5 ml e centrifugar a 10000 xg durante 10 min a 4 ° C para eliminar os detritos celulares. </li><li> sobrenadantes de piscina de todos os tubos de 1,5 ml em um único tubo de 15 ml, e reserve 1/50 e 1/100 do volume do lisado para o RNA e isolamento de proteínas, respectivamente. Nota: Estes irão servir como as amostras de &quot;input&quot; para posterior análise (Figura 1). </li><li> Adicionar 300 ug de avidina por 500 ml de ie cultura de levedura nitial incubar durante 30 minutos (este pode ser reduzido para 10 min) a 4 ° C sob rolamento constante (10 rpm). Nota: Os blocos de avidina e biotina proteínas biotiniladas se liguem às esferas de estreptavidina. </li><li> A pré-lavagem as esferas de estreptavidina antes da incubação com lisado de células. <ol><li> Transferir cerca de 300 ul de estreptavidina grânulos lama para um tubo de microcentrífuga de 1,5 ml e remover o sobrenadante superior. Isto irá resultar em 250 ul de pérolas. Lave as contas duas vezes com 1 ml de tampão de lise rápida. Centrifugar as esferas de estreptavidina a 660 xg durante 2 min a 4 ° C entre cada passo. </li><li> Bloquear as pérolas por incubação durante 1 h com 0,5 ml de tampão de lise rápida, a 0,5 ml de 10 mg / ml de BSA e 10 uL de 10 mg / ml de ARNt de levedura. Grânulos podem ser deixados O / N em solução de bloqueio a 4 ° C com rotação, se necessário. </li><li> Lavar duas vezes com 1 ml de tampão de lise rápida. Nota: As esferas magnéticas pode também ser utilizado para reduzir o tempo e background. Estes, no entanto, tem menor capacidade do que esferas de sepharose. </li></ol></li><li> Adicionar 250 ul das esferas de estreptavidina pré-lavado para o lisado contendo avidina (a partir do passo de 1,14) e incubar 1 h a 4 ° C com rotação constante (10 rpm). Nota: Este tempo de incubação é um compromisso entre a prestação de tempo de ligação suficiente e minimizando as chances de degradação do RNA. </li><li> Centrifuga-se a 660 xg durante 2 min a 4 ° C para eliminar as esferas de estreptavidina e transferir o sobrenadante para um novo tubo de 15 ml. Separe 1/50 e 1/100 do volume do lisado para o isolamento de ARN e proteína, respectivamente. Nota: Estas serão as amostras não ligadas &quot;&quot;. </li><li> Lavam-se as esferas com 1 ml de tampão de lise rápida, agitar, transferir para um novo tubo de microcentrifugação de 1,5 mL, e centrifuga-se tal como no passo 1.17. Repita este passo três vezes. </li><li> Lavam-se as esferas com 1 ml de tampão de lavagem rápida, e esta gire tempo (10 rpm) durante 5 min a 4 ° C. Repita esta etapa duas vezes. </li&gt; <li> Lavam-se as esferas com 1 ml de PBS 1x e centrifugação tal como acima. </li><li> Lavam-se as esferas de novo com 120 ul de PBS 1x. Centrifugar como acima, e tomar todo o sobrenadante para o ARN e a extracção de proteínas como a amostra de &quot;lavagem&quot;. Nota: Esta última amostra de lavagem indica o rigor das lavagens e deve ser do mesmo volume que a amostra de eluição. Isto irá simplificar o processamento e carregamento em ensaios subsequentes. </li><li> Para eluir o ARN e proteínas associadas ao ARN a partir da coluna, adicionar 120 ul de biotina 6 mM em PBS 1x e incubar durante 45 min a 4 ° C com rotação constante (10 rpm). </li><li> Centrifugar as esferas como anteriormente e transferir o material fluido para um novo tubo de 1,5 mL. Girar a amostra eluiu-se novamente e transferir a fase superior para um novo tubo de 1,5 mL para garantir que não haja transição de grânulos. </li><li> Colher amostras para RNA ou extração de proteínas. Nota: O volume ocupado deve depender do nível seguinte ensaio e a expressão do ARN ou proteína de interesse. Como orientação, para análise do Norte 26, tomar 80% da amostra, por Western blot 23,27 ou RT-PCR 28, tome 20%, e por espectrometria de massa 29 para descobrir novas proteínas, tome toda a amostra. </li></ol> 2. Extracção de ARN <ol><li> Adicionar um volume igual de tampão de reticulação 2x Reversão e incubar durante 45 min a 65 ° C. Continue diretamente para a extração de RNA. Nota: A reversão de reticulação contém tampão de ácido etilenodiaminotetracético (EDTA), o que melhora de forma significativa a extracção de RNA 20. </li><li> Use o fenol padrão: Método de clorofórmio 26 para extrair RNA do &quot;input&quot; e amostras &quot;não ligados&quot; (passos de 1,13 e 1,17). Nota: Estas amostras contêm uma grande quantidade de heparina o inibidor de RNase, que pode inibir a ensaios utilizando subsequentes Transcriptase- reversa (RT) (por exemplo, RT-PCR), por conseguinte, LiCl precipitação 30 é aconselhada para removê-lo. O WAsh &quot;e&quot; &quot;amostras de eluição (passos 1.21 e 1.24) contêm pequenas quantidades de ARN e são precipitados através dos seguintes passos. </li><li> Adicionar um volume igual de guanidínio 8M-HCI (GuHCI) e dois volumes de etanol a 100% à &quot;lavagem&quot; e amostras de &quot;eluição&quot;. Incubar a -80 ° C durante pelo menos 2 h. Nota: guanidínio será eficiente desnaturar as proteínas e, assim, inibir as RNases e proteases na amostra. </li><li> Centrifuga-se a 20000 xg, a 4 ° C durante 30 min e desprezar o sobrenadante. Lava-se a pastilha com etanol frio a 80% e centrifuga-se novamente a 20000 xg, 4 ° C durante 15 min. </li><li> Dissolver o sedimento RNA em 400 mL de RNase água livre e precipitar novamente para remover qualquer sobra GuHCl ou outros contaminantes. Adicionar 40 ul de acetato de sódio 3M, pH 5,2 e 800 ul de etanol a 100%, incuba-se a -20 ° C durante pelo menos 1 h. </li><li> Centrífuga e lavar como no passo 2.4 e remover todo o etanol com uma ponta de 10 mL. Ressuspender o Pell RNAet em 10 mL RNase água livre. Tome cuidado extra como o sedimento RNA normalmente não é visível. Nota: As amostras podem ser utilizadas para a análise de Northern como descrito em 23 (Figura 1). </li></ol> 3. Proteína Preparação para Western Blot ou Análise Espectrometria de Massa <ol><li> Adicione 1/3 do volume de tampão de amostra de Laemmli 4x (LSB) para amostras de proteína e incubar 45 min a 65 ° C para inverter a ligação cruzada de proteínas e RNA. Nota: As amostras podem ser armazenadas a -20 ° C. </li><li> Proteínas executado em um gel de poliacrilamida SDS (SDS-PAGE) 31. Ajustar a percentagem de gel de acordo com o tamanho das proteínas a ser analisada. Nota: gel de acrilamida a 10% dá uma vasta gama de separação e está bem como uma primeira aproximação. </li><li> Transferir as proteínas para a membrana como na análise de imunomarcação padrão 27 para controlo da eficiência de isolamento (Figura 1C). Manchar ou com Coomassie Blue R250 ou prata mancha antes da análise de espectrometria de massa. Nota: mancha de prata é a escolha preferida, pois é mais sensível e permite uma melhor detecção (Figura 1B). A coloração pode ser feito com qualquer soluções preparadas manualmente ou kits comerciais. O tempo de incubação do gel na solução final corante deve ser determinada experimentalmente. Longo incubação pode revelar proteínas abundantes de baixo, mas pode causar proteínas altamente expressos tais como MS2-CP-PAS-GFP para ser mais manchado e podem mascarar proteínas circundantes na sua vizinhança. Além disso, pode ocorrer um fundo alta. Siga a reação com cuidado, e adicionar solução de parada no momento apropriado. </li><li> Cortar bandas do gel e transferi-los para tubos de 1,5 ml. Armazenar as amostras a -20 ° C, ou enviar para a extracção de proteína e digestão com tripsina seguida por análise por LC-MS / MS 32. </li><li> Como uma abordagem alternativa para proteómica exclui a etapa de separação em gel, digerir material eluído a partir do passo 1.24 em tãolução utilizando tripsina e sujeita a análise por LC-MS / MS 33. Nota: Omitir a separação SDS-PAGE simplifica o protocolo e aumenta o rendimento. No entanto, as amostras podem conter vestígios de detergente NP-40, que inibe a análise de espectrometria de massa. Para ultrapassar este problema, execute os seguintes passos: <ol><li> Executar proteína em SDS-PAGE a 31 por 10 - 15 min. </li><li> Cortar toda a porção de pista (isto é, onde as proteínas estão localizados). Nota: A amostra de proteína pode incluir uma quantidade significativa de proteína-MS2 CP-PAS que podem obscurecer os sinais de proteínas de baixa abundância. </li></ol></li></ol>

<ol>
	<li>Hogan, D. J., Riordan, D. P., Gerber, A. P., Herschlag, D., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+RNA-binding+proteins+interact+with+functionally+related+sets+of+RNAs,+suggesting+an+extensive+regulatory+system.">Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system.</a> PLoS Biol. 6, e255 (2008).</li><li>Tsvetanova, N. G., Klass, D. M., Salzman, J., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+search+reveals+unexpected+RNA-binding+proteins+in+Saccharomyces+cerevisiae.">Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae.</a> PloS One. 5, (2010).</li><li>Castello, 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=Insights+into+RNA+biology+from+an+atlas+of+mammalian+mRNA-binding+proteins.">Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.</a> Cell. 149, 1393-1406 (2012).</li><li>Gerstberger, S., Hafner, M., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+census+of+human+RNA-binding+proteins.">A census of human RNA-binding proteins.</a> Nat Rev Genet. 15, 829-845 (2014).</li><li>Baltz, A. G., 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+mRNA-bound+proteome+and+its+global+occupancy+profile+on+protein-coding+transcripts.">The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.</a> Mol Cell. 46, 674-690 (2012).</li><li>Mitchell, S. F., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+properties+of+eukaryotic+mRNPs.">Principles and properties of eukaryotic mRNPs.</a> Mol Cell. 54, 547-558 (2014).</li><li>Licatalosi, D. D., Darnell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+processing+and+its+regulation:+global+insights+into+biological+networks.">RNA processing and its regulation: global insights into biological networks.</a> Nat Rev Genet. 11, 75-87 (2010).</li><li>Eliyahu, E., Lesnik, C., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+chaperone+Ssa1+affects+mRNA+localization+to+the+mitochondria.">The protein chaperone Ssa1 affects mRNA localization to the mitochondria.</a> FEBS Lett. 586, 64-69 (2012).</li><li>Ascano, M., Gerstberger, S., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-disciplinary+methods+to+define+RNA-protein+interactions+and+regulatory+networks.">Multi-disciplinary methods to define RNA-protein interactions and regulatory networks.</a> Curr Opin Genet Dev. 23, 20-28 (2013).</li><li>Denman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNPs+take+shape+by+CLIPPING+and+PAIRING.">mRNPs take shape by CLIPPING and PAIRING.</a> BioEssays. 28, 1132-1143 (2006).</li><li>McHugh, C. A., Russell, P., Guttman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+comprehensive+experimental+identification+of+RNA-protein+interactions.">Methods for comprehensive experimental identification of RNA-protein interactions.</a> Genome Biol. 15, 203 (2014).</li><li>Bernstein, D. S., Buter, N., Stumpf, C., Wickens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+mRNA-protein+complexes+using+a+yeast+three-hybrid+system.">Analyzing mRNA-protein complexes using a yeast three-hybrid system.</a> Methods. 26, 123-141 (2002).</li><li>SenGupta, D. J., 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+three-hybrid+system+to+detect+RNA-protein+interactions+in+vivo.">A three-hybrid system to detect RNA-protein interactions in vivo.</a> Proc Natl Acad Sci USA. 93, 8496-8501 (1996).</li><li>Yosefzon, Y., 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=Divergent+RNA+binding+specificity+of+yeast+Puf2p.">Divergent RNA binding specificity of yeast Puf2p.</a> RNA. 17, 1479-1488 (2011).</li><li>Mitchell, S. F., Jain, S., She, M., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+yeast+mRNPs.">Global analysis of yeast mRNPs.</a> Nat Struct Mol Biol. 20, 127-133 (2013).</li><li>Zielinski, J., 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=In+vivo+identification+of+ribonucleoprotein-RNA+interactions.">In vivo identification of ribonucleoprotein-RNA interactions.</a> Proc Natl Acad Sci USA. 103, 1557-1562 (2006).</li><li>Bell, T. J., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Cell+Genomics:+RNA+Exon-Specific+RNA-Binding+Protein+Isolation.">Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.</a> Methods Mol Biol. 1324, 457-468 (2015).</li><li>Leppek, K., Stoecklin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+streptavidin-binding+RNA+aptamer+for+purification+of+ribonucleoprotein+complexes+identifies+novel+ARE-binding+proteins.">An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins.</a> Nucleic Acids Res. 42, e13 (2014).</li><li>Slobodin, B., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mRNA+affinity+purification+technique+for+the+identification+of+interacting+proteins+and+transcripts+in+ribonucleoprotein+complexes.">A novel mRNA affinity purification technique for the identification of interacting proteins and transcripts in ribonucleoprotein complexes.</a> RNA. 16, 2277-2290 (2010).</li><li>Samra, N., Atir-Lande, A., Pnueli, L., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elongation+factor+eEF3+(Yef3)+interacts+with+mRNA+in+a+translation+independent+manner.">The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner.</a> BMC Mol Biol. 16, 17 (2015).</li><li>Loya, 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+3'-UTR+mediates+the+cellular+localization+of+an+mRNA+encoding+a+short+plasma+membrane+protein.">The 3'-UTR mediates the cellular localization of an mRNA encoding a short plasma membrane protein.</a> RNA. 14, 1352-1365 (2008).</li><li>Haim-Vilmovsky, L., Gadir, N., Herbst, R. H., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+integration+method+for+the+simultaneous+visualization+of+endogenous+mRNAs+and+their+translation+products+in+living+yeast.">A genomic integration method for the simultaneous visualization of endogenous mRNAs and their translation products in living yeast.</a> RNA. 17, 2249-2255 (2011).</li><li>Eldad, N., Yosefzon, Y., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+extensive+intra-molecular+associations+between+3'-UTRs+and+their+ORFs.">Identification and characterization of extensive intra-molecular associations between 3'-UTRs and their ORFs.</a> Nucleic Acids Res. 36, 6728-6738 (2008).</li><li>Slobodin, B., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RaPID:+an+aptamer-based+mRNA+affinity+purification+technique+for+the+identification+of+RNA+and+protein+factors+present+in+ribonucleoprotein+complexes.">RaPID: an aptamer-based mRNA affinity purification technique for the identification of RNA and protein factors present in ribonucleoprotein complexes.</a> Methods Mol Biol. 714, 387-406 (2011).</li><li>Schmitt, M. E., Brown, T. A., Trumpower, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+simple+method+for+preparation+of+RNA+from+Saccharomyces+cerevisiae.">A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae.</a> Nucleic Acids Res. 18, 3091-3092 (1990).</li><li>Eldad, N., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ribosomal+density-mapping+procedure+to+explore+ribosome+positions+along+translating+mRNAs.">A ribosomal density-mapping procedure to explore ribosome positions along translating mRNAs.</a> Methods Mol Biol. 419, 231-242 (2008).</li><li>Arava, Y., Seger, R., Fbeta Walker, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GRFbeta,+a+novel+regulator+of+calcium+signaling,+is+expressed+in+pancreatic+beta+cells+and+brain.">GRFbeta, a novel regulator of calcium signaling, is expressed in pancreatic beta cells and brain.</a> J Biol Chem. 274, 24449-24452 (1999).</li><li>Arava, Y., Adamsky, K., Ezerzer, C., Ablamunits, V., Walker, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+gene+expression+in+pancreatic+beta-cells:+cloning+and+characterization+of+differentially+expressed+genes.">Specific gene expression in pancreatic beta-cells: cloning and characterization of differentially expressed genes.</a> Diabetes. 48, 552-556 (1999).</li><li>Bavli-Kertselli, I., Melamed, D., Bar-Ziv, L., Volf, H., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+eukaryotic+initiation+factor+5+rescues+the+translational+defect+of+tpk1w+in+a+manner+that+necessitates+a+novel+phosphorylation+site.">Overexpression of eukaryotic initiation factor 5 rescues the translational defect of tpk1w in a manner that necessitates a novel phosphorylation site.</a> FEBS J. 282, 504-520 (2015).</li><li>Eliyahu, E., Melamed, D., Arava, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+RNA+extracted+from+isolated+mitochondria.">Genome-wide analysis of RNA extracted from isolated mitochondria.</a> Methods Mol Biol. 714, 287-299 (2011).</li><li>Brunelle, J. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+SDS-polyacrylamide+gel+electrophoresis+(1D+SDS-PAGE).">One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).</a> Methods Enzymol. 541, 151-159 (2014).</li><li>Gundry, R. 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=Preparation+of+proteins+and+peptides+for+mass+spectrometry+analysis+in+a+bottom-up+proteomics+workflow.">Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow.</a> Current protocols in molecular biology. Chapter 10, Unit 10.25 (2009).</li><li>Medzihradszky, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-solution+digestion+of+proteins+for+mass+spectrometry.">In-solution digestion of proteins for mass spectrometry.</a> Methods Enzymol. 405, 50-65 (2005).</li><li>Bachler, M., Schroeder, R., von Ahsen, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=StreptoTag:+a+novel+method+for+the+isolation+of+RNA-binding+proteins.">StreptoTag: a novel method for the isolation of RNA-binding proteins.</a> RNA. 5, 1509-1516 (1999).</li><li>Oeffinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+steps+forward--one+step+back:+advances+in+affinity+purification+mass+spectrometry+of+macromolecular+complexes.">Two steps forward--one step back: advances in affinity purification mass spectrometry of macromolecular complexes.</a> Proteomics. 12, 1591-1608 (2012).</li><li>Ross, A. F., Oleynikov, Y., Kislauskis, E. H., Taneja, K. L., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+beta-actin+mRNA+zipcode-binding+protein.">Characterization of a beta-actin mRNA zipcode-binding protein.</a> Mol Cell Biol. 17, 2158-2165 (1997).</li><li>Deshler, J. O., Highett, M. I., Schnapp, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+Xenopus+Vg1+mRNA+by+Vera+protein+and+the+endoplasmic+reticulum.">Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum.</a> Science. 276, 1128-1131 (1997).</li><li>Bertrand, 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=Localization+of+ASH1+mRNA+particles+in+living+yeast.">Localization of ASH1 mRNA particles in living yeast.</a> Mol Cell. 2, 437-445 (1998).</li><li>Aizer, 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=Quantifying+mRNA+targeting+to+P-bodies+in+living+human+cells+reveals+their+dual+role+in+mRNA+decay+and+storage.">Quantifying mRNA targeting to P-bodies in living human cells reveals their dual role in mRNA decay and storage.</a> J Cell Sci. 127, 4443-4456 (2014).</li><li>Gadir, N., Haim-Vilmovsky, L., Kraut-Cohen, J., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+mRNAs+coding+for+mitochondrial+proteins+in+the+yeast+Saccharomyces+cerevisiae.">Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae.</a> RNA. 17, 1551-1565 (2011).</li><li>Lopez de Heredia, ., M, R. P., Jansen, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+integrity+as+a+quality+indicator+during+the+first+steps+of+RNP+purifications+:+a+comparison+of+yeast+lysis+methods.">RNA integrity as a quality indicator during the first steps of RNP purifications : a comparison of yeast lysis methods.</a> BMC Biochem. 5, 14 (2004).</li><li>Lee, J. S., Kallehauge, T. B., Pedersen, L. E., Kildegaard, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+integration+in+CHO+cells+mediated+by+CRISPR/Cas9+and+homology-directed+DNA+repair+pathway.">Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway.</a> Sci Rep. 5, 8572 (2015).</li><li>Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P., Del Bene, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+CRISPR/Cas9-mediated+knock-in+in+zebrafish+by+homology-independent+DNA+repair.">Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.</a> Genome Res. 24, 142-153 (2014).</li><li>Oda, Y., Huang, K., Cross, F. R., Cowburn, D., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+quantitation+of+protein+expression+and+site-specific+phosphorylation.">Accurate quantitation of protein expression and site-specific phosphorylation.</a> Proc Natl Acad Sci USA. 96, 6591-6596 (1999).</li><li>de Godoy, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SILAC+yeast:+from+labeling+to+comprehensive+proteome+quantification.">SILAC yeast: from labeling to comprehensive proteome quantification.</a> Methods Mol Biol. 1156, 81-109 (2014).</li></ol>

The authors declare no competing financial interests.

Vários métodos utilizam o isolamento de ARNm específicos para identificar as suas proteínas associadas 11,34 35. Estes métodos aplicam-se in vitro e in vivo em estratégias para investigar interacções ARN-proteína. Métodos in vitro incubar exogenamente ARN transcrito com lisado celular para capturar RBPs e isolar complexos de RNP 36,37. Uma abordagem eficaz deste tipo foi apresentado recentemente, o que permitiu a identificação de novas proteínas qu...

Proteínas de ligação a ARN (RBPs) representar cerca de 10% de S. proteínas cerevisiae 1,2 e cerca de 15% de proteínas de mamífero 3-5. Eles estão envolvidos em muitos processos celulares, tais como processamento de mRNA pós-transcricional e regulação, tradução, biogênese do ribossomo, tRNA aminoacilaï¿½o e modificação, a remodelação da cromatina, e muito mais. Um subgrupo importante de RBPs é as proteínas de ligação ao ARNm (mRNPs) 6,7. No decurso da maturação do mRNA, diferentes RBPs ligar a transcrição e mediar a transformação nuclear, exportação para fora do núcleo, a localização celular, tradução e degradação 6-8. Assim, o conjunto distinto de RBPs ligados a um transcrito particular, a qualquer ponto de tempo determina o seu processamento e em última análise o seu destino. A identificação de RBPs associados a um mRNA poderia melhorar significativamente a nossa compreensão dos processos subjacentes à sua regulação pós-transcricional. Diverse genética, Métodos microscópicos, bioquímicos e de bioinformática têm sido utilizados para identificar proteínas envolvidas na regulação do ARNm (revisto em 9-11). No entanto, apenas alguns desses métodos permitem a identificação de proteínas associadas a um ARNm alvo particular. Digno de nota é o sistema híbrido de levedura Três (Y3H), que utiliza o mRNA de interesse como isca para pesquisar uma biblioteca de expressão em células de levedura. Os clones positivos são observados geralmente através de uma selecção de crescimento ou repórter expressão 12-14. A principal vantagem deste método é o grande número de proteínas que podem ser digitalizados num ambiente celular e a capacidade para medir a força da interacção RNA-proteína. Desvantagens incluem o número relativamente grande de resultados falsos positivos devido à ligação não específica, eo alto potencial de resultados falsos negativos devido, em parte, a misfolding da presa proteína de fusão ou o RNA isca. Uma alternativa à abordagem genética é purifi afinidadecatião de ARN com as suas proteínas associadas. Poli A contendo ARNm pode ser isolado através da utilização de colunas de oligo dT, e as suas proteínas associadas são detectados por espectrometria de massa. A interacção RNA-proteína é conservado no seu contexto celular por reticulação, o que faz com que as ligações covalentes de curto alcance. A utilização da coluna de oligo dT proporciona uma visão global de todo o proteoma que está associado com qualquer poli A de mRNA contendo 3,5,15. No entanto, este não fornece uma lista de proteínas que estão associadas com um ARNm específico. Muito poucos métodos estão disponíveis para realizar uma tal identificação. O método implica a transfecção PAR de ácido nucleico com complementaridade com o mRNA alvo 16,17. O ácido nucleico está também ligado a um péptido, o que permite a reticulação de RBPs em estreita proximidade ao local de interacção. Após a reticulação, o ácido nucleico RBP-péptido pode ser isolado e submetido a análise proteómica. Recentemente, uma metodologia baseada na aptâmero eraaplicado com sucesso a extractos de linhas de células de mamíferos 18. Um aptâmero de ARN com afinidade melhorada para estreptavidina e foi desenvolvido fundido com uma sequência de interesse (elemento ricos em AU (ARE), neste caso). O ARN aptâmero-ARE foi ligado a esferas de estreptavidina e misturados com o lisado celular. As proteínas associadas a que a sequência são foram purificados e identificados por espectrometria de massa (MS). Embora este método detectado associações que ocorrem fora das definições celulares (isto é, in vitro), é susceptível de ser modificado no futuro, de modo a introduzir os aptâmeros no genoma e, assim, permitir o isolamento de proteínas associadas com o ARNm enquanto que na meio celular (isto é, in vivo). Em leveduras, onde manipulações genéticas estão bem estabelecidos, o método rápido (desenvolvido no laboratório do Prof. Jeff Gerst) fornece uma visão in vivo associações 19. Rápida combina o específico e forte ligação da proteína de revestimento de MS2 (MS2-CP) para a sequência de ARN MS2, e do domínio de ligação a estreptavidina (SBP) a estreptavidina esferas conjugadas. Isso permite a purificação eficiente de mRNAs MS2-marcadas através de contas de estreptavidina. Além disso, a expressão de 12 cópias de MS2 laços permite até seis MS2-CPS de se ligar simultaneamente para o RNA e aumentar a eficiência do seu isolamento. Este protocolo foi, portanto, sugerida para permitir a identificação de novas proteínas associadas à ARNm uma vez que as amostras eluídas são sujeitas a análise por espectrometria de massa proteómica. Recentemente, utilizou rápido para identificar novas proteínas associadas com a levedura Pmp1 ARNm 20. Pmp1 ARNm foi anteriormente mostrado para ser associado com a membrana do RE e do seu 3 &#39;não traduzida (UTR) foi encontrado para ser um dos principais determinantes nesta associação 21. Assim, RBPs que se ligam Pmp1 UTR 3 &#39;são susceptíveis de desempenhar um papel importante na sua localização. Rápida seguida de cromatografia líquiday-espectrometria de massa / espectrometria de massa (LC-MS / MS) resultou na identificação de várias novas proteínas que interagem com Pmp1 20. Aqui, nós fornecemos um protocolo detalhado da metodologia rápida, controles importantes que precisam ser feitas, e dicas técnicas que podem melhorar o rendimento e especificidade.

<table><tbody><tr><td>Tris</td><td>sigma</td><td>T1503</td><td></td></tr><tr><td>SDS</td><td>bio-lab</td><td>1981232300</td><td></td></tr><tr><td>DTT</td><td>sigma</td><td>D9779</td><td></td></tr><tr><td>Acidic Phenol (pH 4.3)</td><td>sigma</td><td>P4682</td><td></td></tr><tr><td>Acidic Phenol: Chloroform (5:1, pH 4.3)</td><td>sigma</td><td>P1944</td><td></td></tr><tr><td>Chloroform</td><td>bio-lab</td><td>3080521</td><td></td></tr><tr><td>Formaldehyde</td><td>Frutarom</td><td>5551820</td><td></td></tr><tr><td>Glycine</td><td>sigma</td><td>G7126</td><td></td></tr><tr><td>NP-40</td><td>Calbiochem</td><td>492016</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>Phenylmethylsulfonyl Flouride (PMSF)</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153</td><td></td></tr><tr><td>Soybean Trypsin Inhibitor</td><td>Sigma</td><td>T9003</td><td></td></tr><tr><td>Pepstatin</td><td>Sigma</td><td>P5318</td><td></td></tr><tr><td>DNase I</td><td>Promega</td><td>M610A</td><td></td></tr><tr><td>Ribonuclease&amp;nbsp; Inhibitor</td><td>Takara</td><td>2313A</td><td></td></tr><tr><td>Glass Beads</td><td>Sartorius</td><td>BBI-8541701</td><td>0.4-0.6mm diameter&amp;nbsp;</td></tr><tr><td>Mini BeadBeater</td><td>BioSpec</td><td>Mini BeadBeater 16</td><td></td></tr><tr><td>Guanidinium</td><td>Sigma</td><td>G4505</td><td></td></tr><tr><td>Avidin</td><td>Sigma</td><td>A9275</td><td></td></tr><tr><td>Streptavidin Beads</td><td>GE Healthcare&amp;nbsp;</td><td>17-5113-01</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Yeast tRNA</td><td>Sigma</td><td>R8508</td><td></td></tr><tr><td>Biotin</td><td>Sigma</td><td>B4501</td><td></td></tr><tr><td>Yeast extract</td><td>Bacto</td><td>288620</td><td></td></tr><tr><td>peptone</td><td>Bacto</td><td>211677</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>1x Phosphate-Buffered saline (PBS)</td><td></td><td></td><td></td></tr><tr><td>0.2 M NaOH</td><td></td><td></td><td></td></tr><tr><td>4x Laemmli Sample Buffer (LSB)</td><td></td><td></td><td>0.2 M Tris-Hcl pH 6.8, 8% SDS, 0.4 M DTT, 40% glycerol, 0.04% Bromophenol-Blue.</td></tr><tr><td>Hot phenol lysis buffer</td><td></td><td></td><td>10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS&amp;nbsp;</td></tr><tr><td>3 M Sodium Acetate pH 5.2</td><td></td><td></td><td></td></tr><tr><td>100% and 70% Ethanol (EtOH)</td><td></td><td></td><td></td></tr><tr><td>RNase-free water</td><td></td><td></td><td></td></tr><tr><td>RaPID lysis buffer</td><td></td><td></td><td>20 mM Tris pH 7.5, 150 mM NaCl, 1.8 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5% NP-40, 5 mg/ml Heparin, 1 mM Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl Flouride (PMSF), 10 &amp;micro;g/ml Leupeptin, 10 &amp;micro;g/ml Aprotinin, 10 &amp;micro;g/ml Soybean Trypsin Inhibitor, 10 &amp;micro;g/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease&amp;nbsp; Inhibitor.</td></tr><tr><td>2x Cross-linking reversal buffer</td><td></td><td></td><td>100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS.</td></tr><tr><td>RaPID wash buffer</td><td></td><td></td><td>20 mM Tris-HCl pH 7.5,&amp;nbsp; 300 mM NaCl, 0.5% NP-40</td></tr><tr><td>0.5 M EDTA pH 8</td><td></td><td></td><td></td></tr><tr><td>Silver Stain Plus Kit</td><td>Bio-Rad&amp;nbsp;</td><td>161-0449</td><td>For detecting proteins in polyacrylamide gels</td></tr><tr><td>SD selective medium&amp;nbsp;</td><td></td><td></td><td>1.7 g/l Yeast nitrogen base with out amino acids and ammonium sulfate, 5 g/l Ammonium sulfate, 2% glucose, 350 mg/l Threonine, 40 mg/l Methionine, 40 mg/l Adenine, 50 mg/l Lysine, 50 mg/l Tryptophan, 20 mg/l Histidine, 80 mg/l Leucine, 30 mg/l Tyrosine, 40 mg/l Arginine</td></tr><tr><td>Anti-eEF3 (EF3A,YEF3)</td><td>Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School)</td><td>1:5,000</td><td></td></tr><tr><td>Anti GFP antibody</td><td>Santa Cruz</td><td>sc-8334</td><td>1:3,000</td></tr><tr><td>Anti rabbit IgG-HRP conjugated</td><td>SIGMA</td><td>A9169</td><td>1:10,000</td></tr></tbody></table>

novel rna-binding proteins isolation by the rapid methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RAPID permite o isolamento de um ARN alvo específico com as suas proteínas associadas. Crítico para o seu sucesso é manter o ARN intacto, tanto quanto possível, obtendo-se assim uma quantidade suficiente de proteínas. Para determinar a eficiência e a qualidade de isolamento de ARN, análise de Northern é realizado (Figura 1A). análise do Norte tem a vantagem de reportar diretamente a eficiência e qualidade dos rápidos. Assim, as quantidades relativas dos produ...

Watch this Scientific Journal Video about Novel RNA-Binding Proteins Isolation by the RaPID Methodology at JoVE.com

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

Novel de Ligação a RNA Proteínas Isolamento pelo rápido Metodologia

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

novel-rna-binding-proteins-isolation-by-the-rapid-methodology

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

8.9K visualizações.  Technion - Israel Institute of Technology. RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell. 

Vídeo: Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Isolamento de complexos de ARN-proteína a partir de células Cognate usando eluição Oligonucleotide-directed

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Bioquímica

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Preparação da amostra para identificação de espectrometria de massa-baseada das regiões de ligação de RNA

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

Um procedimento de isolamento de RNA em Tandem do Oligonucleotide-baseado para recuperar complexos mRNA-proteínas eucariotas

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

Um ensaio para quantificar a ligação de RNA de proteína em bactérias

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Genética

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Um método rápido de alta capacidade para Ribonucleoproteínas Mapping (RNPs) no pré-mRNA Humanos

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Biologia

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Uma Análise Pull-Down Quantitativa Otimizada de Proteínas Ligadoras de RNA Usando RNA Biotinilado Curto

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Método para o isolamento e identificação de mRNAs, microRNAs e componentes proteicos de complexos de ribonucleoproteínas de extratos celulares utilizando RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Correção rápida in Vivo e isolamento de complexos translacionais de células eucarióticas

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

SELEX-Based In Vitro Binding Assay to Identify RNA-Protein Interactions

Source: Singh, R. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59635">Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins.</a> J. Vis. Exp. ( (2019).
In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.