作者要感谢约翰沃尔伍德这种方法的初步基础，TGK是支持由美国国立卫生研究院GM057483和AI076245和中国GM77073支持。

1。 7-47％蔗糖梯度制备<ol><li>准备蔗糖梯度有一天，在使用之前，让渐变成为连续。无菌7％和47％的蔗糖溶液，含50mm 的NH 4 CL，50毫米的Tris -醋酸的pH 值 7.0和12mm MgCl 2的和储存在4 ° C 1。 </li><li> 6梯度组合的7％和47％蔗糖的股票解决方案，使48毫升以下的蔗糖浓度。数码地面电视（1M股票）添加到每个蔗糖溶液至终浓度为1mm。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 终浓度 </td><td> 7％的蔗糖</td><td> 47％的蔗糖 </td></tr><tr><td> 7％ </td><td> 48毫升</td><td> 0 </td></tr><tr><td> 17％ </td><td> 36毫升</td><td> 12毫升</td></tr><tr><td> 27％ </td><td> 24毫升</td><td> 24毫升</td></tr><tr><td> 37％ </td><td> 12毫升</td><td> 36毫升</td></tr><tr><td> 47％ </td><td> 0 </td><td> 48毫升</td></tr></tbody></table></li><li>为了倒梯度，巴斯德吸管附加保障，并用封口膜密封，20毫升注射器或使用长针。 7％的蔗糖添加7毫升1 × 3.5“polyallomer超速离心机试管底部，然后添加7毫升17％，7％的解决方案下的蔗糖溶液，放置巴斯德吸管试管的底部附近的一角，超过0.3吹打没有更快毫升/秒7毫升的27％，37％和47％的蔗糖溶液的重复，您应遵守线条清晰，层与层之间，最小的混合。商店梯度发生在4 ° C过夜连同转子，瓶将用于收获样品管。 </li></ol> 2。酵母提取物的制备<ol><li>增长酵母培养125毫升0.8-1.0外径600。添加酮的文化到终浓度为100微克/毫升，并继续摇动30 ° 15分钟的彗星。 </li><li>同时，准备裂解液中含有80微克/毫升放线菌酮，200微克/ ml肝素，0.2％DEPC，和10毫米的Tris - HCl pH值7.5，0.1M的氯化钠，氯化镁30MM 2。保持冰从这个角度上的一切。 </li><li>倒入一个250毫升离心瓶的文化，并填补了冰。在8000 XG（7250 RPM在SLA - 1500转子）离心5分钟在4 ° C洗涤沉淀一次用10毫升的冰冷的裂解液转移至15 mL聚丙烯管。在5900 XG（〜6000 RPM）在一个SLA - 600TC转子离心3分钟，在4 ° C重悬在0.5 mL裂解缓冲液，转移中的颗粒1.5ml离心管，加入0.5毫升冷冻，烘烤，酸洗玻璃珠。涡4个30秒的间隔，在冰上冷却管，相互之间间隔为30秒的细胞。添加到另一个裂解液0.5毫升1.5毫升管。在微量的Eppendorf 5417R离心（14,000 RPM）的最大速度离心10分钟在4 ° C上清转移到一个新的1.5 ml离心管。取出10μL到一个单独的管，以确定的OD 260 / OD 280比值（见下文步骤3.1）。商店提取物在-80 ° C </li></ol> 3。从哺乳动物细胞提取物的制备<ol><li>对于贴壁细胞，生长在100毫米的菜〜70％汇合。您将需要一个100毫米，每11毫升梯度菜（典型的产量是20 OD 260单位） 。金额较大或较小的梯度大小线性的规模。对于非贴壁细胞，约1 × 10 7细胞都需要每11毫升梯度。 </li><li>裂解之前，添加100μg/ mL的放线菌酮。在37 ° C下15分钟。传输板冰，在冰冷的PBS冲洗细胞两次。将来所有的步骤必须进行在0-4 ° C。 </li><li>的PBS吸删除所有痕迹（让板漏角度的冰床），并添加1 mL裂解缓冲液，刮去，并转移到预冷1.5 mL试管。裂解液成分，将需要的细胞类型和生长条件进行了优化。一个很好的起点缓冲区将20毫米的HEPES - KOH，pH值7.4，15毫米氯化镁2，氯化钾200毫米，1％海卫X - 100（V / V），100微克/毫升放线菌酮，2毫米的数码地面电视服务，和1 mg / ml肝素。的关键变量，镁和氯化钾，以及列入或排除非离子型洗涤剂，以提高提取的骨架或膜约束polysom​​es的浓度。通过两次通过27号注射器针头或5-10 dounce同质化的招用B型杵裂解细胞。应预冷前使用的注射器或匀浆。 </li><li>冷冻离心5分钟旋转14000 XG。将新鲜的裂解蔗糖梯度（哺乳动物细胞裂解缺乏Triton X - 100和肝素的缓冲，但在1.2和1.3所述，否则）或闪光灯以备后用液氮冻结。 </li></ol&gt; 4。渐变离心效果<ol><li>在冰上解冻polyribosome裂解液。保留10μL样品中加入1毫升水，并用分光光度计读取OD 260 / OD 280的比例确定核酸裂解液浓度。从酵母培养成长为描述一个典型的产量是50外径为单位。 </li><li>使用吸管尖端放在对管梯度表面附近的墙壁，轻轻层25 外径 260梯度顶部裂解单位。对于一个35 mL的梯度500μL裂解液通常是加载。可用于裂解缓冲液，以保证所有的梯度与同等体积加载。较低的数额的物质会产生更好的分辨率。使用镊子，小心地降低到一个水平转头桶的梯度。 </li><li>梯度离心4小时100000 XG在Surespin 630转子（23,000 RPM）在4 ° C。该协议可以适用于更小的体积的梯度。 </li></ol> 5。收集的数据和分数<ol><li>大约30分钟前完成4小时旋转针型号184管穿孔。将笔到网上ISCO型号UA - 5吸光度/荧光显示器。对吸光度的电源监控，以便分析样品前，要达到一个稳定的基准。电子收购的polysom​​e轮廓，可以很容易地获得通过附加一个DI - 148U的数据采集单元（DATAQ仪器）图表记录。配置文件可以实时收集和保存以供日​​后使用附带的WinDaq软件的注释。 </li><li>填充Fluorinert使用的逆向流动，迅速设置50毫升的注射泵。确保有没有气泡。从注射器管穿孔的软管连接，并暂时关闭3毫升/分钟的前进方向，以清除任何气泡软管Fluorinert流。 </li><li>放置一个废物管或烧杯系列如果收集馏分流动池下软管收集样品运行。 </li><li>小心取出polyallomer离心管中，含有蔗糖梯度离心转子，放在冰（前形成与冰空管的井，以避免干扰梯度）。 </li><li>为了分析细胞提取物，梯度管，管穿孔。管上方连接到电源插座和安全。提高阶段性底部的离心管，使针头穿透试管底部。一旦针头通过离心管底部的突出的前进方向，开始在6mL/min流Fluorinert。 </li><li>一旦Fluorinert已经开始流入管，监察运动的针上在线吸光度监测。一旦针开始移动，在图表上的运动，以60的速度设定。监视器将记录与40S核糖体亚基检测，并继续通过polyribosome络合物自由材料的外径254起。如果你正在收集RNA或蛋白质分析的分数，通常是0.2分的分数是（1毫升）。 RNA分析，分数应该是直接收集到含有Trizol试剂（Invitrogen公司），苯酚或SDS /蛋白酶K管上的首选方法提取而定。 </li><li>当年底的polyribosome轮廓已经达成，关闭流Fluorinert离心管和停止的吸光度/荧光显示器中的图表运动。 </li><li>缩回到注射器Fluorinert开始在相反的方向流动快速，请务必不要从蔗糖进入注射器管率从离心管。拧开管穿孔的离心管中，低针，取出管。 </li><li>重复步骤5.8 5.5每个蔗糖梯度。 </li><li>当所有的蔗糖梯度进行了分析，收回Fluorinert从软管进入注射器。删除每个分馏部分，包括在上方废物软管（管穿孔和流动池），并用清水冲洗干净。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/1948/1948fig1.jpg" alt="图1" /> 图1代表核糖体中提取的痕迹的酵母菌株BY4741中存在的放线菌酮（垫HIS3ΔLEU2Δ0 met15ΔΔ0 0 URA3）的准备。核糖体的提取物被分割利用7 47％的蔗糖梯度和分析，使用一台泵注射器仪器连接到紫外探测器。含有多聚核糖体80S，60S和40S核糖体的峰表示。

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, 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+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, 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=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

从polyribosome配置文件中获得的信息，可以进入翻译状态的细胞提供有价值的见解。此外，可确定自己的核糖体亚基组装的状态2 。例如halfmers或80S和较大的山峰有轻微的肩膀上个人资料的权利的存在表明绑定的40S亚基等待60S亚基加入。执行在没有任何附加的放线菌酮或其他伸长抑制剂实验可以分析的流失率，这表明与否的伸长率是改变3。的分数本身是为一个特定的基因或蛋白质与核糖体的亚群，北方或西方的分数印迹协会随后决定的试剂的丰富来源。可确定的积极核糖体mRNA的总池通过相关的芯片 4或深度测序分析 5 。也可以稳定蛋白质协会由适当的另外一个交叉连接试剂6。 从哺乳动物细胞的Polyribosome分析也值得一提的，在不同的协议在建立所需的条件，以获得稳定的polysom​​es明显的困难。文献报道的缓冲区系统广泛变量7-10，从而有可能为细胞裂解液的具体优化的要求，或者是同样合理的缓冲系统，是不是只要与热切关注的重要新鲜裂解液在低温下保存。据我们所知，系统评价的影响哺乳动物polysom​​e形成的参数尚未见报道。

<table><tbody><tr><td>polyallomer ultracentrifuge tube</td><td>Beckman Coulter Inc.</td><td>326823</td><td>1 x 3.5 in. (25x89mm)</td></tr><tr><td>Fluorinert</td><td>Sigma-Aldrich</td><td>F9755</td><td></td></tr><tr><td>Cycloheximde</td><td>Sigma-Aldrich</td><td>C-7698</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393</td><td></td></tr><tr><td>BY4741</td><td>Open Biosystems</td><td>YSC1048</td><td>Other strains work equally well</td></tr></tbody></table>

eukaryotic polyribosome profile analysis

蛋白质合成是一个复杂的细胞的过程，是多层次的监管。例如，全球翻译，可以抑制在开始阶段，或由多种氨基酸饥饿或生长因子撤出的细胞，如强调伸长阶段。另外，个别的mRNA的翻译，可调节mRNA的本地化或同源的microRNA存在。蛋白质合成的研究经常使用polyribosome的分析，阐明翻译调控机制或蛋白质合成的缺陷。在这个实验中，mRNA /核糖体的复合物是由真核细胞中分离出。蔗糖密度梯度分离势必被称为绑定到一个单一的核糖体或monosome的mRNA的多聚核糖体的多个核糖体mRNA的。分馏梯度允许不同的核糖体的人口及其相关的mRNA或蛋白质分离和量化。多聚核糖体的比例在规定条件下的差异，以monosomes可以在任的翻译起始或伸长/终止的缺陷指示。目前的mRNA在polyribosome分数的考试可以揭示是否对个人的mRNA队列被翻译与实验条件的变化。此外，核糖体的组装可以监测分析小核糖体大亚基梯度分离峰。在这段视频中，我们目前的原油从酵母细胞中，蔗糖梯度的提取分离和对结果的解释的核糖体中提取的准备方法。此过程是哺乳动物细胞容易适应。

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

本文介绍了协议的真核细胞的核糖体翻译的提取。一旦提取，核糖体是由蔗糖梯度分馏分离到monosomes和多聚核糖体，允许不同的核糖体的人群进行分析。因此，这种方法是研究翻译调控的黄金标准。

真核Polyribosome剖面分析

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

52.5K 次观看.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. 本文介绍了协议的真核细胞的核糖体翻译的提取。一旦提取，核糖体是由蔗糖梯度分馏分离到monosomes和多聚核糖体，允许不同的核糖体的人群进行分析。因此，这种方法是研究翻译调控的黄金标准。

视频: Eukaryotic Polyribosome Profile Analysis

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

科研

生物化学

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.

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

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 ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

神经科学

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.

无需梯度测定器或分馏系统的多聚体分析

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

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.

翻译起始的分析在应力条件由多聚核糖体剖析

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

生物学

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.

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

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

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.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

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

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.

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.

植物多聚核糖体剖面的简单方法

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

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

通过选择性核糖体分析对共翻译相互作用网络进行全局鉴定

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Identification of open reading frames (ORFs), especially those encoding small peptides and being actively translated under specific physiological contexts, is critical for&#160;comprehensive annotations of context-dependent translatomes. Ribosome profiling, a technique for detecting the binding locations and densities of translating ribosomes on RNA, offers an avenue to rapidly discover where translation is occurring at the genome-wide scale. However, it is not a trivial task in bioinformatics to efficiently and comprehensively identify the translating ORFs for ribosome profiling. Described here is an easy-to-use package, named RiboCode, designed to search for actively translating ORFs of any size from distorted and ambiguous signals in ribosome profiling data. Taking our previously published dataset as an example, this article provides step-by-step instructions for the entire RiboCode pipeline, from preprocessing of the raw data to interpretation of the final output result files. Furthermore, for evaluating the translation rates of the annotated ORFs, procedures for visualization and quantification of ribosome densities on each ORF are also described in detail. In summary, the present article is a useful and timely instruction for the research fields related to translation, small ORFs, and peptides.

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Translating ribosomes decode three nucleotides per codon into peptides. Their movement along mRNA, captured by ribosome profiling, produces the footprints exhibiting characteristic triplet periodicity. This protocol describes how to use RiboCode to decipher this prominent feature from ribosome profiling data to identify actively translated open reading frames at the whole-transcriptome level.

真核Polyribosome剖面分析

摘要

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此视频中的章节

核糖体剖面对芽殖酵母中翻译的全基因组定量

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

在中枢神经系统Translatome由多核糖体分馏体内拷问

无需梯度测定器或分馏系统的多聚体分析

翻译起始的分析在应力条件由多聚核糖体剖析

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

植物多聚核糖体剖面的简单方法

通过选择性核糖体分析对共翻译相互作用网络进行全局鉴定

从头使用核糖体分析数据识别主动翻译的开放阅读框

真核Polyribosome剖面分析

摘要

探索更多视频

此视频中的章节

核糖体剖面对芽殖酵母中翻译的全基因组定量

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

在中枢神经系统Translatome由多核糖体分馏体内拷问

无需梯度测定器或分馏系统的多聚体分析

翻译起始的分析在应力条件由多聚核糖体剖析

多聚核糖体分离和哺乳动物Translatomes分析在基因组范围内

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

植物多聚核糖体剖面的简单方法

通过选择性核糖体分析对共翻译相互作用网络进行全局鉴定

从头 使用核糖体分析数据识别主动翻译的开放阅读框

从头使用核糖体分析数据识别主动翻译的开放阅读框