Biz yararlı tartışmalar için Tuschl laboratuvar üyeleri teşekkür ederiz. MH Deutscher Akademischer Austauschdienst (DAAD) tarafından desteklenmektedir. Bu çalışma, İsviçre Ulusal Fonu Hibe MZ # 3100A0-114.001 tarafından desteklenen; TT bir HHMI araştırmacı, ve onun laboratuvarda çalışma NIH hibe GM073047 ve MH08442 ve Starr Vakfı tarafından desteklenmiştir.

Aşağıdaki protokol HEK293 hücreleri ifade BAYRAK / doksisiklin ile indüksiyon üzerine IGF2BP1 HA-etiketli PAR-Klip prosedürü açıklamaktadır. Biz anti-BAYRAK antikor immunoprecipitation için kullanacaktır. PAR-Klip immunoprecipitation için etkin bir antikor varsa, herhangi bir hücre hattı ilgi RNA bağlayıcı protein (RBP) etiketsiz endojen saptanabilir düzeyleri, ifade çalışacaktır. Genişleyen Hücreler <ol><li> Besi FlpIn-HEK293/TO/FLAG/HA-IGF2BP1 hücreleri genişletin. Biz 100-400 x 10 6 hücre (yaklaşık 10-40 15 cm hücre kültürü plakaları) arasında bir başlangıç ​​noktası olarak kullanmanızı öneririz. Yaklaşık% 80 confluency büyütün. </li><li> Crosslinking 14 saat önce doğrudan hücre kültür ortamına 100 mcM bir final konsantrasyonu ((1:1000) 1 M 4-thiouridine stok solüsyonu v / v) a) 4-thiouridine eklemek ve b) BAYRAK / HA ifadesi neden doksisiklin (10 mg / ml doksisiklin stok solüsyonu 1:10,000 v / v) 1 mcg / ml ilave edilerek IGF2BP1 etiketli. NOT: 4-thiouridine yerine 6-thioguanosine 100 mcM kullanabilirsiniz. </li></ol> UV-Crosslinking <ol><li> Plaka başına 10 ml buz gibi soğuk PBS ile hücreler bir kez yıkayın ve tamamen kaldırmak PBS. </li><li> Buz ve Stratalinker 2400 0.15 J / cm 2 365 nm UV ışığı (Stratagene) veya benzeri bir cihaz ile ortaya çıkarılan ışın tedavisi ile bir tepsi yerleştirin plakalar. </li><li> Plaka, 50 ml santrifüj tüplerine transferi başına 1 ml PBS içinde lastik bir polis memuru ile hücreleri kazıyın ve 4 ° C&#39;de 5 dakika boyunca 500 xg&#39;de santrifüj yoluyla toplamak ve supernatant atın. 100 x 10 6 HEK293 hücreleri (10 x 15 cm plakaları) yaklaşık verecektir. Islak hücre pelet 1 ml. </li><li> (Isteğe bağlı) hücre parçalama ile doğrudan devam etmek istemiyorsanız, şok -80 sıvı azot ve mağaza hücre pelletini ° C dondurmak Hücre pelletleri en az 12 ay süreyle saklanabilir. </li></ol> Hücre parçalama ve RNaseT1 sindirimi <ol><li> 1x NP40 lizis tamponu 3 cilt halinde çapraz hücreleri hücre pelletini alın ve 10 dakika buz üzerinde inkübe. </li><li> 15 dakika 13.000 xg&#39;de santrifüj 4 Berrak hücreli lizat ° C </li><li> 0.2 mikron membran şırınga filtresi (Pall Acrodisc veya eşdeğeri) üzerinden filtreleme lizat temizleyin. </li><li> 1 U / ul ve 15 dakika için bir su banyosunda inkübe at 22 ° C arasında nihai bir konsantrasyona RNaz T1 (Fermentas, 10.000 U / ml) Devam etmeden önce 5 dakika boyunca buz üzerinde sonradan Cool tepki. </li></ol> Immunoprecipitation kurtarma ve çapraz hedef RNA parçaları Manyetik ayırıcı Manyetik boncuklar kurumasını önlemek için numune hazırlama boyunca bu yönergeleri izleyin. <ol><li> 1 2 dakika boyunca manyetik standında boncuklar içeren tüp yerleştirin. </li><li> Tüp tüp manyetik ayırıcı açıkken tampon ekleyin. </li><li> Tüp kapağı, manyetik ayırıcı kaldırmak ve boncuklar tekrar süspansiyon. Tüp parmağınızla hafifçe vurarak boncuklar tekrar süspansiyon veya 5 6 vortexer kullanabilirsiniz. </li><li> Tüp kap kalır herhangi bir boncuk toplamak için kısaca santrifüjleyin. </li><li> Gerektiği gibi 1 ile 4 arasındaki adımları tekrarlayın. </li></ol> Manyetik boncuk hazırlanması <ol><li> 10 ul (Invitrogen) Dynabeads Protein G manyetik partiküller ml başına hücre lizat Devret (olması gereken tipik bir deney için yaklaşık 40 50 boncuk ul) 1.5 ml mikrofuge&#39;de tüp. Boncuk iki kez 1 ml sitrat-fosfat tamponu ile yıkayın. </li><li> Sitrat-fosfat tamponu boncuk süspansiyonu orijinal hacmine göre iki kat daha fazla hacim olarak yeniden süspanse edin. </li><li> 0.25 mikrogram anti-BAYRAK M2 monoklonal antikor (Sigma), oda sıcaklığında 40 dakika süreyle dönen bir tekerlek başına ml süspansiyon ve inkübe ekleyin. </li><li> Bağlanmamış antikor kaldırmak için 1 ml sitrat-fosfat tamponu boncuk iki kez yıkayın. </li><li> Boncuk boncuk süspansiyonu orijinal hacmine göre sitrat-fosfat tamponu iki kez hacmi tekrar </li></ol> Immunoprecipitation (IP), ikinci RNaz T1 sindirim, ve Defosforilasyon <ol><li> 20 ml başına kısmi RNaz T1 tedavi hücre lizat taze hazırlanmış antikor konjuge manyetik boncuk ul ekleyin ve 4, 1 saat için dönen bir tekerlek üzerinde 15 ml santrifüj tüpleri inkübe ° C </li><li> 15 ve 50 ml santrifüj tüpleri (Invitrogen) bir manyetik parçacık toplayıcı manyetik boncuk toplayın ve 1,5 ml mikrofuge&#39;de tüpler transfer. </li><li> IP yıkama tamponu 1 ml boncuk, 3 kere yıkayın. </li><li> 100 U / ml bir final konsantrasyon RNaseT1 (Fermentas, 10.000 U / ml) ekleyin ve 15 dakika süreyle su banyosunda 22 boncuk süspansiyon inkübe ° C. Buz üzerinde daha sonra soğutun5 dk. </li><li> 1 ml yüksek tuz yıkama tamponu boncuk, 3 kere yıkayın. </li><li> Defosforilasyon tampon 1 hacmi içinde süspanse edin boncuk </li><li> 0.5 U / ml nihai konsantrasyonu buzağı intestinal alkalen fosfataz (CIAP NEB) ekleyin ve 10 dakika süreyle askıya inkübe 37 ° C </li><li> Fosfataz yıkama tamponu 1 ml boncuk iki kez yıkayın </li><li> DTT (DTT konsantrasyonu enzimatik reaksiyon için gerekli olan manyetik boncuklar zarar verecek kadar yüksek) olmadan iki kez polinükleotid kinaz (PNK) tampon boncuk yıkayın. </li><li> PNK tampon bir orijinal boncuk hacmi yeniden süspanse boncuk </li></ol> Immunoprecipitated proteinlerin çapraz RNA segmentleri, Radyoaktif <ol><li> Yukarıda açıklanan boncuk süspansiyonu, tek bir özgün boncuk hacmi 1 U / ul 0.5 μCi / ml ve T4 PNK nihai konsantrasyonu (NEB) Υ-32 P-ATP eklemek . 37 30 dakika süspansiyon inkübe ° C </li><li> 100 mcM son bir konsantrasyon elde etmek ve 37 başka bir 5 dakika boyunca inkübe non-radyoaktif ATP ° C </li><li> DTT olmadan PNK tampon 800 ul manyetik boncuk 5 kez yıkayın. </li><li> SDS-PAGE yükleme tamponu 70 ul boncuk süspanse edin. </li></ol> SDS-PAGE jel dilimleri ve çapraz RNA-protein kompleksleri, electroelution <ol><li> Radyoaktif süspansiyon bir ısı bloğu 5 dakika 95 ° C&#39;de denatüre ve çapraz bağlı RNA ve vorteks immunoprecipitated RBP bırakın. </li><li> Ayırıcı manyetik boncuk çıkarın ve temiz bir 1.5 ml mikrofuge&#39;de tüp süpernatantı transfer. </li><li> 40 süpernatantı ul için iyi bir Novex Bis-Tris% 4-12 (Invitrogen) prekast poliakrilamid jel yerleştirin ve 55 dakika süreyle 200 jel çalıştırmak V. </li><li> Jel odasının sökün ve bir plaka üzerine monte bırakarak, jel sökmeye. Phosphorimager kağıt çıktısı jel uyum kolaylaştırmak için, jel dört bir köşesinden üç asimetrik üç küçük radyoaktif jel parçaları implante tavsiye ederiz. Radyoaktif jel parçaları önceden radyoaktif sentetik oligonükleotidler arındırmak için kullanılan jeller toplanabilir. Kontaminasyonu önlemek için plastik film (örneğin, Saran wrap) jel sarın. </li><li> 1 saat blanked phosphorimager ekran jel ortaya çıkarmak ve bir phosphorimager görselleştirmek. </li><li> Yönelim implante jel parçaları kullanılarak phosphorimager çıktının üst jel hizalayın. RBP beklenen boyutu (IGF2BP1, yaklaşık 75 kDa) karşılık gelen ve D-Tube diyalizer Midi Tüp transfer ve 800 ul 1x SDS çalışan tampon bantları kesin. </li><li> 2 saat için 100 V tampon çalışan 1x SDS çapraz RNA RBP kompleksi Electroelute jel eksize ve üreticinin talimatlarına göre 800 ul SDS çalışan tampon D-Tube diyalizer Midi (Novagen) electroeluted. </li></ol> Proteinaz K sindirimi <ol><li> Proteinaz K (Roche), son konsantrasyonu 1.2 mg / ml &#39;lik bir ek tarafından takip electroeluate açısından eşit bir ses, 2x Proteinaz K Tampon ekleyin. 55 az 30 dakika boyunca inkübe ° C. </li><li> Asidik fenol / kloroform / IAA çıkarımı (25:24:1, pH 4.0), kloroform ekstraksiyon takip RNA kurtarın. 1 ul glikojen (10 mg / ml stok) 3 hacim etanol eklenerek RNA hızlandırabilir. Pelet 10.5 ul suda eritin. </li></ol> cDNA kütüphanesi hazırlama ve derin sıralama Başlangıçta küçük düzenleyici RNA&#39;lar 19 klonlama açıklanan standart bir cDNA kütüphanesi hazırlık protokolü aracılığıyla kurtarılan RNA taşır. Kurtarılan RNA 10.5 ul kullanarak 20 ul ölçekte açıklandığı gibi ilk adım, 3 &#39;adaptörü ligasyonu, gerçekleştirildi. Solexa sıralama adaptörü açıklanan ayarlar kullanın. Iyileşti RNA miktarını bağlı olarak, ekler olmadan 5&#39;-adaptör-3&#39;-adaptör ürünleri, ek PCR bantları gibi cDNA amplifikasyonu sonra tespit edilebilir. Bu gibi durumlarda, beklenen büyüklüğü% 3 NuSieve uzun PCR ürününün tüketim düşük erime noktası agaroz jel, Solexa teknolojisini kullanarak GelElute kiti (Qiagen) ve sırası ile jel parçalarından PCR ürünü Zehir. Bir Solexa sıralama çalıştırmak genellikle sırası 6 ve 10 milyon arasında tanıyor, RNA bağlayıcı proteinlerin bağlanma yerleri bir transcriptome geniş kapsama alanı için yeterli olduğunu okur. Biyoinformatik analizi Dikkatli biyoinformatik analiz dizisi dizisi vs çevrilmemiş kodlama ihtiyaçlarına incelenen RBP, RNA tanıma elemanı, RBP tercih edilen bağlayıcı bölgeleri (exonic vs intronik gibi RNA bağlayıcı siteleri, anlamlı anlayışlar elde etmek için yapılması gereken okur dizisi). Okuma sırası genom ve EST veritabanlarının karşı uyumlu hale getirilmesi gerekir. Biz genellikle okuma mappibir uyumsuzluk, ekleme veya silme sırası kümeleri oluşturmak için benzersiz genom ng daha sonra analiz edilebilir okur. Kümelenmiş sıralı frekans karakteristik mutasyonlar, C geçişler 6-SG kullanırken bir geçişler 4-SU ve G kullanırken, T okur başarıyla çapraz dizilerinin göstergesidir. Bizim tecrübelerimize göre bir arka plan mutasyon oranı yaklaşık% 20 4-SU ile etiketlenmiş uncrosslinked RNA&#39;lar göstermektedir. Bu oran yaklaşık artar. Crosslinking üzerine% 50-80. Biyoinformatik analizi ayrıntılı bir açıklama Hafner ve arkadaşları tarafından yayın Ek malzeme bulundu. 18 İsteğe bağlı adımlar Toplam RNA içine 4-thiouridine dahil düzeylerinin belirlenmesi 100 mcM hasattan önce 4SU 16 saat ile desteklenmiş ortamda büyüyen sonra stabil bir şekilde ilgi RBP ifade hücre hattı toplam RNA izole edin. Kontrol olarak, hasat hücreler 4SU ilave edilmeden büyüdü. Ayrıca üreticinin talimatlarına izleyerek yıkanmış hücre granül Trizol reaktifi (Sigma) 3 cilt toplam RNA izole edin. Ayrıca üreticinin protokole göre Qiagen RNeasy kullanarak toplam RNA arındırmak oldu. 4SU, RNA izolasyonu ve analizi sırasında oksitlenmeyi önlemek için, yıkama tamponları ve enzimatik adımdan sonraki 0.1 mM dithiothreitol (DTT) ekleyin. Digest ve HPLC analizi için tek nükleosidlerin A&#39;ya defosforilize toplam RNA 20 önce nitelendirdi. Kısaca, 30 ul hacmi, 37, 16 saat, toplam RNA saflaştırılmış 40 mikrogram idi inkübe ° C 0.4 U bakteriyel alkalin fosfataz (Biyokimyasal Worthington) ve 0.09 U yılan zehiri fosfodiesteraz (Worthington Biyokimyasal). Bir referans standart olarak, 4SU etiketli sentetik RNA (standart CGUACGCGGAAUACUUCGA (4SU) U) ve enzimatik sindirimi tamamlamak için maruz kullanın. Bir Supelco Discovery C18 (gümrüklü faz silika 5 mcM parçacık, 250 x 4.6 mm) ters faz kolon (Bellefonte PA, USA) HPLC ile ribonucleosides çıkan karışımları ayırın. HPLC tamponlar% 3 asetonitril (A) ve% 90 su asetonitril (B) 0,1 M TEAA. 15 dakika, 20 dakika 0 -% 10 B, 30 dakika 10 ila% 100 B: 0% B izokratik degrade kullanın. Arasında uygulanan yıkama HPLC kolon temizlemek için çalışan bir 5 dakika% 100 B uygulayın. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2034/2034fig1.jpg" alt="Şekil 1" /> Şekil 1 (sağ panel) hücre hatları BAYRAK / 4-SU ve 6-SG ile IGF2BP1 HA etiketli ifade ile yapılan bir PAR-Klip temsili bir sonuç göstermektedir. IGF2BP1 6-SG crosslinking verimliliği 4-SU crosslinking verimliliği daha düşük olduğunu unutmayın. Düşük crosslinking verimliliği daha yüksek bir arka planda bol hücresel RNA parçaları elde dizilerinin neden olacaktır ve bu nedenle daha az verimli photoreactive nükleosidlerin kullanırken deneme ölçeklendirme düşünmelisiniz. Şekil 1 sol panelindeki potansiyel geleneksel UV 254 nm crosslinking göre PAR-Klip için kullanılan olabilir farklı photoreactive üridin analogları kullanarak bir karşılaştırma gösterir. Doğru uzunlukta radyoaktif bant yoğunluğu PAR-Klip deney küçük bir RNA sıralama protokolü (küçük cDNA kütüphanesi hazırlamak için adım adım açıklaması ile çalıştı ve taşımak için yeterli RNA izole olup olmadığını iyi bir fikir verir RNA&#39;lar sıralama) 19 bulunabilir. 6-SG kullanarak bir geçişler 4-SU ve G ile C geçişler için sıralı okuma, T karakteristik mutasyonlar, frekans, başarılı bir şekilde çapraz dizilerinin göstergesidir. Bizim tecrübelerimize göre bir arka plan mutasyon oranı yaklaşık% 20 4-SU ile etiketlenmiş uncrosslinked RNA&#39;lar göstermektedir. Bu oran yaklaşık artar. Crosslinking üzerine% 50-80.

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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 Alnylam İlaç kurucularından ve bilimsel danışmanı ve Regulus Therapeutics bir danışman.

Tamponlar ve reaktifler Büyüme orta HEK293 hücreleri <ul><li> DMEM </li><li> % 10 FBS </li><li> 2 mM L-Glutamine </li><li> 100 U / ml penisilin </li><li> 100 U / ml streptomisin </li><li> 100 mg / ml hygromycin </li><li> 15 mcg / ml blasticidin </li></ul> 4-thiouridine stok solüsyonu (1 M) <ul><li> 260,27 mg 4-thiouridine </li><li> 1 ml DMSO </li></ul> Doksisiklin stok (10 mg / ml) <ul><li> 10 mg doksisiklin </li><li> 1 ml DMSO </li></ul> 1x NP40 lizis tamponu DTT ve proteaz inhibitörleri olmadan 5x tampon stok hazırlayın. DTT ve proteaz inhibitörü doğrudan deneyden önce ekleyin. <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> tam EDTA içermeyen proteaz inhibitörü kokteyl (Roche) </li></ul> Sitrat-Fosfat tampon <ul><li> 4.7 g / L sitrik asit </li><li> 9.2 g / l Na 2 HPO 4 </li><li> pH 5.0 </li></ul> IP-yıkama tamponu <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 (deneyden önce doğrudan eklemek) </li><li> tam EDTA içermeyen proteaz inhibitörü kokteyl (Roche) (doğrudan deney eklemeden önce) </li></ul> Yüksek tuz yıkama tamponu <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 (deneyden önce doğrudan eklemek) </li><li> tam EDTA içermeyen proteaz inhibitörü kokteyl (Roche) (deneyden önce doğrudan eklemek) </li></ul> Defosforilasyon tampon <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> Fosfataz yıkama tamponu <ul><li> 50 mM Tris-HCl, pH 7.5 </li><li> 20 mM EGTA </li><li> % 0.5 (v / v) NP40 </li></ul> DTT olmadan polinükleotid kinaz (PNK) tampon <ul><li> 50 mM Tris-HCl, pH 7.5 </li><li> 50 mM NaCl </li><li> 10 mM MgCl 2 </li></ul> PNK tampon <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 yükleme tamponu <ul><li> % 10 gliserol (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 Bromophenol Mavi </li></ul> 2x Proteinaz K tampon <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

RNA transkript yüzlerce RNA bağlayıcı proteinler (RBPs) ve microRNA içeren, genellikle bağımlı bir hücre tipi ifade ribonükleoprotein kompleksleri (miRNPs) ile etkileşim transkripsiyon sonrası gen düzenlemeye tabi Anlamak için bu RNA bağlayıcı faktörlerin etkileşimi bireysel transkript, in vivo protein-RNA etkileşimleri 1 gerekli olan yüksek çözünürlüklü haritalar yönetmelik nasıl etkiler. Genetik, biyokimyasal ve hesaplama yaklaşımları bir arada genellikle RNA RBP veya RNA-RNP etkileşimleri belirlemek için uygulanır. Immunopurified RBPs ile ilişkili RNA&#39;lar mikroarray (RIP-Chip) 2 transcriptome bir seviyede hedefler tanımlar, ancak uygulaması, kinetik istikrarlı etkileşimler ve sadece nadir durumlarda 3,4 RBP tanıma elemanı (RRE tanımlamanıza olanak sağlar karakterizasyonu ile sınırlıdır Uzun hedef RNA içinde). Daha doğrudan RBP hedef site bilgileri, in vivo UV crosslinking 5,6 çapraz RNA segment ve cDNA sıralama (CLIP) 10 izolasyonu takip immunoprecipitation 7-9 ile birleştirerek elde edilir . CLIP 11-17 RBPs bir takım hedefleri tanımlamak için kullanılır oldu. Ancak UV 254 nm RNA-protein çapraz bağlanma, düşük verimlilik, CLIP sınırlıdır ve çapraz bağ yerini zor arka planı olmayan-çapraz UV-çapraz hedef RNA segmentlerinde ayrı, sıralı çapraz parçaları içinde kolayca tanımlanabilir. RNA aynı zamanda örnek mevcut parçaları. Biz, yüksek çözünürlük ve hücresel RBPs ve miRNPs transcriptome çapında bağlayıcı siteleri belirlemek için güçlü bir hücre tabanlı crosslinking yaklaşım geliştirdi ki dönem PAR-Klip (Photoactivatable ribonükleozit-Geliştirilmiş Crosslinking ve Immunoprecipitation) (Şekil 1A anahat yöntemi). Bu yöntem, canlı hücreler tarafından 4-thiouridine (4-SU) ve 6-thioguanosine (6-SG) olarak photoreactive ribonükleozit analogları, doğmakta olan RNA transkript içine eklenmesi dayanmaktadır. 365 nm UV ışık hücreleri tarafından Işınlama etkileşim RBPs photoreactive nükleozid etiketli hücresel RNA&#39;lar verimli crosslinking neden olur. Ilgi RBP Immunoprecipitation çapraz ve coimmunoprecipitated RNA izolasyonu ile takip edilmektedir. Izole RNA, cDNA kütüphanesi dönüştürülür ve derin Solexa teknoloji kullanarak sıralı. CDNA kütüphaneleri PAR-Klip tarafından hazırlanan bir karakteristik özelliği de crosslinking kesin konumunu sıralı cDNA ikamet eden mutasyonların tespit edilebilir olmasıdır. sitidinin geçiş mutasyonlar adenozin guanozin 6-SG sonuçlarını kullanarak ise 4-SU kullanarak, çapraz dizileri timidin,. Çapraz dizileri mutasyonların varlığı mümkün bol hücresel RNA&#39;lar türetilen dizilerinin arka plan onları ayırmak için yapar. Çeşitli RNA bağlayıcı proteinler bir dizi yöntem Uygulama Hafner ve ark bildirilmiştir. 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

RNA transkript trans etkili RNA bağlayıcı proteinler (RBPs) çok sayıda aracılığı ile geniş posttranskripsiyonel düzenlemeye tabidir. Burada hassas ve transcriptome geniş bir ölçekte RBPs, RNA bağlayıcı siteleri tanımlamak için genellenememektedir bir yöntem mevcut.

PAR-Klip - RNA Cilt Proteinler Transcriptome geniş Cilt Siteleri Belirlemek Amacıyla Bir Yöntem

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

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Biology

53.2K Görüntüleme.  Rockefeller University. RNA transkript trans etkili RNA bağlayıcı proteinler (RBPs) çok sayıda aracılığı ile geniş posttranskripsiyonel düzenlemeye tabidir. Burada hassas ve transcriptome geniş bir ölçekte RBPs, RNA bağlayıcı siteleri tanımlamak için genellenememektedir bir yöntem mevcut.

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

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

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

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

Aktin Co-Sedimantasyon Testi; F-Aktin Bağlayıcı Protein Analizi

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

Biyoloji

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.

P2X2 Ligandla Gated Katyon Kanalları ile etkileşimde Proteinler tanımlamak için Proteomik

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.

Methyltransferases, profilleme ve Diğer S Adenosil- L</subYakalama Bileşik Kütle Spektrometresi (CCMS)> homosistein bağlayıcı Proteinler

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.

Cilt Mekanizmaları belirleme Sipariş Değişken konsantrasyon İzotermal Titrasyon Kalorimetre Veri Kümeleri toplanıyor

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 - Bireysel Nükleotid Çözünürlük protein-RNA Etkileşimleri Transcriptome geniş Haritalama

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.

Kalmodulin bağlayıcı Proteinler, Pull-down

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.

Büyüme Faktörü Progranulin ile etkileşimde Proteinler tanımlamak Modifiye Maya-İki Hybrid Sistemi

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.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

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.

Mikro Termoforosis tarafından Bağlama Affinity belirlenmesi Protein Saflaştırma-ücretsiz Yöntemi

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.

Elektrokemiluminesan immunoassay Serum Kardiyak Miyozin Bağlayıcı Protein-C belirlenmesi için hassas ve özel miktar tayini yöntemi

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