Este trabalho foi financiado pela Swiss National Science Foundation Sinergia concessão CRSII3 147697, Stiftung für Angewandte Krebsforschung e Zürich Krebsliga.

1. preparação da fração de proteína citosólica e Nuclear
<ol><li>Placa 4 x 106 ACC-MESO-4 células num balão de cultura de célula T150 cultivadas em RPMI - 1640 meio suplementado com 10% FBS, 1 x penicilina/estreptomicina (100 x) e 2 mM L-glutamina (200 mM). Quando as células alcançam uma confluência de 80-90% (~5-5.5 x 106 células), prosseguir com a extração da proteína da fração nuclear e citosólica. Nota: ACC-MESO-4 linha de celular foi obtida o RIKEN BioResource centro11.</li>
	<li>Aspire médio, lavar as células adicionando-se 15 mL de 1X PBS, incline a placa suavemente algumas vezes e aspire a PBS 1x. Adicionar 3 mL de 0,25% Trypsin-EDTA e incubar o frasco de cultura a 37 ° C por 3 min. Nota: Veja as células sob o microscópio para destacamento; se ainda ligado a torneira o frasco contra a palma de sua mão 3 vezes.</li>
	<li>Adicionar 7 mL de RPMI-1640, suplementado com 10% FBS, 1x penicilina/estreptomicina (100 x) e L-glutamina (200 mM) de 2 mM, coletar as células em um tubo de 15 mL e spin para baixo a 200 x g por 5 min.</li>
	<li>Médio, Aspire e resuspenda o pellet de células com 1,5 mL de 1X PBS e, em seguida, transferir as células para um tubo de microcentrifugadora de 1,5 mL. Spin para baixo a x 500 g e a 4 ° C por 5 min. descartar o sobrenadante. Nota: A partir deste passo em diante, executar todas as etapas de centrifugação a 4 ° C e manter todos os reagentes no gelo.</li>
	<li>Ressuspender as células com gelada 1,5 mL de 1X PBS e spin-down as células a x 500 g e a 4 ° C por 3 min. descartar o sobrenadante. Nota: As quantidades necessárias de CER I, II CER e reagentes NER são 10, 0,55 e 5 volumes de centrifugado estimado embalado volume, respectivamente; o protocolo seguinte assume um volume de 20 µ l. Recomendamos preparar apenas a quantidade necessária de reagente CER eu.</li>
	<li>Adicionar 200 µ l de reagente de extração citoplasmática gelada (CER eu) suplementado com 2 µ l de 1x do inibidor da protease, por exemplo, diluir 1 comprimido de inibidor de Protease (EDTA-livre) em 500 µ l de água para obter 100 ações de x. Esta quantidade de reagente CER chega a lyse 20 µ l de uma pelota de célula embalado volume.<ol>
<li>Para estimar o volume de sedimento embalado de célula, compare o tubo contendo o centrifugado com um tubo de 1,5 mL cheio com 10, 20, 50 ou 100 µ l de PBS 1x.</li>
	</ol>
</li>
	<li>Resuspenda o pellet de células por vigorosamente num Vortex o tubo por 15 s e incubar o tubo no gelo por 10 min. Adicionar 11 µ l de reagente de extração citoplasmática gelada II (II CER) para o tubo, vórtice vigorosamente por 5 s e incubar 1 min no gelo.</li>
	<li>Vórtice vigorosamente durante 5 s e centrifugar o tubo a 16.000 x g e 4 ° C por 5 min. Transfira o sobrenadante no tubo limpo previamente refrigerados no gelo. O sobrenadante é a fração citoplasmática que é mais usado no experimento de RNA-pulldown.</li>
	<li>Ressuspender o restante em 100 µ l de reagente de extração nuclear gelada (NER) suplementado com 1 µ l de inibidor de proteinase (100x) e vigorosamente vórtice por 15 s. Recomendamos preparar apenas a quantidade necessária de reagente NER suplementado com inibidores de protease.</li>
	<li>Incubar a amostra no gelo por 40 min com ocasional num Vortex por 15 s (a cada 10 min).</li>
	<li>Centrifugar o tubo a 16.000 x g durante 10 min. transferência do sobrenadante (isto é a fração de proteína nuclear) para um tubo limpo previamente refrigerado.</li>
	<li>Proceder imediatamente por medir a concentração de proteína usando o método bicinchoninic (BCA) (ver Tabela de materiais). Para controlar a pureza da proteína de cada fração, execute immunoblotting (Figura 1) contra a α-tubulina (detecção positivos somente na fração citosólica) e poli ADP-ribose polimerase - PARP (detecção positivos somente na fração nuclear). Para visualização de proteína, consulte a seção 4.</li>
	<li>A fim de manter a atividade de proteína e seu estado nativo, prepare 25 µ l de alíquotas dos extratos citosólico e nucleares. Snap-congelar essas alíquotas, colocando-os em nitrogênio líquido para 5 s e loja diretamente a-80 ° C.</li>
</ol>2. rotulagem do RNA com Desthiobiotin
<ol><li>Sondas de RNA sintetizado comercialmente e HPLC purificado. Resuspenda com água livre de nuclease a 10 µM. Nota: Sonda sequências estão listadas na tabela 1.</li>
	<li>Use 50 pmol de RNA por reação de RNA-pulldown. Descongelar e manter todos os reagentes no gelo exceto para PEG-30%, usado para rótulo 3' terminus de RNA oligo.</li>
	<li>Transferência de 5 µ l de 10 sondas de RNA µM, rotulado como CALB2 3' UTR (são), CALB2 3' UTR (mtARE) e independentes-RNA (IRE), em 0,5 mL microcentrifuga de parede fina tubos e incubá-los em uma máquina PCR a 85 ° C por 5 min. Coloque os tubos imediatamente no gelo. Nota: Este passo é importante, como promove o relaxamento e a acessibilidade da sonda para a rotulagem do RNA.</li>
	<li>Para uma única reação de 30 µ l, adicionar ao tubo de RNA, contendo a mistura de 10 µ l a seguir: 3 µ l de água livre de nuclease, 3 µ l de RNA Ligase Buffer de reação, 1 µ l de RNase inibidor de 10x (40 U / µ l), 1 µ l de Desthiobiotinylated citidina bisfosfato (1 mM) e 2 µ l de RNA T4 ligase do (20 U / µ l). Nota: Para preparar A mistura de mestre 3 amostras em excesso (3.2), misturar 9,6 µ l de nuclease água livre, 9,6 µ l de RNA Ligase Buffer de reação, 3,2 µ l de RNase inibidor de 10x (40 U / µ l), 3,2 µ l de Desthiobiotinylated citidina bisfosfato (1 mM) e 6.4 µ l de RNA T4 ligase do (20 U / µ l).</li>
	<li>Adicione cuidadosamente 15 µ l de PEG-30% para a reação. Use outra dica de misturar a reação. Incubar a reação durante a noite a 16 ° C.</li>
	<li>No dia seguinte, prepare-se o seguinte: 5 M NaCl (fresco/nuclease-free), clorofórmio: álcool isoamílico na proporção 24:1 (por exemplo, por reação - 96 µ l clorofórmio e 4 µ l de álcool isoamílico), gelada 100% de etanol e gelada 70% de etanol.</li>
	<li>Adicione 70 µ l de água livre de nuclease aos tubos de reação de RNA-rotulagem. Adicione 100 µ l de clorofórmio: isoamílico álcool e vórtice brevemente. Desativação da rotação a 13.000 x g por 3 min. retire apenas a fase superior e transferência para um novo tubo isento de nuclease 1,5 mL; Evite tocar a fase inferior.</li>
	<li>Adicione 10 µ l de NaCl 5m, 1 µ l de glicogênio e 300 µ l de etanol gelado a 100%. Coloca o tubo a-20 ° C durante 2 h. Nota: Neste passo, o experimento pode ser continuado no dia seguinte.</li>
	<li>Cuidadosamente, centrifugar 13.000 x g e 4 ° C por 15 min. desprezar o sobrenadante sem perturbar o sedimento. Adicione 300 µ l de etanol a 70% gelado e centrifugar novamente a 13.000 x g e 4 ° C por 5 min.</li>
	<li>Descartar o sobrenadante completamente e deixe secar naturalmente a pelota (15 min). Resuspenda o pellet em 20 µ l de água livre de nuclease. Nota: Proceda com RNA-pulldown no mesmo dia.</li>
	<li>Incubar o RNA a 90 ° C por 2 min e colocar no gelo. Coloque o RNA rotulados no gelo durante a etapa seguinte de pre-lavagem dos grânulos magnético estreptavidina. Nota: Um tempo de incubação pode danificar a sonda de RNA.</li>
</ol>3. RNA-proteína Pulldown
<ol><li>
Pré-lavagem grânulos streptavidin-magnético e incubação com desthiobiotin-RNA
	<ol>
<li>Use 50 µ l de estreptavidina grânulos magnético por 50 pmol de RNA. No entanto, isto deve ser otimizado dependendo do experimento.</li>
		<li>Vórtice com grânulos streptavidin-magnético para 15 s e rapidamente remover 200 µ l (mestre mistura; suficiente para reações de 3 + 1) em um limpa 1,5 mL tubo cofre-bloqueio usando o tubo de corte pontas de pipetas. Coloque o tubo num suporte magnético para que os grânulos recolher ao lado do tubo e esperem 1 min. Retire o líquido de ressuspensão.</li>
		<li>Lave os grânulos, remover o tubo do suporte magnético, adicione 400 µ l de NaOH 0,1 M, 0,05 M NaCl solução e suavemente pipeta subindo e descendo várias vezes. Coloque o tubo de volta a depor magnético. Espere 1 minuto e recolher o sobrenadante. Repita este passo novamente.</li>
		<li>Lave os grânulos com 200 µ l de 100 mM de NaCl. Remova o sobrenadante.</li>
		<li>Adicione 200 µ l de 20 mM Tris (pH 7,5), resuspenda grânulos pipetando, colocar o tubo no stand magnético, esperar 1 min e remover o sobrenadante. Repita a etapa.</li>
		<li>Retire o tubo do suporte magnético, adicionar 200 µ l de buffer de captura do RNA de 1x e ressuspender streptavidin grânulos magnético vortexing brevemente. Remover 50 µ l de grânulos magnético streptavidin e adicionar a cada tubo rotulado-RNA usando uma ponta de pipeta de corte. Incube os tubos por 30 min à temperatura ambiente em um rolo.</li>
	</ol>
</li>
	<li>
Ligação de proteínas de RNA
	<ol>
<li>Coloque os tubos em um carrinho magnético, esperar 1 min e retirar o sobrenadante.</li>
		<li>Adicionar 50 µ l de 20 mM Tris (pH 7,5) para as contas e ressuspender pipetando. Coloque os tubos em um carrinho magnético, esperar 1 min e retirar o sobrenadante. Repita este passo.</li>
		<li>Adicionar 100 µ l de tampão de ligação proteína-RNA 1x aos talões e ressuspender pipetando.</li>
		<li>Entretanto, prepare a seguinte mistura: 10 µ l de 10x buffer de ligação proteína-RNA, 30 µ l de glicerol a 50%, 50 µ g de proteínas citoplasmáticas e livre de nuclease até 100 µ l de água. Nota: Preparar mistura mestre B para 3 amostras em excesso (3.3) da seguinte forma: 33 µ l de 10x buffer de ligação proteína-RNA, 99 µ l de glicerol a 50%, 165 µ g de proteínas citoplasmáticas e água livre de nuclease até 330 µ l. Mantenha o tubo de mistura mestre B no gelo.</li>
		<li>Coloque os tubos no suporte magnético, espere 1 minuto e recolher o sobrenadante. Adicionar 100 µ l do mix master B, resuspenda suavemente pipetando acima e para baixo e evitar a criação de bolhas. Incube os tubos de reação para 60 min a 4 ° C em um rolo.</li>
	</ol>
</li>
	<li>
Lavagem e eluição
	<ol>
<li>Coloque os tubos em um carrinho magnético, recolher o sobrenadante (que é o fluxo através de - FT) e transferir para um tubo novo. Nota: Mantenha esta FT no gelo para análise posterior.</li>
		<li>Pipetar 100 µ l de 1x tampão de lavagem sobre os grânulos, Ressuspender delicadamente, colocar de volta no carrinho magnético, esperar 1 min e descartar o sobrenadante. Repita essa etapa 2 vezes.</li>
		<li>Adicionar 40 µ l de tampão de eluição para as esferas magnéticas, misturar pipetando para cima e para baixo e incubar a 37 ° C num agitador de tubo (950 rpm) por 30 min.</li>
		<li>Coloque os tubos em um carrinho magnético, esperar 1 min e coletar amostra de eluted (que é eluído - E). Coloque no gelo e usar para análise a jusante. Nota: Neste passo, cada sonda RNA testada consiste em uma passagem (FT) e amostra de eluato (E).</li>
	</ol>
</li>
</ol>4. análise do borrão ocidental e eletroforese em poliacrilamida
Nota: Ver tabela 2 receitas para os buffers usados nesta seção. Realizar um Western blot de acordo com o método de Laemmli12.
<ol><li>Handcast 12% geles de SDS-PAGE (10-poços, espaçador de 1,5 mm, 66 µ l). Preparar uma mistura de gel de execução: 4 mL de solução 30% do acrilamido-bisacrylamide, 2,4 mL de Tris inferior do buffer, 3,2 mL de dH2O, 9,6 µ l de TEMED, 96 µ l da solução de persulfato de amónio de 10% (APS). Preparar a mistura de gel de empilhamento: 3,25 mL de dH2O, 0,5 mL de acrilamida 30% - solução bisacrylamide, 1,3 mL de tampão Tris superior, 10 µ l de TEMED, 20 µ l de 10% APS.</li>
	<li>Adicione 16,6 µ l de tampão de x Laemmli 6 (por exemplo, 14 mL de pH Tris/HCl/SDS 6,8 (tampão Tris superior), 2 g de SDS, 1,86 g de TDT, 6 mL de glicerol e 0,012% bromofenol; armazenado a-20 ° C) para a amostra FT e 6.6 µ l de buffer de x Laemmli 6 em amostra E. Incubar as amostras durante 5 min à 95 ° C.</li>
	<li>Carregar as amostras (20 µ l de FT e toda a eluir ~ 40 µ l) e executá-los por 30 min a 60 V, continuando com 20 V durante a noite.</li>
	<li>Usando um sistema eletroblotting semi-seco, transferência de proteínas para uma membrana PVDF para 80 min em 15 V. Nota: A membrana PVDF deve ser ativada antes de executar a transferência de proteína. Incube a membrana em metanol 100% por 1 min, seguido de uma incubação de 2 min em água ultrapura.</li>
	<li>Após a transferência da proteína, incube o gel de SDS-PAGE com Coomassie solução para 3 h, seguido por três etapas de lavagem com água ultrapura, a cada 30 min.</li>
	<li>No final da transferência de proteína, deixe-a membrana PVDF secar por 1 h. reativar a membrana como indicado no ponto 4.5. Bloquear a membrana 1 h com 5% BSA em 1 x TTBS.</li>
	<li>Incubar a membrana com o primeiro anticorpo: HuR ou mesothelin, ambos diluído 1: 1000 em 5% BSA em 1 x TTBS por 4 horas em temperatura ambiente ou durante a noite a 4 ° C.</li>
	<li>Lavar a membrana três vezes por 7 min em 1 x TTBS e incubar com peroxidase de coelho anti-rato rábano (HRP)-anticorpo secundário conjugado diluído 1:10,000 em 5% BSA, 1 x TTBS.</li>
	<li>Visualize as proteínas utilizando quimioluminescência aprimorada e um sistema de imagem digital.</li>
</ol>

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Os autores declaram que eles têm não tem interesses financeiro concorrente.

3' UTRs pertencem ao não-codificantes do genoma3, e todos os RNAs não codificantes podem interagir com as proteínas a fim de exercer sua função7. Quando o genoma de mamíferos foi encontrado para ser comodamente transcrito e produzido uma parcela significativa de tempo não-codificante RNAs16, emergentes evidências demonstraram que estes RNAs long-não-codificante função na regulação da expressão gênica como eles interagem com cromatina-r...

Expressão gênica e o nível final do produto do gene podem ser fortemente regulamentados por afetar a estabilidade do mRNA e o mRNA tradução taxa1. Estes mecanismos de regulação pós-transcricional são exercidos através de interações de não-codificantes do RNA e/ou ligação de RNA proteínas (RBPs) com mRNA alvo. Geralmente é a região untranslated 3' do mRNA (3' UTR - pertencentes à porção não-codificantes do genoma2) que contém específica cis-elementos reguladores (CRE), que são reconhecidos pelo trans-agindo fatores como miRNA ou RBPs 3. o mais bem estudadas cis-elemento dentro do 3' UTR, é motivo de adenina-rico elemento (são), que é reconhecido por proteínas específicas de AU-ligação (AUBP) e, por sua vez, induz qualquer degradação do mRNA/deadenylation (decaimento são-mediada) ou de estabilização do mRNA4.
O tamanho da 3' UTR de desnutridos mRNA (CALB2) é 573 bp longa e contém um pentâmero AUUUA putativo, como previsto pelos AREsite2, uma ferramenta de bioinformatic5. Consistente com a presença de um putativo são motivo, o ensaio de vetor-repórter pmirGLO demonstrado um papel de estabilização deste elemento dentro do mRNA CALB26. Finalmente, o em vitro RNA-pulldown foi usado para identificar a AUBP que estabiliza desnutridos mRNA através do motivo de são.
Desde que todos os RNAs não codificantes interagirem com proteínas7, o em vitro RNA-pulldown é um ensaio de primeira escolha para a identificação de RNA-interactianos e boa maneira para ajudar a decifrar os mecanismos moleculares. Neste método de RNA-pulldown, sondas de RNA sintetizadas comercialmente, que foram rotuladas com uma forma modificada de biotina (desthiobiotin) no terminal 3' da strand, RNA, foram usadas. O RNA-desthiobiotin está imobilizado através da interação com estreptavidina em grânulos magnéticos, que são usados para puxar para baixo de proteínas que interagem especificamente com o limite-RNA de interesse. Proteínas não-desnaturado e ativas da fracção citosólica de células mesotelioma são utilizadas como fonte de proteínas. Essas proteínas RNA-limite são eluídas dos grânulos magnéticos, executado através de um gel de SDS-PAGE 12%, transferido para uma membrana e sondado com anticorpos diferentes.
O procedimento de purificação padrão estreptavidina-biotina afinidade, dura desnaturação requeridas para romper o vínculo forte irreversível biotina-streptavidin para Eluir o limite proteínas8, que pode levar a dissociação de complexos de proteínas. Ao contrário de biotina, desthiobiotin reversível vincula streptavidin e competitivos é deslocada com uma solução tamponada de biotina, permitindo a eluição suave de proteínas e evitando o isolamento de naturalmente biotinilado moléculas9, sugerindo que a técnica é ideal para isolar os complexos da proteína nativa sob condições nativas.
In vitro as condições de vinculação e o rigor, que são determinados pela concentração de sal, agentes redutores e porcentagem de detergente, devem ser próximo aos presentes no contexto celular a fim de identificar interações de verdade na vivo . As condições de ligação implementadas neste documento foram anteriormente demonstradas conforme apropriado para a identificação da HuR como uma proteína ligadora AU10. Esta abordagem poderia poupar tempo, desde que a otimização das condições de vinculação adequada pode ser demorada e desafiador. Além disso, esse método poderia ser usado como um protocolo de partida para qualquer experiência de RNA-pulldown e pode ser otimizado progressivamente alterando a concentração de sais e detergentes, mudando a porcentagem de glicerol, e adicionando outros sais. Além disso, demonstrar que mesmo uma curta 25-nucleotídeo do RNA-sonda abrigando um pentâmero são-motivo poderia ser usado para demonstrar a interação com um específico AUBP.

<table><tbody><tr><td>NE-PER Nuclear and cytoplasmic extraction kit</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>78833</td><td></td></tr><tr><td>Pierce Protease Inhibitor Tablets, EDTA-free</td><td>Thermo Fisher Scientific</td><td>A32965</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>Pierce Magnetic RNA-protein Pull-Down Kit&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>20164</td><td>Includes magnetic beads and therefore the kit need to be stored at 4 &amp;deg;C</td></tr><tr><td>Pierce RNA 3&amp;rsquo; End Desthiobiotinylation Kit&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>20163</td><td>The kit is a part of the &quot;Pierce Magnetic RNA-protein Pull-Down Kit&quot;, and needs to be stored at -20 &amp;deg;C unlike the pull-down kit (4 &amp;deg;C).</td></tr><tr><td>DynaMag-2,&amp;nbsp; magnetic stand</td><td>Thermo Fisher Scientific</td><td>12321D</td><td></td></tr><tr><td>Anti - HuR (MOUSE) monoclonal antibody</td><td>Thermo Fisher Scientific</td><td>-</td><td>The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit&amp;nbsp; - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td></tr><tr><td>Anti - &amp;alpha; - tubulin (MOUSE) monoclonal antibody</td><td>Santa Cruz</td><td>8035</td><td>1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td></tr><tr><td>Anti - PARP (RABBIT) Polyclonal antibody</td><td>Cell Signaling</td><td>9542</td><td>1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS</td></tr><tr><td>Anti - Mesothelin (MOUSE) Monoclonal Antibody&amp;nbsp;</td><td>Rockland</td><td>200-301-A87</td><td></td></tr><tr><td>Secondary goat anti-rabbit antibody</td><td>Cell Signaling</td><td>7074</td><td></td></tr><tr><td>Secondary rabbit anti-mouse antibody</td><td>Sigma-Aldrich</td><td>A-9044</td><td></td></tr><tr><td>RPMI - 1640 medium</td><td>Sigma-Aldrich</td><td>R8758</td><td></td></tr><tr><td>FBS - Filtrated Bovine Serum</td><td>Pan Biotech&amp;nbsp;</td><td>P40-37500</td><td></td></tr><tr><td>Penicillin - streptomycin (100x)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P4333</td><td></td></tr><tr><td>L-Glutamine solution (200 mM)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G7513</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Gibco by Life technologies&amp;nbsp;</td><td>25200-056</td><td></td></tr><tr><td>Mini-PROTEAN 3 Cell</td><td>Bio-Rad</td><td>1653301</td><td></td></tr><tr><td>Mini Protean System Glass Plates&amp;nbsp;</td><td>Bio-Rad</td><td>1653308</td><td></td></tr><tr><td>Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer</td><td>Bio-Rad</td><td>1653312</td><td></td></tr><tr><td>Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 &amp;micro;L</td><td>Bio-Rad</td><td>1653365</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell Casting Modules</td><td>Bio-Rad</td><td>1658050</td><td></td></tr><tr><td>RNaseZap RNase Decontamination Solution</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1</td><td>Roth</td><td>3029</td><td></td></tr><tr><td>20% SDS&amp;nbsp;</td><td>PanReac AppliChem</td><td>A3942</td><td></td></tr><tr><td>PVDF transfer membrane&amp;nbsp;</td><td>Perkin Elmer</td><td>NEF1002</td><td>Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min&amp;nbsp;</td></tr><tr><td>Trans-Blot SD semi-dry transfer cell&amp;nbsp;</td><td>Bio-Rad</td><td>1703940</td><td></td></tr><tr><td>Clarity Western ECL Substrate</td><td>Bio-Rad</td><td>1705061</td><td></td></tr><tr><td>FusionFX&amp;nbsp; Digital Imager</td><td>Vilber</td><td>-</td><td></td></tr><tr><td>RNA oligo synthesis</td><td>Mycrosynth AG, Switzerland</td><td>-</td><td>the synthesis scale - 0.04 &amp;micro;mol - HPLC purified</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7030</td><td></td></tr><tr><td>Hydrochloric Acid (HCl) 6 M</td><td>PanReac AppliChem</td><td>182883.1211</td><td></td></tr><tr><td>Ultra Pure 1 M Tris, pH 7.5&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>15567027</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>17904</td><td>50% sterile aliquots to be stored at -20&amp;deg;C</td></tr><tr><td>Pierce Streptavidin magnetic beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>88817</td><td>Please note that these magnetic beads have an average diameter of 1 &amp;micro;m (range from 0.5 - 1.5 &amp;micro;m). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 &amp;micro;m, are also used in RNA-pulldown but be aware that this may affect purification process.</td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Ammonium persulfate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>Coomassie G-250</td><td>Applichem</td><td>A3480</td><td></td></tr><tr><td>Aluminiumsulfate-(14-18)-hydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>368458</td><td></td></tr><tr><td>ortho-phosphoric acid&amp;nbsp;</td><td>Applichem</td><td>A0637</td><td></td></tr></tbody></table>

desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells

O em vitro RNA-pulldown é ainda amplamente usado nos primeiros passos de protocolos que visam identificar RNA-proteínas que reconhecem estruturas de RNA e motivos específicos. Neste protocolo de RNA-pulldown, sondas de RNA sintetizadas comercialmente são rotuladas com uma forma modificada de biotina, desthiobiotin, no terminal 3' da strand, RNA, que se liga reversivelmente para streptavidin e permite assim a eluição das proteínas sob mais fisiológica condições. O RNA-desthiobiotin está imobilizado através da interação com estreptavidina em grânulos magnéticos, que são usados para puxar para baixo de proteínas que interagem especificamente com o RNA de interesse. Proteínas não-desnaturado e ativas da fracção citosólica de células mesotelioma são utilizadas como fonte de proteínas. O método descrito aqui pode ser aplicado para detectar a interação entre proteínas de ligação de RNA conhecidas e uma 25-nucleotide (nt) longo RNA sonda contendo uma sequência de interesse. Isso é útil para completar a caracterização funcional de estabilização ou desestabilizar os elementos presentes em moléculas de RNA alcançadas usando um ensaio de vetor de repórter.

Neste experimento, utilizou-se um fragmento de tempo 25-nt de desnutridos 3' UTR abrigando são motivo (CALB2) 3' UTR (são) 25-nt para testar se liga-se especificamente à proteína antígeno humano-R (HuR), um conhecido estabilizador de mRNA. Para testar a especificidade do elemento são, um RNA 25-nt sonda CALB2 3' UTR (mtARE) contendo uma mutação são-motivo, que anteriormente foi mostrada para abolir o efeito de estabilização do motivo são, foi usado6. O ...

Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin-Streptavidin-afinidade mediada purificação de proteínas RNA-interagindo em células mesotelioma

Desthiobiotin rotulagem de um sintético 25-nucleotide oligo de RNA, que contém um motivo de adenina-rico elemento (são), permite a ligação específica da proteína de ligação são citosólica.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

desthiobiotin-streptavidin-affinity-mediated-purification-rna

Research

JoVE Journal

Cancer Research

7.9K visualizações.  University Hospital Zurich. Desthiobiotin rotulagem de um sintético 25-nucleotide oligo de RNA, que contém um motivo de adenina-rico elemento (são), permite a ligação específica da proteína de ligação são citosólica.

Vídeo: Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

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.

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 ...

Genética

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

Pulldown biotina-baseado do ensaio para validar mRNA miRNAs alvos de celular

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabólica de rotulagem com 4-thiouracil e a quantificação dos recém sintetizado mRNA como um Proxy para a RNA polimerase II atividade

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

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 ...

Bioquímica

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Ensaios bioquímicos in vitro utilizando rótulos de biotina para estudar interações ácido proteína-nucleico

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

Purificação de etapa única de complexos macromoleculares usando RNA ligado à biotina e um foto-cleavable vinculador

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

Transcriome-Wide Perfil de Interações Protein-RNA por Cross-Linking e Imunoprecipitação Mediada pela Flag-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

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 ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Isolamento de Ribossomos Traduzindo Contendo peptidil-tRNAs para análises funcionais e estruturais

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Biologia

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Detecção de proteínas de ligação a ARN pela<em&gt; In Vitro</em&gt; RNA Pull-down em Cultura adipócito

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...