Name: an easy method for plant polysome profiling
Uploaded: 2016-08-28T14:00:00.000+00:00
Duration: 11 min 9 s
Description: Watch this Scientific Journal Video about An Easy Method for Plant Polysome Profiling at JoVE.com

Bu çalışma Fransız Ulusal Araştırma Ajansı (ANR-14-CE02-0010) tarafından desteklenmiştir. Biz yazının eleştirel okuma Dr Benjamin Field ve Dr. Elodie Lanet teşekkür ederim. Biz video düzenleme ile yaptığı yardım için Bay Michel Terese teşekkür ederim.

% 20 ila 50 (ağırlık / hacim) sukroz dereceleri hazırlanması 1. Not: Gradyan 13,2 ml&#39;lik bir ultra santrifüj tüpüne sükroz 4 kat (% 50,% 35 ve% 20 2 kat) imal edilmiştir. Bizim tecrübelerimize göre, iki ayrı katmanlar halinde% 20 sukroz dökme ölçüde Polizom hazırlıkları kalitesini artırır. <ol><li> stok çözümleri hazırlayın. tüm çözümler RNAse ve DNAse serbest olduğundan emin olun. <ol><li> 400 mM Tris-HCI pH 8.4, 200 mM KCI, 100 mM MgCl2: 10X tuz solüsyonu. </li><li> 200 ml 1X tuz çözeltisi içinde sakrozun 137 g çözülür: 2 M sukroz çözeltisi hazırlayın. </li></ol></li><li> gradyanlar hazırlamak için, Tablo 1 &#39;de tarif edildiği gibi, tuz çözeltisi içinde sukroz çözeltisi ile seyreltilir. Verilen miktarlar altı geçişlerini içindir. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Nihai sakaroz konsantrasyonu </td><td> sukroz 2M (mi) </td><td> Tuz çözeltisi 1X (mi) </td><td> Nihai Vol. (mi) </td></tr><tr><td> % 50 </td><td> 8.8 </td><td> 3.2 </td><td> 12 </td></tr><tr><td> % 35 </td><td> 12.9 </td><td> 12.1 </td><td> 25 </td></tr><tr><td> % 20 </td><td> 7.4 </td><td> 17.6 </td><td> 25 </td></tr><tr><td> % 20 </td><td> 5.8 </td><td> 14.2 </td><td> 20 </td></tr></tbody></table> Tablo Sukroz Çözüm 1. dilüsyonlar altı geçişlerini hazırlamak. <ol start="3"><li> Tablo 2&#39;deki gibi katmanları dökün. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> sukroz katmanıdır </td><td> % 50 </td><td> % 35 </td><td> % 20 </td><td> % 20 </td></tr><tr><td> Vol. (Mi) </td><td> 1.85 </td><td> 3.65 </td><td> 3.65 </td><td> 1.35 </td></tr></tbody></table> Katman başına sukroz çözeltisi Tablo 2. Cilt. <ol start="4"><li> Her katman döküldükten sonra, bir -40 ° C tüpler tutmak veya -80 ° C derin dondurucuda tam donma kadar bir sonraki sakaroz katmanı eklemeden önce. Donma, genellikle -40 ° C de 2 saat sonra elde edilir, ancak bir sonraki tabaka dökülmeden önce yaklaşık 6 saat bekleyen önerilir. Son 20% tabaka donduruldu ve deney gününde taze olarak ilave edilebilir. Not: Bir sonraki bir eklemeden önce her bir katmanı Donma tabakaların herhangi karıştırma veya rahatsızlık önler. Gradyanlar bir -40 ° C&#39;de saklanır ya edilebilir -80 ° C sıcaklıkta dondurucuya en az altı ay süreyle. </li><li> Zaten bitmiş değilse, deney günü degradeler son% 20 katman ekleyin. Sonra, degradeler soğuk bir odada ya da bir buzdolabı çözülme sağlar. </li></ol> Sitosolik özleri 2. Hazırlık Not: tavsiyesinebiyolojik numune başına iki gradyanları kullanılarak sona erer. 300 mg 6 günlük, Arabidopsis thaliana fide çalışırken iki gradyan hazırlanması için bitki malzemesi uygun bir miktardır. az translationally aktif dokularda çalışırken, bitki materyali miktarı 600 mg kadar artırılabilir. <ol><li> ½ Murashige ve Skoog yetiştirilen Hasat altı gün eski fideler 8 orta sıvı nitrojen içinde hızlı dondurulması ile% 1 sükroz veya eşdeğer medya ile desteklenmiş </li><li> sıvı nitrojen ile önceden soğutulmuş havan ve tokmak taşlama bitki malzemesi. </li><li> önceden soğutulmuş tartım tabağına toz malzemenin 300 mg tartılır. Numunenin çözülme önlemek için hızlı bir şekilde bu adımı gerçekleştirin. Not: -80 ° C derin dondurucuda en fazla bir hafta dondurulmuş örnekleri tutun. </li><li> Dallanmış önceden soğutulmuş polisom tamponu 2.4 ml (Tuz Çözeltisi 4X, 5.26 mM EGTA,% 0.5 (h / h) oktilfenoksi poli (etilenoksi) etanol, 50 μg.ml -1 sikloheksimid, ekleme 50 μg.ml 1kloramfenikol) ve pipet ile karıştırılarak homojenize. iki adet 1,5 ml tüpler içine aktarın. ısınma örnekleri önlemek için hızlı bir şekilde çalışın. </li><li> bir mikrosantrifüj 4 ° C&#39;de 15 dakika boyunca 16,000 x g&#39;de santrifüjleyin yıkıntıları pelet haline getirildi. Not: Her zaman, RNA bozulmasını önlemek 4 ° C&#39;de örnekleri tutmak için, RNaz / DNaz ücretsiz koşullarda RNaz / DNaz ücretsiz çözümler ve çalışmayı kullanma. Heparin RNA korumasını geliştirmek için, 300 μg.ml -1 nihai konsantrasyona kadar, polisom tampon eklenebilir. Ancak, aşağı analizi engelleyebilir heparin beri, bu lityum klorür yağış yerine etanol yağış (bkz not 4.7) gerçekleştirerek RNA yağış adımı sırasında kaldırılması gerekir. </li></ol> 3. Polizom Profil <ol><li> Dikkatle pelet bozmadan süpernatant pipetle. Bitki parçaları pipetle varsa, adımı 2.5 tekrarlayın. hafifçe pipettin tarafından degrade üstünde yüzer yükleyinsabit bir akışı tüpün yan duvar üzerine g. Her 1.5 ml tüp için bir degrade kullanın. </li><li> (A SW41 rotor kullanıldığında, örneğin 32.000 rpm) bir ultrasantrifüjdeki, 4 ° C&#39;de 2 saat 45 dakika süre ile 175,000 x g&#39;de kepçe ve santrifüj soğutulmuş aktarın. </li><li> Şekil 1&#39;de açıklandığı gibi degrade toplama sistemi kurmak. UV küvet 1 mm yol uzunluğuna sahiptir. </li></ol><img alt="Şekil 1" src="/files/ftp_upload/54231/54231fig1.jpg" /> Şekil 1. Gradient toplama sistemi. UV küvet degrade altına inen bir cam kılcal tüp polivinil klorür boru ile bağlanır. degrade bir peristaltik pompa sistemi sayesinde yoluyla ilerler. OD 260 sürekli okunur ve 2 ml&#39;lik fraksiyonlar toplanır. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">Büyük halini görmek için tıklayınız</a>bu figür. <ol start="4"><li> Oda sıcaklığına fraksiyon toplayıcısı ayarlayın ve 2 mi fraksiyon koleksiyonu sağlayacak bir hızda döner bandı ayarlayın. Önceden soğutulmuş toplama tüpleri kullanın. </li><li> gradyan toplama sistemine polivinil klorür boru ile bağlı bir cam kılcal tüp kullanarak alttan üste doğru kesirler toplayın. polivinil klorür boru ve cam kılcal boru arasındaki bağlantıyı sabitlemek için plastik bir parafin filmi kullanın. </li><li> degrade yukarıya doğru 260 nm&#39;de absorbansı okumak. Tüm gradyanı elde edildiğinde, RNA ekstraksiyonu önce buz üzerinde toplama tüpleri. </li></ol> 4. RNA Ekstraksiyon <ol><li> aşağıdaki gibi, santrifüj tüpleri şapkalı 50 ml iki geçişlerini toplanan kesirler, havuz: Polizom: fraksiyonlar 1-3 (12 mi) Monosomes: fraksiyon 4 (4 mi) Süpernatant: fraksiyonlar 5 ve 6 (8 mi) </li><li> Her fraksiyon için, 1 vol ekleyin. 8 M guanidin hidroklorür,50 ug akril taşıyıcısı ve 1.5 hac. izopropanol. Not: Akrilik taşıyıcı alkol çökeltme sırasında, nükleik asitlerin geri kazanımını arttırmak için coprecipitant olarak kullanılan lineer akrilamid vardır. </li><li> tüpler ters çevirerek karıştırınız ve -20 ° C &#39;de O / N hızlandırabilir. </li><li> bir ultrasantrifüjdeki 4 ° C&#39;de 1 saat, (a SW32 rotor içinde 32.000 rpm) 175,000 x g&#39;de santrifüjleyin. Çökeltme aşaması, ultra-santrifüj ile uyumlu olmayan bir tüp içinde gerçekleştirilir, uygun bir tüp aktarın. </li><li> Süpernatant atılır ve 200 mL RNaz içermeyen TE tamponu (10 mM Tris-HCI, pH 7,5-1 mM EDTA) içinde pelet çözülür. yeni bir 1.5 ml&#39;lik tübe transfer edin. </li><li> 1 vol ekleyerek RNA ayıklayın. suyla doyurulmuş fenol (pH 6.6) ve 1 hacim. kloroform: izoamil alkol (24: 1). kuvvetlice karıştırın. 4 ° C&#39;de 20 dakika boyunca 15,000 x g&#39;de santrifüjleyin. yeni bir 1.5 ml&#39;lik tübe sulu faz transfer. Not: asit fenol (pH 4.5), DNA kontaminasyonu en aza indirmek için de kullanılabilir. </li><li>1/10 vol ekleyerek RNA hızlandırabilir. 3 M sodyum asetat ve 3 hac. % 100 etanol eklenmiştir. -20 ° C&#39;de 20 dakika veya O / N -80 ° C&#39;de çökelmesine izin verin. 4 ° C&#39;de 15 dakika boyunca 15,000 x g&#39;de santrifüjleyin. Not: polisom tamponu (bakınız Not 2.5) içinde heparin kullanılarak, uygun bir şekilde alt analizi 9 müdahale edebilecek heparin arındırıldı etanol çökeltme yerine lityum klorür (LiCİ) çökelmesini gerçekleştirin. Ekstraksiyon işleminden sonra, 4 ° C&#39;de 20 ° C veya O / N 30 dakika çöktürülmüştür 2.5 M bir son konsantrasyona kadar LiCI ekleyin. 4 ° C&#39;de 15 dakika boyunca 15,000 x g&#39;de santrifüjleyin. </li><li> 500 ul% 75 etanol ile pelet yıkayın. Hava pelet kurutulur ve 30 ul TE tampon maddesi içinde çözülür. Bir RNazsız% 1.2 agaroz jeli (100V, 15 dakika) ya da kapiller elektroforez yöntemlerle elektroforez RNA kalitesini değerlendirmek. </li></ol> 5. Veri Analizi <ol><li> Bir grafik ve veri analizi yazılımı İhracat ham veriler. NotŞekil 2A ve B de gösterildiği gibi, ham profil niteliksel bilgileri vermek: görülebileceği polisom tepe sayısı monosome tepe yüksekliği ve serbest ribozom alt birimlerine karşılık gelen bir omuz varlığı. </li><li> örnekler arasındaki profillerin karşılaştırılması izin profilleri Birleştirme. Her numune için monosome zirve X değerini belirlemek için ekran okuyucu aracını kullanın ve eğrilerin başında ekstra veri noktaları kaldırarak monosome doruklarına hizalayın. eğrinin en alçak noktası belirlemek ve Y değeri (ekran okuyucu aracını kullanarak) belirler. &quot;Set sütunları değer&quot; fonksiyonunu kullanarak 0&#39;a en alçak noktası Y değerini ayarlayın. </li><li> Ham bulunmaktadır (Şekil 3C) için eğri altındaki alanların integrasyonu ile belirlenmişlerdir polisomlann / monosomes oranını belirler. alanın sınır entegre edilecek tanımlamak için veri seçici aracını kullanın. Sonra entegre fonksiyonu (Analiz-Matematik) kullanın. </li><li> normalleştirmekmonosome tepe zirvesine Y değerine göre bir eğrinin tüm veri noktalarını bölerek monosome zirve eğrileri (ekran okuyucu aracını kullanarak belirlenir). sütunu seçin, ardından Analiz-Matematik altında, Normale seçin ve &quot;belirli bir değere bölünmesi&quot; yöntemleri seçin. Bu adım, polizom göreli seviyesi (Şekil 3D) karşılaştırılmasına olanak sağlar. </li></ol><img alt="şekil 2" src="/files/ftp_upload/54231/54231fig2.jpg" /> Şekil 2. Örnek polisom profilleri., Arabidopsis thaliana, vahşi tip (wt, ekotip col-0) ve mutant fideleri uzun gün fotoperiyodların (16 saat ışık, 8 saat karanlık) altında yarım Murashige ve Skoog ortamı üzerine, altı gün süre ile büyütülmüştür. A: wt fideler ham Polizom profilleri. B: mutant fideler ham Polizom profilleri. C: eğri D altındaki alanın entegrasyonu ile polizom ve monosomes yüzdesinin belirlenmesi: Polizom profiles monosome zirveye normalize. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

<ol>
	<li>Nelson, C. J., Millar, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+turnover+in+plant+biology.">Protein turnover in plant biology.</a> Nat Plants. 1 (3), 15017 (2015).</li><li>Warner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+economics+of+ribosome+biosynthesis+in+yeast.">The economics of ribosome biosynthesis in yeast.</a> Trends Biochem Sci. 24 (11), 437-440 (1999).</li><li>Amthor, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+McCree-de+Wit-Penning+de+Vries-Thornley+Respiration+Paradigms:+30+Years+Later.">The McCree-de Wit-Penning de Vries-Thornley Respiration Paradigms: 30 Years Later.</a> Ann Bot. 86 (1), 1-20 (2000).</li><li>Preiss, T., W Hentze, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starting+the+protein+synthesis+machine:+eukaryotic+translation+initiation.">Starting the protein synthesis machine: eukaryotic translation initiation.</a> BioEssays news and reviews in molecular, cellular and developmental biology. 25 (12), 1201-1211 (2003).</li><li>Vogel, C., Marcotte, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses.">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nat Rev Genet. 13 (4), 227-232 (2013).</li><li>Baerenfaller, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+proteomics+reveals+Arabidopsis+thaliana+gene+models+and+proteome+dynamics.">Genome-scale proteomics reveals Arabidopsis thaliana gene models and proteome dynamics.</a> Science. 320 (5878), 938-941 (2008).</li><li>Zanetti, E., Chang, I., Gong, F., Galbraith, D. W., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopurification+of+Polyribosomal+Complexes+of+Arabidopsis+for+Global+Analysis+of+Gene+Expression.">Immunopurification of Polyribosomal Complexes of Arabidopsis for Global Analysis of Gene Expression.</a> Plant physiol. 138 (2), 624-635 (2005).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bioassays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bioassays with tobacco tissue cultures.</a> Physiol plant. 15 (3), 473-497 (1962).</li><li>del Prete, M. J., Vernal, R., Dolznig, H., M&#252;llner, E. W., Garcia-Sanz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+polysome-bound+mRNA+from+solid+tissues+amenable+for+RT-PCR+and+profiling+experiments.">Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments.</a> RNA. 13 (3), 414-421 (2007).</li><li>Salinas, J., Sanchez-Serrano, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Arabidopsis protocols. , (2006).</li><li>Piques, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+and+transcript+copy+numbers,+polysome+occupancy+and+enzyme+dynamics+in+Arabidopsis.">Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis.</a> Mol syst biol. 5 (314), 314 (2009).</li><li>Morita, M., Alain, T., Topisirovic, I., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+Profiling+Analysis.">Polysome Profiling Analysis.</a> bio-protocol. 3 (14), 3-8 (2013).</li><li>Y&#225;ng&#252;ez, E., Castro-Sanz, A. B., Fern&#225;ndez-Bautista, N., Oliveros, J. C., Castellano, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genome-wide+changes+in+the+translatome+of+Arabidopsis+seedlings+subjected+to+heat+stress.">Analysis of genome-wide changes in the translatome of Arabidopsis seedlings subjected to heat stress.</a> PloS One. 8 (8), (2013).</li><li>Sormani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublethal+cadmium+intoxication+in+Arabidopsis+thaliana+impacts+translation+at+multiple+levels.">Sublethal cadmium intoxication in Arabidopsis thaliana impacts translation at multiple levels.</a> Cell Physiol. 52 (2), 436-447 (2011).</li><li>Lanet, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+evidence+for+translational+repression+by+Arabidopsis+microRNAs.">Biochemical evidence for translational repression by Arabidopsis microRNAs.</a> Plant cell. 21 (6), 1762-1768 (2009).</li><li>Jabnoune, M., Secco, D., Lecampion, C., Robaglia, C., Shu, Q., Poirier, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rice+cis-Natural+Antisense+RNA+Acts+as+a+Translational+Enhancer+for+Its+Cognate+mRNA+and+Contributes+to+Phosphate+Homeostasis+and+Plant+Fitness.">A Rice cis-Natural Antisense RNA Acts as a Translational Enhancer for Its Cognate mRNA and Contributes to Phosphate Homeostasis and Plant Fitness.</a> Plant cell. 25 (10), 4166-4182 (2013).</li><li>Ingolia, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+translational+profiling+by+ribosome+footprinting.">Genome-wide translational profiling by ribosome footprinting.</a> Methods Enzymol. 470 (10), (2010).</li><li>Juntawong, P., Girke, T., Bazin, J., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+dynamics+revealed+by+genome-wide+profiling+of+ribosome+footprints+in+Arabidopsis.">Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis.</a> PNAS. 111 (1), 203-212 (2013).</li><li>Ingolia, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling:+new+views+of+translation,+from+single+codons+to+genome+scale.">Ribosome profiling: new views of translation, from single codons to genome scale.</a> Nat Rev Genet. 15 (3), 205-213 (2014).</li></ol>

The authors have nothing to disclose

The protocol we present here is an easy and cheap method for generating polysome profiles and isolating mRNAs associated with polysomes, single ribosomes or free of ribosomes. A wide range of different polysome fractionation methods is described in the literature. The method we have described here has been optimized to keep only the necessary compounds and has been adapted for plant material. In particular, we reduced the amount of detergent11 and added chloramphenicol to the buffer to fix the chloroplastic ri...

Protein sentezi, bütün hücreler 1 çok önemli ve enerjik olarak masraflı bir işlemdir. Her şeyden önce, hücreler için makine, ribozom üretiminde enerji yatırım gereklidir. Örneğin bir aktif bölünmesi maya hücre Dakikada kadar 2.000 olarak ribozomlar üretir. Böyle bir üretimi toplam transkripsiyonel aktivitenin% 60 ve hücrenin 2 toplam birleştirme etkinliğinin% 90 kadar gerektirmektedir. Ayrıca, enerji amino asitler, aminoasil-tRNA ve peptid bağlarının sentezi için gereklidir. Bitkilerde, ATP 3 4.5 5.9 moleküllerden bir peptid zinciri maliyetlerine bir amino asit eklenerek. Nedenle, protein mRNA&#39;nın çeviri bunun değişen çevre koşullarına ile ilgili söz konusu, özellikle düzenlemenin önemli bir yer olması şaşırtıcı değildir. Ribozom bir mRNA&#39;nın birliğidir çeviri başlangıç ​​aşaması, düzenlenmesi ana hedefidirçeviri 4. Bir tercüme düzenlemenin bir sonucu olarak ve diğer transkripsiyon sonrası düzenleme adım olarak, protein konsantrasyonuna varyasyonları sadece% 40 mRNA bolluğu 5,6 ile açıklanabilir. Böylece, toplam mRNA çalışma protein bolluğu hakkında nispeten zayıf bilgi verir. Öte yandan, ribozom ile mRNA&#39;nın dernek çeviri katılanların mRNA erişim sağlayan protein bolluğu içine daha iyi fikir verir. Aktif tercüme mRNAlar polisomlann adlandırılan yapılar çeşitli ribozom ile ilişkilidir. Tersine, kötü çevrilmiş mRNA&#39;ların tek Ribozom (monosome) ile ilişkili olacaktır. Sonuç olarak, bir mRNA&#39;nın translasyon durumu ribozom 7 ile ilişkisi izleyerek değerlendirilebilir. Bu protokol altı gün eski Arabidopsis thaliana fide, RNA sonraki izolasyonu polizom izolasyonunu ve sonuçların analizini açıklar. polysomes ve monosomes bir sükroz yoğunluk gradyanı ile ayrılmıştır. Gradyanlar altı fraksiyon halinde toplanır. Polisomlar, monosomes ve ribozomlar ile ilişkili olmayan serbest 60S ve 40S ribozomal alt birimleri ve mRNA içeren hafif kısmını (bundan sonra adlandırılan yüzer madde): kesirler Bazı üç iyi ayrılmış kesirler elde etmek için bir araya getirilir. Küresel için etkinliği ve polisomlann profilleri karşılaştırarak eğri altında kalan alan entegrasyonu tarafından belirlenen bir polisom / monosome oranı üretilmesiyle tahmin edilebilir. mRNA ve proteinlerin daha sonra farklı fraksiyonlardan çıkarılır ve RT-PCR, qRT-PCR, Northern blot, mikrodizi, Western blot veya proteomik ile analiz için kullanılmıştır. Bu protokol, diğer bitkiler ve dokular için onaylanmıştır. Bu protokol gerçekleştirmek için gerekli ekipman yaygın çoğu laboratuvarlarda bulunur: bir degrade üreticisi için gerek yoktur. Bir sonraki önlemek eklemeden önce her katmanı Donmatabakaların herhangi bir karışımını ya da rahatsızlık dan. Resim boru delicisi gradyanı içinde bir cam kılcal tüp daldırma ile elde edilebilmektedir gradyan toplanması için kullanılır. Bu nedenle, yüksek maliyetli ultrasantrifüj borular hasarsız kalır ve birçok kez yeniden kullanılabilmektedir. Topluca, bu mevcut protokol Polizom profil için kolay ve ucuz bir yöntemdir yapar.

<table><tbody><tr><td>Ultracentrifuge tube, thinwall, polyallomer - 13.2 ml</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>Ultracentrifuge tube, thinwall, polyallomer - 38.5 ml</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>Glass capillary tube</td><td>Drummond Scientific</td><td>1-000-1000</td><td></td></tr><tr><td>Ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>Optima series</td><td></td></tr><tr><td>Ultracentrifuge Rotor SW41</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>Ultracentrifuge Rotor SW32</td><td>Beckman Coulter</td><td>369650</td><td></td></tr><tr><td>Peristaltic pump</td><td>Any</td><td></td><td></td></tr><tr><td>Tygon R3607 polyvinyl chloride tubing&amp;nbsp;</td><td>Fisher Scientific</td><td>070534-22</td><td>Polyvinyl chloride tubing, 2.29 mm&amp;nbsp;</td></tr><tr><td>Fraction collector Model 2110</td><td>Bio-Rad</td><td>731-8120</td><td></td></tr><tr><td>UV cuvette</td><td>Hellma</td><td>170.700-QS</td><td>Quartz flow-through cuvette</td></tr><tr><td>UV Spectrophotometer</td><td>Varian</td><td>Cary50</td><td>Read every 0.0125 sec</td></tr><tr><td>All chemicals</td><td>Any</td><td></td><td>Use only Molecular Biology Grade</td></tr><tr><td>Murashige and Skoog Basal Salt Mixture (MS)</td><td>Sigma-Aldrich</td><td>M5524</td><td></td></tr><tr><td>Rnase-Free water</td><td>Any</td><td></td><td></td></tr><tr><td>Petri Dishes</td><td>Fisher Scientific</td><td>10083251&amp;nbsp;</td><td></td></tr><tr><td>Octylphenoxy poly(ethyleneoxy)ethanol, branched (Nonidet P40)</td><td>Euromedex</td><td>UN3500</td><td></td></tr><tr><td>Linear acrylamide (acryl carrier)</td><td>ThermoFischer scientific</td><td>AM9520</td><td>RNA precipitation carrier</td></tr><tr><td>OriginPro 8</td><td>OriginLab</td><td></td><td>Analysis software</td></tr></tbody></table>

an easy method for plant polysome profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

Literatürde, polisom profilleri genellikle örneğin, yukarıdan aşağıya doğru, gradyanlar toplanır şeklinin bir sonucu olarak, ağır fraksiyonuna hafif fraksiyondan gösterilmiştir. Burada anlatılan protokolde geçişlerini alttan üste doğru toplanır, biz göstermek profilleri ağır fraksiyon (Polisomlar) ile başlar ve hafif fraksiyon (serbest ribozom alt birimlerinin ve RNA&#39;lar) (Şekil 2A) gidin. Daha sonra, altı, 2 ml fraksiyonlar her gradyanı to...

Watch this Scientific Journal Video about An Easy Method for Plant Polysome Profiling at JoVE.com

An Easy Method for Plant Polysome Profiling

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Bitki Polizom Profiling için bir Kolay Yöntem

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

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Research

JoVE Journal

Biology

12.5K Görüntüleme.  Aix-Marseille Université. This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Video: An Easy Method for Plant Polysome Profiling

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

Bitki Protoplastlarından mRNA İnteraktom Yakalama

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biyokimya

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Polysome LEISHMANIA, insan hücreleri ve fare Testis profil oluşturma

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Dinamik proteomik ve miRNA analiz polizomlar izole fare kalp sonra Langendorff perfüzyon üzerinden

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Degrade Yapıcılar veya Fraksiyonasyon Sistemleri Olmadan Polizom Profilleme

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Ökaryotik Polyribosome Profil Analizi

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Biyoloji

Polysome Purification from Soybean Symbiotic Nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

Soya Fasulyesi Simbiyotik Nodüllerinden Polizom Saflaştırma

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

Gelişim Biyolojisi

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Polizom profilleme Stres Durumlardaki Çeviri başlatılması Analizi

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Bir genom Ölçeği Polizom Fraksiyonu ve Memeli Translatomes Analizi

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

Polisome Profilleme Kullanarak Gelişen Fare Beyninde Çeviri Analizi

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

Nörobilim

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polizom Profil Yöntemi ile memeli hücrelerine selektif mRNA dönüşümüyle Değerlendirilmesi

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...