Agradecemos a membros do laboratório Tuschl para discussões úteis. MH é suportado pelo Deutscher Akademischer Austauschdienst (DAAD). Este trabalho foi financiado pelo Fundo Nacional Suíço Grant # 3100A0-114001 a MZ; TT é um investigador HHMI, e trabalhar em seu laboratório foi apoiado pelo NIH concede GM073047 e MH08442 ea Fundação Starr.

O protocolo a seguir descreve o procedimento PAR-clip para HEK293 células que expressam FLAG / HA-tagged IGF2BP1 após indução com doxiciclina. Vamos usar um anticorpo anti-FLAG para imunoprecipitação. PAR-Clip irá funcionar com qualquer linha de células expressando níveis detectáveis ​​do endógeno, untagged RNA binding protein (RBP) de juros, se um anticorpo eficiente para imunoprecipitação está disponível. Células expansão <ol><li> Expandir FlpIn-HEK293/TO/FLAG/HA-IGF2BP1 células em meio de crescimento. Recomendamos o uso de entre 100-400 x 10 6 células (aprox. 10-40 15 centímetros placas de cultura de células) como ponto de partida. Cultivá-las a aproximadamente 80% confluência. </li><li> 14 h antes de adicionar um crosslinking) 4 Tiouridina a uma concentração final de 100 mM (1:1000 v / v de um M-1 4 Tiouridina solução estoque) diretamente para o meio de cultura celular e b) induzir a expressão do FLAG / HA tagged IGF2BP1 pela adição de 1 mg / ml de doxiciclina (1:10.000 v / v da solução estoque 10 mg / ml doxiciclina). NOTA: em vez de 4-Tiouridina você também pode usar 100 mM de 6 thioguanosine. </li></ol> UV-Crosslinking <ol><li> Lave as células uma vez com 10 ml gelada PBS por placa e remover completamente PBS. </li><li> Placas lugar em uma bandeja com gelo e irradiar descoberto com 0,15 J / cm 2 de 365 nm de luz UV em um Stratalinker 2400 (Stratagene) ou dispositivo similar. </li><li> Raspe as células com um policial de borracha em 1 ml de PBS por transferência de placas, para 50 tubos de centrifugação ml e recolher por centrifugação a 500 xg por 5 min a 4 ° C e desprezar o sobrenadante. 100 x 10 6 células HEK293 (10 15 pratos cm) vai render aprox. 1 ml de pellet celular molhado. </li><li> (Opcional) menos que você queira continuar diretamente com a lise celular, choque congelar o pellet celular em nitrogênio líquido e armazenar a -80 ° C. Pellets de células podem ser armazenadas por pelo menos 12 meses. </li></ol> Lise celular e RNaseT1 digest <ol><li> Tome-se pellet celular de células reticuladas em 3 volumes de 1x tampão de lise NP40 e incubar no gelo por 10 min. </li><li> Lisado de células claras por centrifugação a 13.000 xg por 15 min a 4 ° C. </li><li> Limpar o lisado ainda filtrando-a através de uma membrana de 0,2 mm seringa filtro (Acrodisc Pall ou equivalente). </li><li> Adicionar RNase T1 (Fermentas, 10.000 U / mL) para uma concentração final de C. ° 1 U / mL e incubar em banho-maria por 15 min a 22 Reação fria posteriormente por 5 min no gelo antes de prosseguir. </li></ol> Imunoprecipitação e recuperação de fragmentos de RNA alvo reticulado Usando o separador magnético Siga estas orientações ao longo da preparação da amostra para evitar a esferas magnéticas de secar. <ol><li> Coloque o tubo que contém as contas sobre o suporte magnético para 1 2 minutos. </li><li> Adicione o buffer para o tubo enquanto que o tubo está no separador magnético. </li><li> Tampe o tubo, remova-o do separador magnético, e ressuspender as esferas. Você pode voltar a suspender as contas sacudindo o tubo com o dedo ou usar um vortexer fixado em 5 6. </li><li> Centrifugar brevemente para coletar todas as pérolas que podem permanecer na tampa do tubo. </li><li> Repita os passos 1 a 4, conforme necessário. </li></ol> Preparação de esferas magnéticas <ol><li> Transferência de 10 ml de partículas de proteínas Dynabeads G magnética (Invitrogen) por lisado celular ml (para uma experiência típica que deve ser aprox. 40 50 ul de contas) para um microtubo de 1,5 ml. Lavar contas duas vezes com 1 ml de tampão citrato-fosfato. </li><li> Ressuspender em duas vezes o volume de tampão citrato-fosfato em relação ao volume original de suspensão de esferas. </li><li> Adicionar 0,25 mg de anticorpo anti-FLAG M2 monoclonal (Sigma) por ml de suspensão e incubar em uma roda giratória por 40 min em temperatura ambiente. </li><li> Lavar contas duas vezes em 1 ml de tampão citrato-fosfato para remover anticorpos não ligados. </li><li> Ressuspender contas em duas vezes o volume de tampão citrato-fosfato em relação ao volume original de suspensão de esferas. </li></ol> Imunoprecipitação (IP), segundo a digestão RNase T1 e desfosforilação <ol><li> Adicionar 20 l de recém-preparados anticorpos conjugados esferas magnéticas por ml de RNase T1 parcial lisado celular tratados e incubar em 15 tubos de centrifugação ml em uma roda giratória por 1 hora a 4 ° C. </li><li> Colete esferas magnéticas em um coletor de partículas magnéticas para 15 e 50 tubos de centrifugação ml (Invitrogen) e transferir para tubos de 1,5 ml de microcentrífuga. </li><li> Lavar contas 3 vezes em 1 ml de tampão de lavagem IP. </li><li> Adicionar RNaseT1 (Fermentas, 10.000 U / mL) a uma concentração final de 100 U / mL e incubar a suspensão de esferas em banho-maria por 15 min a 22 ° C. Legal posteriormente no gelopor 5 min. </li><li> Lavar contas 3 vezes em 1 ml de alto-sal tampão de lavagem. </li><li> Contas ressuspender em 1 volume de tampão desfosforilação </li><li> Adicionar fosfatase alcalina intestinal bezerro (CIAP, NEB) para uma concentração final de 0,5 U / mL, e incubar a suspensão por 10 min a 37 ° C. </li><li> Lavar contas duas vezes em 1 ml de fosfatase tampão de lavagem </li><li> Lavar contas duas vezes em polinucleotídeo quinase buffer (PNK) sem TDT (televisão digital terrestre a concentração necessária para a reação enzimática é alta o suficiente para danificar a esferas magnéticas). </li><li> Contas ressuspender em um volume original talão de PNK tampão </li></ol> Radiomarcação dos segmentos de RNA reticulado às proteínas immunoprecipitated <ol><li> À suspensão de esferas descrito acima, adicione Υ-32 P-ATP para uma concentração final de 0,5 PNK μCi / mL e T4 (NEB) a 1 U / mL em um volume original talão. Incubar a suspensão por 30 min a 37 ° C. </li><li> Adicionar não-radioativo ATP para obter uma concentração final de 100 mM e incubar por mais 5 minutos a 37 ° C. </li><li> Lavar a esferas magnéticas 5 vezes com 800 mL de tampão PNK sem DTT. </li><li> Volte a suspender as contas em 70 mL de SDS-PAGE tampão de carregamento. </li></ol> SDS-PAGE e eletroeluição reticulado de RNA-proteína complexos de fatias de gel <ol><li> Incubar a suspensão radiolabeled por 5 min em um bloco de aquecimento a 95 ° C para desnaturar e solte o RBP immunoprecipitated com RNA reticulado e vortex. </li><li> Remova a esferas magnéticas sobre o separador e transferir o sobrenadante para um tubo limpo de microcentrífuga de 1,5 ml. </li><li> Carga 40 mL do sobrenadante por poço de uma Novex Bis-Tris 4-12% (Invitrogen) em gel de poliacrilamida pré-moldados e executar o gel por 55 min em 200 V. </li><li> Desmonte a câmara de gel e desmantelar o gel, deixando-a montada em uma placa. Para facilitar o alinhamento do gel para a impressão em papel phosphorimager, recomendamos implantar três minúsculos pedaços gel radioativos assimetricamente menos três dos quatro cantos do gel. Peças gel radioativos podem ser coletados a partir de géis que foram anteriormente utilizadas para purificar radiolabeled oligonucleotídeos sintéticos. Embrulhe o gel em filme plástico (por exemplo, Saran wrap) para evitar a contaminação. </li><li> Expõe o gel a uma tela phosphorimager blanked para 1 h e visualizar em um phosphorimager. </li><li> Alinhar o gel na parte superior da impressão phosphorimager usando as peças gel implantado para orientação. Cortar as bandas que correspondem ao tamanho esperado de RBP (IGF2BP1, aprox. 75 kDa) e transferir para um D-Tube Midi dialisadores e adicionar 800 mL 1x tampão SDS execução. </li><li> Electroelute o reticulado RNA-RBP complexo em 1x SDS tampão de corrida a 100 V por 2 h. excisadas do gel e electroeluted em uma D-Tube de dialisadores Midi (Novagen) em 800 mL de buffer SDS execução de acordo com as instruções do fabricante. </li></ol> Digestão por proteinase K <ol><li> Adicionar um volume igual de tampão 2x proteinase K em relação ao electroeluate, seguido pela adição de proteinase K (Roche) para uma concentração final de 1,2 mg / ml. Incubar por 30 min a 55 ° C. </li><li> Recuperar o RNA por ácido fenol / clorofórmio / extração IAA (25:24:1, pH 4,0), seguido por uma extração de clorofórmio. Adicionar 1 ml de glicogênio (estoque 10 mg / ml) precipitar o RNA, adicionando 3 volumes de etanol. Dissolver o sedimento em 10,5 mL de água. </li></ol> preparação biblioteca de cDNA e sequenciamento de profundidade Levar o RNA recuperados através de um protocolo de preparação de cDNA da biblioteca padrão originalmente descrita para a clonagem de pequenos RNAs reguladores 19. O primeiro passo, a ligadura 3 &#39;adaptador, foi realizado como descrito em uma escala de 20 l utilizando 10,5 mL do RNA recuperado. Use o seqüenciamento Solexa adaptador conjuntos descritos. Dependendo da quantidade de RNA recuperado, 5&#39;-adaptador-3&#39;-adaptador produtos sem inserções podem ser detectados após a amplificação do cDNA como faixas adicionais PCR. Em tais casos, o produto mais impostos PCR de tamanho esperado de um NuSieve 3% de baixo ponto de fusão agarose gel, eluída do produto da PCR do gel peças usando o kit GelElute (Qiagen) e seqüência usando a tecnologia Solexa. Um Solexa seqüenciamento executar normalmente oferece, entre 6 e 10 milhões seqüência lê-se que são suficientes para uma cobertura ampla do transcriptoma sítios de ligação das proteínas RNA vinculativo. Análise bioinformática Análise bioinformática cuidadosa da seqüência lê precisa ser feito para obter insights significativos para os locais de ligação para o RNA RBP examinados, como o elemento de reconhecimento de RNA, as regiões preferenciais de ligação a RBP tem (exônico vs intrônicos, codificação seqüência vs não traduzidas seqüência). A seqüência lê precisam ser alinhados contra as bases de dados do genoma e EST. Nós usamos geralmente lê mapping exclusivamente ao genoma com até um desencontro de inserção ou exclusão de construir clusters de seqüência de leituras que podem então ser analisados. A freqüência de mutações características na seqüência cluster lê, T a transições C ao usar 4-SU e G para A transição quando utilizando 6-SG, são indicativos de seqüências de sucesso reticulado. Em nossa experiência RNAs uncrosslinked rotulados com 4-SU mostram uma taxa de mutação de fundo de cerca de 20%. Esta taxa é aumenta para aprox. 50-80% em cima de reticulação. A descrição detalhada da análise bioinformática pode ser encontrado no material suplementar da publicação por Hafner et al. 18 Passos opcional Determinação dos níveis de incorporação de 4-Tiouridina em RNA total Isolar RNA total a partir da linha celular estável expressando a RBP de interesse depois de crescer em meio suplementado com 100 mM 4SU 16 h antes da colheita. Como controle, as células cultivadas sem adição de colheita 4SU. Isolar RNA total através da adição de 3 volumes de reagente Trizol (Sigma) para as pelotas de células lavadas seguindo as instruções do fabricante s. Além disso foi purificar RNA total usando Qiagen RNeasy acordo com o protocolo do fabricante. Para evitar oxidação da 4SU durante RNA isolamento e análise, adicionar 0,1 mM ditiotreitol (TDT) para os buffers de lavar e subseqüentes passos enzimáticos. Digerir e desfosforilada RNA total de nucleosídeos único para a análise de HPLC como descrito anteriormente 20. Brevemente, em um volume de 30 mL, incubar 40 mg de RNA total foram purificados por 16 horas a 37 ° C com 0,4 U de fosfatase alcalina bacteriana (Worthington Bioquímica) e 0,09 veneno de cobra U fosfodiesterase (Worthington Bioquímica). Como padrão de referência, use um RNA sintético 4SU marcado, (nós standardly uso CGUACGCGGAAUACUUCGA (4SU) U) e também submetê-lo para completar a digestão enzimática. Separar os misturas de ribonucleosídeos por HPLC em uma descoberta Supelco C18 (ligado partículas de sílica de fase M 5, 250 x 4,6 mm) coluna de fase reversa (Bellefonte PA, EUA). Buffers HPLC são 0,1 M TEAA em acetonitrila 3% (A) e acetonitrila 90% em água (B). Use um gradiente isocrático: 0 B% por 15 min, 0-10 B% por 20 min, 1-10 B% por 30 min. Aplicar a 5 min B 100% lavar aplicada entre corre para limpar a coluna de HPLC. Resultados representante <img src="/files/ftp_upload/2034/2034fig1.jpg" alt="Figura 1" /> Figura 1 (painel da direita) mostra um resultado representativo de um PAR-Clip realizado com linhagens de células expressando FLAG / HA-tagged IGF2BP1 com 4 e 6-SU-SG. Note-se que a eficiência de reticulação de 6-SG para IGF2BP1 é menor do que a eficiência de reticulação para 4-SU. A baixa eficiência de reticulação irá resultar em uma maior fundo de seqüências derivadas de fragmentos de RNAs celulares abundantes e, portanto, você deve considerar a expansão da experiência ao usar menos eficiente nucleosídeos fotorreativas. O painel esquerdo da Figura 1 mostra uma comparação do uso de diferentes análogos uridina fotorreativas que poderia ser potencialmente usada para PAR-Clip em comparação com crosslinking nm tradicional UV 254. A intensidade da banda radioativo do comprimento correto lhe dá uma boa idéia se o experimento PAR-Clip tem trabalhado e tem isolado RNA suficiente para realizar um pequeno RNA seqüenciamento protocolo (passo-a-passo para a preparação descrição biblioteca de cDNA de pequenas seqüenciamento RNAs podem ser encontrados em 19). A freqüência de mutações características lê na seqüência, T a transições C ao usar 4-SU e G para A transição quando utilizando 6-SG, são indicativos de seqüências de sucesso reticulado. Em nossa experiência RNAs uncrosslinked rotulados com 4-SU mostram uma taxa de mutação de fundo de cerca de 20%. Esta taxa é aumenta para aprox. 50-80% em cima de reticulação.

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

TT é um conselheiro co-fundador e científicos para Alnylam Pharmaceuticals e um assessor de Regulus Therapeutics.

Tampões e reagentes Crescimento a médio HEK293 células <ul><li> DMEM </li><li> 10% FBS </li><li> 2 mM L-Glutamina </li><li> 100 U / ml de penicilina </li><li> Estreptomicina 100 U / ml </li><li> Higromicina 100 mg / ml </li><li> 15 mcg / ml blasticidin </li></ul> 4 Tiouridina solução estoque (1 M) <ul><li> 260,27 mg de 4 Tiouridina </li><li> 1 ml DMSO </li></ul> Doxiciclina estoque (10 mg / ml) <ul><li> 10 mg doxiciclina </li><li> 1 ml DMSO </li></ul> 1x tampão de lise NP40 Prepare um estoque de tampão 5x sem TDT e os inibidores da protease. Adicionar TDT e inibidor da protease diretamente antes do experimento. <ul><li> 50 mM HEPES, pH 7.5 </li><li> 150 mM KCl </li><li> 2 mM EDTA </li><li> 1 mM NaF </li><li> 0,5% (v / v) NP40 </li><li> 0,5 mM DTT </li><li> completa EDTA livre coquetel inibidor de protease (Roche) </li></ul> Tampão citrato-fosfato <ul><li> 4,7 g / l de ácido cítrico </li><li> 9,2 g / l Na 2 HPO 4 </li><li> pH 5,0 </li></ul> IP lave-buffer <ul><li> 50 mM HEPES-KOH, pH 7,5 </li><li> 300 mM KCl </li><li> 0,05% (v / v) NP40 </li><li> 0,5 mM DTT (adicionar diretamente antes de experiência) </li><li> completa EDTA livre coquetel inibidor de protease (Roche) (adicionar diretamente antes da experiência) </li></ul> Alto-sal tampão de lavagem <ul><li> 50 mM HEPES-KOH, pH 7,5 </li><li> 500 mM KCl </li><li> 0,05% (v / v) NP40 </li><li> 0,5 mM DTT (adicionar diretamente antes do experimento) </li><li> completa EDTA livre coquetel inibidor de protease (Roche) (adicionar diretamente antes do experimento) </li></ul> Buffer de desfosforilação <ul><li> 50 mM Tris-HCl, pH 7,9 </li><li> 100 mM NaCl </li><li> 10 mM MgCl 2 </li><li> 1 mM DTT </li></ul> Fosfatase tampão de lavagem <ul><li> 50 mM Tris-HCl, pH 7,5 </li><li> 20 mM EGTA </li><li> 0,5% (v / v) NP40 </li></ul> Polinucleotídeo quinase buffer (PNK) sem TDT <ul><li> 50 mM Tris-HCl, pH 7,5 </li><li> 50 mM NaCl </li><li> 10 mM MgCl 2 </li></ul> PNK tampão <ul><li> 50 mM Tris-HCl, pH 7,5 </li><li> 50 mM NaCl </li><li> 10 mM MgCl 2 </li><li> 5 mM DTT </li></ul> SDS PAGE tampão de carregamento <ul><li> Glicerol 10% (v / v) </li><li> 50 mM Tris-HCl, pH 6,8 </li><li> 2 mM EDTA </li><li> 2% SDS (w / v) </li><li> 100 mM DTT </li><li> 0,1% azul de bromofenol </li></ul> 2x tampão proteinase K <ul><li> 100 mM Tris-HCl, pH 7,5 </li><li> 150 mM NaCl </li><li> 12,5 mM EDTA </li><li> 2% (w / v) SDS </li></ul>

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

Transcrições de RNA são submetidos a pós-transcricional de regulação gênica, interagindo com centenas de RNA-proteínas de ligação (RBPs) e microRNA contendo complexos de ribonucleoproteínas (miRNPs) que muitas vezes são expressos em uma célula do tipo dependente. Para entender como a interação desses fatores RNA-binding afeta a regulação da transcrição individual, mapas de alta resolução de in vivo da proteína-RNA interações são necessárias 1. Uma combinação de fatores genéticos, bioquímicos e abordagens computacionais são tipicamente aplicados para identificar RNA-RBP ou RNA-RNP interações. Microarray perfis de RNAs associados RBPs immunopurified (RIP-Chip) 2 define metas em nível de transcriptoma, mas sua aplicação é limitada para a caracterização das interações cineticamente estável e somente em casos raros 3,4 permite identificar o elemento de reconhecimento de RBP (RRE ) dentro do RNA alvo de comprimento. Mais direta RBP informações do site-alvo é obtido através da combinação in vivo crosslinking UV 5,6 imunoprecipitação com 7-9 seguido pelo isolamento de segmentos de RNA reticulado e seqüenciamento de cDNA (CLIP) 10. CLIP foi usado para identificar alvos de uma série de RBPs 11-17. No entanto, CLIP é limitada pela baixa eficiência de UV 254 nm crosslinking RNA-proteína, e da localização do crosslink não é facilmente identificável dentro dos fragmentos seqüenciados reticulado, o que torna difícil separar segmentos de UV-reticulado RNA alvo de fundo não-reticulado RNA também fragmentos presentes na amostra. Nós desenvolvemos uma abordagem crosslinking poderosa baseada em células para determinar em alta resolução e transcriptoma-wide os sítios de ligação de celular e RBPs miRNPs que chamamos de PAR-Clip (Photoactivatable-ribonucleosídeo-Enhanced Crosslinking e imunoprecipitação) (ver fig. 1A para um esboço do método). O método baseia-se na incorporação de análogos de ribonucleosídeo fotorreativas, tais como 4-Tiouridina (4-SU) e 6-thioguanosine (6-SG) em transcrições de RNA nascente por células vivas. Irradiação das células pela luz UV de 365 nm induz crosslinking eficiente de fotorreativas nucleosídeos marcado RNAs celulares para RBPs interagindo. Imunoprecipitação da RBP de interesse é seguido pelo isolamento do RNA reticulado e coimmunoprecipitated. O RNA isolado é convertida em uma biblioteca de cDNA e profundo seqüenciado usando a tecnologia Solexa. Um traço característico de bibliotecas de cDNA preparados por PAR-Clip é que a posição precisa de reticulação podem ser identificados por mutações que residem no cDNA seqüenciado. Ao usar 4-SU, timidina seqüências reticulado para a transição da citidina, enquanto usando 6-SG resulta em guanosina à adenosina mutações. A presença das mutações em seqüências reticulado torna possível separá-las do fundo de seqüências derivadas de abundantes RNAs celulares. Aplicação do método a um número de diversas proteínas de ligação RNA foi relatada em Hafner et al. 18

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

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

Transcrições RNA estão sujeitos à regulamentação posttranscriptional extensa que é mediada por uma multidão de proteínas RNA-binding trans-acting (RBPs). Aqui apresentamos um método generalizado para identificar com precisão e em escala transcriptoma-wide os sites ligação do RNA RBPs.

PAR-Clip - um método para identificar em toda a Transcriptoma Sítios de Ligação de Proteínas RNA Binding

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

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

Research

JoVE Journal

Biology

53.2K visualizações.  Rockefeller University. Transcrições RNA estão sujeitos à regulamentação posttranscriptional extensa que é mediada por uma multidão de proteínas RNA-binding trans-acting (RBPs). Aqui apresentamos um método generalizado para identificar com precisão e em escala transcriptoma-wide os sites ligação do RNA RBPs.

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

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

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

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

Actina Ensaio Co Sedimentação; para a Análise da proteína de ligação a F-Actina

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

Biologia

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

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

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

Proteômica para identificar proteínas Interagindo com P2X2 Canais Ligand-Gated Cation

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

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

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

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

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

Perfil de metiltransferases e Outros S-Adenosil- L Homocisteína-Proteínas de Ligação de captura por Espectrometria de Massa Compound (CCMS)

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

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

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

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

Coleta variável concentração Datasets Calorimetria Isotérmica de Titulação a fim de determinar mecanismos de ligação

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

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

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

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

iCLIP - Transcriptoma-wide Mapeamento das Interações Proteína-RNA com a Resolução Nucleotide Individual

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

Pull-down of Calmodulin-binding Proteins

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

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

Pull-down da Calmodulina Proteínas de Ligação

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

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

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

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

Modificado Sistema Yeast-Two-Hybrid para identificar proteínas Interagindo com o Fator de Crescimento progranulina

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

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

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

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

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

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

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

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

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

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

Método de purificação de proteína livre de Determinação Affinity Binding por termoforese Microscale

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

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

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

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

Um método de quantificação sensível e específico para Determinação do Serum miosina cardíaca Binding Protein-C por Imunoensaio electroquimioluminescência

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