Name: assessment of selective mrna translation in mammalian cells by polysome profiling
Uploaded: 2014-10-28T15:00:00.000+00:00
Duration: 10 min
Description: Watch this Scientific Journal Video about Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling at JoVE.com

We are grateful to the past and present members of the laboratory, in particular Urszula Liwak, for critical discussion regarding polysome profiling protocols and sharing of data. This work was supported by operating grants from the Canadian institutes of Health Research, Cancer Research Society, and the National Science and Engineering Research Council of Canada to MH. MDF was supported by the Vanier Canada Graduate Scholarship Doctoral Award and the Ontario Graduate Scholarship, and this work forms part of her Ph.D. dissertation.

注 : 注与RNA的工作:采取标准预防措施，以防止RNA降解RNA酶。使用手套，防护吸管头，无RNase的塑料制品和化工产品中的所有步骤的协议。使用超纯或DEPC处理过的水用于所有的解决方案。净化任何怀疑表面用按照制造商的协议RNA酶失活的溶液。 1.准备解决方案。 <ol style="margin-left: 40px;"><li>制成500毫升之基本溶液（0.3 M氯化钠，15毫摩尔的MgCl 2·6H 2 O; 15毫摩尔的Tris-HCl，pH 7.4）中。混合使用搅拌棒，直到溶液澄清并通过0.45μm的过滤器。在室温下储存。 </li><li>制备10和50％蔗糖溶液和60％大通解如下: <ol><li>对于10％的蔗糖溶液（W / V），在50 mL离心管中加入5克蔗糖，并填写到50毫升的基本解决方案。为50％的蔗糖溶液（重量/体积），在50毫升的离心管中的广告D 25克蔗糖，并填写到50毫升的基本解决方案。 </li><li> 60％（W / V）大通解决方案，在50毫升离心管中加30克蔗糖，并填写到50毫升的基本解决方案。加入100μl饱和溴酚蓝溶液（添加溴酚蓝水，直到没有更多的会溶解，离心沉淀不溶解的粉末），以60％追的解决方案。混合使用一个旋涡，并确认完全溶解。 </li><li>商店溶液在4℃下直到需要。 </li></ol></li><li>准备RNA裂解缓冲液。准备这个缓冲区新鲜的实验当天。制备500微升的RNA裂解缓冲液对每个样品。到1ml的基本解的加10微升的Triton X-100（1％[体积/体积]最后），1微升100mg / ml的放线菌酮的DMSO（CHX，0.1毫克/毫升终浓度）和3.5微升的RNasin（140Ú /毫升）。混合使用涡旋。保持在冰上。 注:放线菌酮是剧毒的，并可能导致突变。避免皮肤接触和吸入。为了避免处理粉末状过于频繁，准备在DMSO中，等分试样较大的量为100 mg / ml储备，并保持在-80℃下长期贮存。保持在-20℃下工作的股票。 </li><li>在实验的当天，通过添加放线菌酮（0.1毫克/毫升终浓度）为10％的所需量和50％的蔗糖溶液（〜7毫升每梯度每种溶液）制备蔗糖+ CHX解。 </li></ol> 2.制备蔗糖梯度的使用自动梯度机<ol style="margin-left: 40px;"><li>打开渐变壶&#39;&#39;ON&#39;和水平板，其将持有的梯度（criticalstep！）点击&#39;完成&#39;&#39;当板拉平。 </li><li>在菜单上选择渐变程序来运行: <ol><li>按&#39;&#39;GRAD&#39;&#39;菜单，选择&#39;LIST&#39;。 </li><li>选择"SW41"。 </li><li>滚动列表，然后选择"&#39;10 - 50％的坡度 - 11步&#39;&#39;计划通过压荷兰国际集团的&#39;RUN&#39;&#39;按钮。 </li></ol></li><li>放置一个锥形离心管（SW-41管，14×89毫米）插入标记块并使用所提供的细管标记物跟踪沿着标记块的上面步骤中的管的标记。将管在一个稳定的表面装配架。 注:此标志将是填充指南关于分层的10和50％的蔗糖溶液。 </li><li>将两个提供给用套管210毫升注射器和通过上下抽吸用含有RNA酶失活溶液温蒸馏水洗净。一定要去除水中的所有痕迹之后。 </li><li>填充有10％蔗糖+ CHX注射器中的一个，并插入套管，在超速离心管的底部。轻轻地分配10％的蔗糖溶液，直到它进入刚刚过去制成的管子的标记。 注意:要避免气泡。如果形成气泡，轻轻拍打管来取代它们。 </li><li>填补其他的注射器用50％蔗糖+ CHX，擦去多余的液体关闭与在超速离心管的底部上的擦拭并轻轻插入插管。轻轻地分配的50％蔗糖溶液，直到达到完全的标记。快速，顺利地取出套管。 注:10％蔗糖应该上升管中形成一个弯月面在顶部。如果没有，则添加更多的10％蔗糖溶液，在表面上。 </li><li>通过倾斜超速离心管稍微取代所有气泡将提供的盖子。在帽的水库有微量吸管去除多余的液体。 </li><li>擦拭沿筒壁和地点上的梯度机板的剩余液体。 </li><li>按&#39;&#39;RUN&#39;&#39;按钮。 注:该板块将在倾斜指定角度，停顿5秒，旋转，形成梯度将开始。 </li><li>当梯度的形成是完整的（约2分钟，机器会发出哔哔声），轻轻取下超速离心机（S），放在架上，并迅速在向上运动瓶盖取下，以避免干扰gradie新台币。 </li><li>保持梯度冰（S）。请勿打扰！另外，梯度保持一夜之间在4℃。 </li><li>另外，使用两腔梯度制造商进行渐变，如下所示: <ol><li>冲洗管道和梯度机用RNA酶失活溶液和无RNA酶的水。 </li><li>加入5 ml的50％蔗糖+ CHX对梯度制造者的近端腔室，并确保该两个腔室之间的任何空气被除去。 </li><li>加入5毫升10％蔗糖+ CHX对梯度制造商的远端室。 </li><li>添加0.5毫升50％蔗糖+ CHX对底部的锥形离心管（SW-41管，14×89毫米），并装在管保持架。 </li><li>打开搅拌器放在近端室，开停，公鸡和存款梯度速度设置为"3"（约1ml /分钟）。 </li><li>处的梯度（多个）上的冰。请勿打扰。 注释:梯度可以保持过夜，在4℃。 </li></ol></li></ol> 3.细胞裂解和超速离心。 <ol style="保证金左:40PX;"&gt; <li>放置SW-41转子和轮叶在4℃下，并设置离心机至4℃。 注:细胞裂解过程被优化为2150毫米汇合（〜70-80％汇合）每梯度HEK293细胞的板和所用的量可能需要调整为不同的细胞系。 </li><li>板的细胞在生长培养基中至少24小时之前，裂解的合适的细胞密度。 </li><li>制备含有0.1mg / ml的放线菌酮的生长培养基的等分试样（每150毫米的钢板20ml）中。 </li><li>孵育细胞CHX +媒体在37℃/ 5％CO 2（每150毫米的钢板20ml）中3分钟，以逮捕和稳定多聚核糖体。 </li><li>工作迅速，吸媒体从板，在冰上代替板，用10ml冰冷的磷酸盐缓冲盐水洗一次细胞（PBS:137mM的氯化钠，2.7毫摩尔的KCl，10mM的磷酸氢二钠，1.8毫摩尔KH 2 PO 4），补充用放线菌酮（0.1毫克/毫升终浓度）小心缓慢地吸取下来的sidE中的盘子壁的以最小化细胞单层的升降。 </li><li>吸出PBS中，并加入另外的10毫升的PBS + CHX到每个板，刮去细胞与细胞挺杆和从两板的总体积转移至50ml离心管置于冰上。保留1毫升细胞悬浮液在冰上下游蛋白分析微量离心管中。 </li><li>离心机的50ml管中的细胞在300×g离心在4℃下进行10分钟。 </li><li>删除所有PBS重悬沉淀在500μl冰冷的RNA裂解液。 </li><li>转移细胞悬液至2ml微量离心管中。 </li><li>移液管上下裂解细胞并在冰上孵育10分钟，偶尔涡旋。 </li><li>离心机在2000×g离心在4℃下进行5分钟以沉淀细胞核。 </li><li>转移上清液至新的2ml微量离心管中。 </li><li>离心机以17000×g离心在4℃下进行5分钟以沉淀细胞碎片。 </li><li>转移上清液至新的2ml微量离心管中，并使用2微升测量在260nm处的光密度（用裂解缓冲液作为空白）。 </li><li>从每个样品中总RNA的分析去除50微升（共10％）。注裂解物可以储存在-80℃直到需要。 </li><li>等于A260单位轻轻放入到每个蔗糖梯度（10个OD，最佳25 OD，在500微升的最大音量;如果需要稀释浓缩的裂解额外的裂解缓冲液）。 </li><li>确保梯度内0.1克彼此之前超速离心。 </li><li>梯度离心在39000转（260343 XG）1.5小时，在4℃。 </li><li>在降速过程保持转子制动器和减速时将其关闭，当它达到1000转。 注:此步骤最大限度地减少了加速度的突然变化造成的刷头在制动过程中与转子上的梯度的任何干扰。 </li></ol> 4.梯度分馏系统仪器设置。 <ol style="margin-left: 40px;"><li>设置仪器和空白的S用水pectrophotometer如下: <ol><li>打开组件和允许灯预热至少15分钟。 </li><li>确保馏分收集器被设置为"核糖体"的方法（按文件夹图标的两倍;返回箭头两次，返回到主屏幕）。 </li><li>确保在馏分收集器的阀设置为浪费（箭头指向垃圾桶图标）。把这个包含废物收集管下蒸馏水的250ml锥形瓶中。 </li><li>选择在图表记录进行以下设置:设置感光度转盘"SET灯和光学的;设置噪声滤波器开关&#39;1.5&#39;;山顶设置拨号器，以"OFF";设置图表单键拨号&#39;30厘米/小时"（5厘米/ 10分钟）;设置基线调节旋钮（在分光光度计单元的顶部），以"最大打开&#39;。 注:可选配:数字录音机软件，可以用来代替图表记录。在计算机上，单击"开始"。采集窗口将打开:下个ê选项菜单，取消暂停图形。在缩放，设置图形的限制-10〜100（可改变运行期间的即时）。点击保存按钮来创建一个新的文件，并记录运行。 </li><li>放置在所述泵的注射器和枪管和拧入位置（设置使用螺钉和六角扳手）。 注意:不要拧得过紧。 </li><li>通过使用&#39;&#39;反转&#39;&#39;命令的速度快装满大通解决方案的注射器。放置在垂直位置，泵以及使用该&#39;&#39;前进&#39;的命令通过管道追空气。仅使用RAPID此功能和洗涤。 </li><li>安装一个超速离心管填充有不含RNA酶的水。 </li><li>皮尔斯的超速离心管与套管直到两个黑色标记是可见的（分配孔位于这两个标记之间），并开始注射泵上&#39;&#39;手动&#39;&#39;为6.0毫升/分钟。 </li><li>当水通过流动池传递（当cHASE溶液填充该管的1/3），开关速度变量和设定为1.0毫升/分钟。 </li><li>以1.0ml / min的流速，调整基线调整拨盘上的分光光度计直到图表上的电压是零。（+/- 0.5单位）。 注:观察从废液管出口处的水进入锥形瓶中。 </li><li>设置所需的灵敏度为&#39;0.5&#39;或&#39;1.0&#39;AU。 注:1.0 AU在10ml的梯度最适合10的OD单位。如果OD值低于10时，&#39;0.5&#39;的灵敏度，可以用于增强信号。 </li><li>按下自动基线按钮，通过旋转记录的偏移拨号（使用数字录像机选购IF）调整基线设置，在图表记录了10％大关。 </li><li>一旦稳定的基线实现，如果使用模拟图表记录仪或&#39;OFF&#39;，如果使用数字录音机打​​开泵开关为"OFF"和图表快速拨号至60厘米/小时。 </li><li>通过切换泵&#39;再恢复大通解决方案V&#39;的位置（如果需要的话可以增加流量回至6.0毫升/分钟。请不要使用RAPID），直到它到达的穿刺插管的第一个标志。 </li><li>取出离心管中，并通过其上运行的解决方案大通一点点追套管内的任何气泡。擦拭穿孔阶段干净。 注:某些残留的水可能泄漏从流动池。 </li></ol></li></ol> 5.梯度分离和收集样品。 <ol style="margin-left: 40px;"><li>安装和刺穿含有蔗糖梯度的离心管中。 </li><li>将112毫升离心管中馏分收集器托盘位置1-11这样的帽子不向上突出。更换&#39;&#39;管＃1&#39;&#39;现在有一个&#39;废管&#39;来收集遗留在收集管的液体。 </li><li>泵控制转盘设置为"手动"和流速的注射泵，以"6.0毫升/分钟" </l我&gt; <li>请按馏分收集器"播放"图标。一旦臂到达第一管中，按&#39;暂停&#39;按钮（这将定位臂和打开分数分配阀）。 </li><li>启动泵使用&#39;前进&#39;的命令和监控图表记录笔。当它开始向上偏转（表示样品通过分光光度计的流通池），快速更换&#39;废物管&#39;&#39;与&#39;&#39;管＃1&#39;&#39;。 </li><li>当第一滴收集，快速切换命令"远程启动/停止&#39;，&#39;&#39;变量（1.0毫升/分钟），&#39;&#39;，按&#39;播放&#39;图标。 注:该泵现在由馏分收集器接口控制和1ml的级分被收集每一分钟。 </li><li>从这一点上，A260读数将出现在数字记录器接口（或模拟图表recorde上R）。确保&#39;&#39;记录&#39;上的软件按钮被按下！ </li><li>蓝色大通溶液进入收集管或最后管（通常管11）后，按"停止"图标。 注:分配阀应切换到排污阀。 </li><li>保持分数上的冰，直到所有的梯度被分级。在一天的部分的端部可存放在-80℃之前，蛋白质或RNA分离。如果需要的蛋白质和RNA的分析，对半分割的每个级分（每500微升的蛋白质和RNA分析）。 </li></ol> 6.洗注:（这是要只进行一个可选的步骤，如果多个梯度将被收集）。 <ol style="margin-left: 40px;"><li>淹没废液管到含有锥形瓶中〜50毫升的超纯水和用6毫升/分钟的流速反向泵浦。 </li><li>停泵时，大通溶液到达的第一个标记0n中的穿孔套管。 </li><li>取下离心管中，追任何空气从套管和清洁穿孔阶段。 注:某些残留的水可能泄漏从流动池。 </li><li>重复分馏过程始于步骤5.1。 </li></ol> 7.最后的清理。 <ol style="margin-left: 40px;"><li>断开从穿孔器的底部泵管和泵的大通解成使用快速流量设定在50ml离心管中。存储解决方案大通在4℃，因为它可重复使用。 </li><li>泵管连接到一个3路管道系统，并关闭阀通向注射器。连接一个管到50ml注射器中加入蒸馏水和另一管的穿孔器的底部。 </li><li>安装用于冲裁步骤中的超速离心管，并通过分光光度计和收集管（切换到集合菜单的界面上通过点击""星传递至少50毫升蒸馏水T /暂停""图标）。 </li><li>取出试管，注射器（用内六角扳手卸下螺丝）和所有其它可移动部件和用温自来水彻底清洗，然后冲洗用超纯水。 注意:清洗柱塞，桶，油管和套管刺入时要特别小心。 注:处理玻璃注射器针筒小心！存储所有组件远离灰尘。 </li><li>擦拭流动池的底部与软组织蘸有蒸馏水。擦拭馏分收集器托盘和那里的蔗糖可能已经洒任何其他地区。 </li></ol> 8.隔离从蔗糖分数的RNA。 <ol style="margin-left: 40px;"><li>制备50微升蛋白酶K溶液，每1毫升蔗糖级分（37.5微升10％SDS，7.5微升0.5M EDTA，1微升Glycoblue，加入4μl20mg / ml的蛋白酶K）。 </li><li>在2毫升平底管中，孵育1毫升蔗糖级分用50μl的蛋白酶K溶液在55℃下1小时（如果使用500μl的级分调整相应的蛋白酶K溶液体积）。 </li><li>添加的苯酚用等体积氯仿:异戊醇（125:24:1，优选酸性的（pH值4.5），以尽量减少DNA污染）的蔗糖级分。 注意:添加一个额外的200微升氯仿馏分7-10，因为他们是重，阶段可以得到反转。 注:酚/氯仿可能造成接触和吸入灼伤。穿白大褂，防护眼镜，并使用通风柜。 </li><li>涡流〜30秒，以最大速度离心5分钟，在室温下进行。 </li><li>删除〜水相的80-90％（不碰相间其中可能包含的基因组DNA），并将其放置在新的试管中。 </li><li>加入氯仿并重复步骤8.4等体积。 </li><li>除去水相要小心，以避免相间。 </li><li>加一点10体积的3M乙酸钠，pH为5.2（可替换的5M乙酸铵可以使用）。 </li><li>加入1.5倍体积的冷藏，绝对完美德乙醇，涡旋15秒。 </li><li>一夜沉淀在-20°C（或1小时，在-80℃，如果赶时间）。 注:样品可在-20留下 °下，如果不再需要。 </li><li>以最大速度离心30分钟，在4℃下。 </li><li>用1毫升冷冻的不含RNA酶的70％乙醇洗涤沉淀。 </li><li>空气干燥沉淀，并重新悬浮于20μl不含RNA酶的水。 </li><li>通过分光光度法定量的总RNA，以确保有足够的产率和纯度（基于A260 / 280的比例），并使用每个级分的特定的感兴趣的基因（多个）相同体积和PCR引物进行标准的RT-qPCR的分析14。 </li></ol> 9.隔离从蔗糖馏分蛋白。 <ol style="margin-left: 40px;"><li>除了RNA，蛋白质可被分离和鉴定在单个多核糖体级分也是如此。使用可用于蛋白质分离许多优秀的协议， 如 18之一。 </li></ol>

<ol>
	<li>Holcik, M., 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=Translational+control+in+stress+and+apoptosis.">Translational control in stress and apoptosis.</a> Nat Rev Mol Cell Biol. 6 (4), 318-327 (2005).</li><li>Obrig, T. G., Culp, W. J., McKeehan, W. L., Hardesty, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+by+which+cycloheximide+and+related+glutarimide+antibiotics+inhibit+peptide+synthesis+on+reticulocyte+ribosomes.">The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes.</a> J Biol Chem. 246 (1), 174-181 (1971).</li><li>Tscherne, J. S., Pestka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+protein+synthesis+in+intact+HeLa+cells.">Inhibition of protein synthesis in intact HeLa cells.</a> Antimicrob Agents Chemother. 8 (4), 479-487 (1975).</li><li>Damgaard, C. K., Lykke-Andersen, 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+coregulation+of+5'TOP+mRNAs+by+TIA-1+and+TIAR.">Translational coregulation of 5'TOP mRNAs by TIA-1 and TIAR.</a> Genes Dev. 25 (19), 2057-2068 (2011).</li><li>Thakor, N., Holcik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRES-mediated+translation+of+cellular+messenger+RNA+operates+in+eIF2alpha-+independent+manner+during+stress.">IRES-mediated translation of cellular messenger RNA operates in eIF2alpha- independent manner during stress.</a> Nucleic Acids Res. 40 (2), 541-552 (2012).</li><li>McKinney, C., 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=Global+reprogramming+of+the+cellular+translational+landscape+facilitates+cytomegalovirus+replication.">Global reprogramming of the cellular translational landscape facilitates cytomegalovirus replication.</a> Cell Rep. 6 (1), 9-17 (2014).</li><li>Jivotovskaya, A. V., Valasek, L., 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=Eukaryotic+translation+initiation+factor+3+(eIF3)+and+eIF2+can+promote+mRNA+binding+to+40S+subunits+independently+of+eIF4G+in+yeast.">Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast.</a> Mol Cell Biol. 26 (4), 1355-1372 (2006).</li><li>Gross, J. D., 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+Loading+onto+the+mRNA+Cap+Is+Driven+by+Conformational+Coupling+between+eIF4G+and+eIF4E.">Ribosome Loading onto the mRNA Cap Is Driven by Conformational Coupling between eIF4G and eIF4E.</a> Cell. 115 (6), 739-750 (2003).</li><li>Graber, T. E., Baird, S. D., Kao, P. N., Mathews, M. B., Holcik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF45+functions+as+an+IRES+trans-acting+factor+that+is+required+for+translation+of+cIAP1+during+the+unfolded+protein+response.">NF45 functions as an IRES trans-acting factor that is required for translation of cIAP1 during the unfolded protein response.</a> Cell Death Differ. 17 (4), 719-729 (2010).</li><li>Spriggs, K. A., Stoneley, M., Bushell, M., Willis, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-programming+of+translation+following+cell+stress+allows+IRES-mediated+translation+to+predominate.">Re-programming of translation following cell stress allows IRES-mediated translation to predominate.</a> Biol Cell. 100 (1), 27-38 (2008).</li><li>Uniacke, J., 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=An+oxygen-regulated+switch+in+the+protein+synthesis+machinery.">An oxygen-regulated switch in the protein synthesis machinery.</a> Nature. 486 (7401), 126-129 (2012).</li><li>Liwak, U., 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=Tumor+suppressor+PDCD4+represses+internal+ribosome+entry+site-mediated+translation+of+antiapoptotic+proteins+and+is+regulated+by+S6+kinase+2.">Tumor suppressor PDCD4 represses internal ribosome entry site-mediated translation of antiapoptotic proteins and is regulated by S6 kinase 2.</a> Mol Cell Biol. 32 (10), 1818-1829 (2012).</li><li>Occhi, G., 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=A+novel+mutation+in+the+upstream+open+reading+frame+of+the+CDKN1B+gene+causes+a+MEN4+phenotype.">A novel mutation in the upstream open reading frame of the CDKN1B gene causes a MEN4 phenotype.</a> PLoS Genet. 9 (3), (2013).</li><li>Faye, M. D., 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=Nucleotide+composition+of+cellular+internal+ribosome+entry+sites+defines+dependence+on+NF45+and+predicts+a+posttranscriptional+mitotic+regulon.">Nucleotide composition of cellular internal ribosome entry sites defines dependence on NF45 and predicts a posttranscriptional mitotic regulon.</a> Mol Cell Biol. 33 (2), 307-318 (2013).</li><li>Zong, Q., Schummer, M., Hood, L., Messenger Morris, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+translation+state:+the+second+dimension+of+high-throughput+expression+screening.">RNA translation state: the second dimension of high-throughput expression screening.</a> Proc Natl Acad Sci U S A. 96 (19), 10632-10636 (1999).</li><li>Bunnik, E. 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=Polysome+profiling+reveals+translational+control+of+gene+expression+in+the+human+malaria+parasite+Plasmodium+falciparum.">Polysome profiling reveals translational control of gene expression in the human malaria parasite Plasmodium falciparum.</a> Genome Biol. 14 (11), (2013).</li><li>Choudhuri, A., Maitra, U., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+initiation+factor+eIF3h+targets+specific+transcripts+to+polysomes+during+embryogenesis.">Translation initiation factor eIF3h targets specific transcripts to polysomes during embryogenesis.</a> Proc Natl Acad Sci U S A. 110 (24), 9818-9823 (2013).</li><li>Corbin, F., 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=The+fragile+X+mental+retardation+protein+is+associated+with+poly(A)++mRNA+in+actively+translating+polyribosomes.">The fragile X mental retardation protein is associated with poly(A)+ mRNA in actively translating polyribosomes.</a> Hum Mol Genet. 6 (9), 1465-1472 (1997).</li><li>Riley, A., Jordan, L. E., Holcik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+5'+UTRs+regulate+XIAP+expression+under+normal+growth+conditions+and+during+cellular+stress.">Distinct 5' UTRs regulate XIAP expression under normal growth conditions and during cellular stress.</a> Nucleic Acids Res. 38 (14), 4665-4674 (2010).</li><li>Thermann, R., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+miR2+induces+pseudo-polysomes+and+inhibits+translation+initiation.">Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation.</a> Nature. 447 (7146), 875-878 (2007).</li><li>Darnell, J. C., 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=FMRP+stalls+ribosomal+translocation+on+mRNAs+linked+to+synaptic+function+and+autism.">FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism.</a> Cell. 146 (2), 247-261 (2011).</li><li>Graber, T. 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=Reactivation+of+stalled+polyribosomes+in+synaptic+plasticity.">Reactivation of stalled polyribosomes in synaptic plasticity.</a> Proc Natl Acad Sci U S A. 110 (40), 16205-16210 (2013).</li><li>Wang, Y., Ringquist, S., Cho, A. H., Rondeau, G., Welsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+polyribosome+fractionation.">High-throughput polyribosome fractionation.</a> Nucleic Acids Res. 32 (10), (2004).</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 (5924), 218-223 (2009).</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><li>Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M., 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=The+ribosome+profiling+strategy+for+monitoring+translation+in+vivo+by+deep+sequencing+of+ribosome-protected+mRNA+fragments.">The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.</a> Nat Protoc. 7 (8), 1534-1550 (2012).</li></ol>

The authors have nothing to disclose.

多核糖体谱是一个功能强大的技术，其允许特定的转录物的翻译的状态的不同处理条件下的定量。它在原理上是一种非常简单的技术但它需要多个步骤执行的，其中的一些是用于制造好的多核糖体轮廓的关键。这些关键的步骤是:1）使用无RNA酶的化学品和塑料制品，2）准备好蔗糖梯度（以我们的经验，10％-50％的蔗糖梯度最适合有效解决40 / 60S亚单位的mRNA，并与核糖体结合）和3 ）使用，以便细胞被成倍增长的和不大于80％汇合捕获翻译率最高。这后一步骤应做长细胞的治疗，如基因敲除或过表达时加以考虑，使细胞与板的量将是多少的细胞将在端点被汇合的功能。 在结果presen泰德这里，多核糖体信息分析是用于评估在IRES-携带的mRNA XIAP和Bcl-xL 12的翻译调控PDCD4的作用的一个重要工具。我们没有观察到一般多核糖体轮廓时PDCD4击倒（ 图1B）任何改变的事实使我们得出这样的结论PDCD4没有实验条件下影响全球蜂窝翻译。我们接下来使用的RT-qPCR的分析，以确定XIAP和Bcl-xL的mRNA与PDCD4或对照siRNA处理的细胞中的分布。 qPCR的是所选择的用于分析多核糖体剖面，相对于标准的RT-PCR或Northern印迹的方法，因为它允许进行定量mRNA的分布，而不是半定量分析，并在治疗之间的翻译效率的微妙变化的检测。使用这种技术，我们能够显示，XIAP和Bcl-xL mRNA的分布（表示为总目标mRNA的跨越克的百分比adient）溶液显著移入重型多聚核糖体（mRNA表达与后PDCD4的敲低（ 图1D，E），有大量的核糖体与它们相关联），从而表明增加这些mRNA的翻译。这是通过增加XIAP和Bcl-xL蛋白水平而不是在它们的稳态RNA水平（ 图1F，G）进一步证实。重要的是，XIAP的非IRES变体的多核糖体分布处理和未处理的细胞中（ 图1C）之间变化。或者，翻译效率可以表示为mRNA的含量在多聚核糖体（通常是馏分5〜10）与monosomes的比率（通常是馏分2〜4）14。 在整个协议中使用的控件是验证任何多核糖体分析的结果很重要，因为从吸光度读数观察到在254nm处的峰不能自行归因于核糖体RNA（洗涤剂S，如Triton X-100强烈地吸收在光谱的该区域，并可能掩盖40 / 60S峰梯度的顶部）。空白梯度而不裂解物和/或与溶胞产物缓冲液需要运行，以确定在254nm处的缓冲器，以吸光度的相对贡献。假象高阶结构也可导致多核糖体，其中有实际上没有（ 例如，"伪多核糖体"20）的错误的解释。螯合镁（用于整个过程，以稳定80S核糖体）与二价阳离子的螯合剂乙二胺四乙酸加入到裂解物中和至梯度缓冲液（20mM，15分钟）将导致核糖体分离成其组成小的（40S）和大的（60S ）亚基。因此，如果记录在260nm处的吸收峰是确实的多核糖体，EDTA处理就会崩溃使得单个最大将邻近梯度对应于释放mRNA和核糖体单元的顶部观察到的信息（数据未示出）。一致与轮廓的EDTA诱导的崩溃，所有的通过qPCR分析的具体目标，现在应该在梯度的顶部中。 与mRNA的关联核糖体的数目不总是对如何有效地特定的mRNA被翻译的指示。事实上，有其中在多聚核糖体活性翻译成为推迟21,22，读者应该知道的特定上下文。确定是否转录物都在积极地与翻译机器可以通过完成）处理样品嘌呤，它不像的EDTA，使"径流"核糖体正在积极跨越的mRNA转运的，或b）处理所述样品与高三尖杉酯抑制只在起始密码子的第一个核糖体的移位，再次造成任何下游易位核糖体&#39;流失&#39;。 对于定量PCR分析利用控制的成绩单也很关键。该SELEC转录物的灰即不受不同的处理条件，如某些管家基因（GAPDH，肌动蛋白，微管蛋白，核仁），核糖体的mRNA（18S，RPL13A），或在这种情况下，XIAP的IRES的非IRES变体中，是在重要验证是否对翻译的治疗的效果是特定的或普遍。这些控制转录物可以用来归一化分析的mRNA作为在这里所做的分布（XIAP和Bcl-xL的mRNA进行标准化为GAPDH mRNA和表达为总mRNA含量的通过mRNA的含量的总和在每一个分数表示的百分比）或可选地，目标和控制的mRNA可以被表示为绝对值，并分别示出。输入裂解物（通常为10％总体积的）的定量PCR分析是另一个步骤，将控制特定的mRNA的分布。理想情况下，在输入裂解特定转录的mRNA的含量应相当于在所有10级分析mRNA表达量的总和。最后，一个可选的步骤，以控制对苯酚:氯仿提取步骤中，每穗多核糖体级分用体外转录的报道基因（如CAT，氯霉素乙酰转移酶），将通过qPCR随后被量化，并甚至可以用来归一化的数据14。 与任何技术中，将多核糖纹呈现一定的局限性。通常至少10分（在翻译效率更微妙的变化，可能需要20个或更多）需要收集，以便有很好的多核糖体分布的事实使得这种步骤限制性的，在这个意义上，最多只有4个样本可以是在同一时间进行分析，以便能够一次轻松的处理RNA提取和40个样本和4个输入控件的qPCR分析。样本大小可以很容易地添加了qPCR的多少转录物进行分析和多少重复做的，这可能是昂贵的。的定量PCR为基础的多核糖体分析一个又一个的限制nalysis是，它只能在一个候选为基础的方法（其中，将待分析的转录物是预先已知的），并不能适用于翻译的全基因组分析来完成。然而，在21 世纪初，研究人员就开始询问自己的多核糖体RNA在使用DNA基因组水平最近15芯片和新一代测序16,17。在这种强大的方法，多核糖体分析已在本协议中所述不同的是，而不是执行单个组分的定量PCR分析进行，monosomes分数（通常是2-4分）和多核糖体部分（通常是分数）汇集在一起​​，在变化的全球分析了翻译通过DNA微阵列或全基因组RNA测序。因此，使用这种方法，可以评估所有的mRNA，其分布的变化，从多聚核糖体来monosomes（减少翻译），反之亦然（提高翻译）在一个特定的治疗换货。验证特定mRNA的多核糖体的分布与定量然后可以通过定量PCR来完成。一个更强大的使用的多核糖体分析的原则是核糖体分析。这种技术的进一步高吞吐量的扩展最近有报道称，允许同时监控上百个核糖体部分23的。这提供了明显的优势探测的突变体集合或在大规模RNAi筛选转化效率时。核糖体分析是一种技术，采用新一代测序的优势，通过映射核糖体（核糖体的足迹）保护的RNA片段从事蛋白质合成24,25。在该技术中，核糖体易位可以抑制与放线菌酮（可逆）或依米丁（不可逆），随后加入细胞裂解和RNA酶消化，得到的核糖体脚印的群体。核糖体富集，总RNA分离和污染核糖体和线粒体核糖体RNA被除去。约26-28个核苷酸的RNA脚印是孤立的，反转录，转化成cDNA文库进行测序26。不像标准的多核糖体分析，这种方法不仅标识了翻译调控特异mRNAs但也产生了在密码子的第mRNA的特定信息，如精确占领和核糖体的密度沿特定转录物。这是特别的mRNA的翻译与特定的模式，如IRES-窝藏mRNA的利益，因为它可以给哪里的成绩单核糖体招募正在发生的更多信息。

蛋白质的合成（翻译）的调节是其密切相关的细胞存活的一个关键的细胞过程。鉴于翻译消耗细胞的能量的50％以上，这是不足为奇的翻译是被严格调控，并且在翻译扰动没有得到很好的耐受性。相反，许多异常的细胞过程需要翻译机器翻译输出（ 即蛋白质），必须进行修改。这是常常在各种应激反应和疾病状态的情况下，和可能是最好的例证中的癌症。翻译控制的一个主要优点是电池的快速重新编程的蛋白质输出响应于各种外部和内部触发的能力，从而微调机制，提示细胞存活和死亡1之间的平衡。 翻译控制的两个方面是特别令人感兴趣的;监管MEC的本质的认识hanism（S）和控制元件，使特定的翻译，并识别特定的mRNA及其蛋白质产物参与到引起选择性的翻译在首位的特定触发的细胞应答。多核糖体分析是一种技术，它允许两个进程的有效的学习。 多核糖体分析技术的总体目标是研究和量化不同的细胞条件下的特定mRNA的翻译状态。该技术的原理是捕捉mRNA的翻译由&#39;上的蔗糖梯度&#39;冻结&#39;&#39;上的不同的转录积极翻译的核糖体，并分离得到的多聚核糖体通过超速离心，从而允许高度翻译转录物之间的区别（受几个核糖体）和差的翻译那些（结合被一个或两个核糖体）。在这个特定的协议，该抗生素放线菌酮，其结合到所述60S核糖体亚基和块脱乙酰化的tRNA从核糖体E盘2的释放，被用来抑制核糖体易位和失速的核糖体上翻译的mRNA。然而，其他的封端剂，如吐根碱也阻挡翻译延伸3，都可以使用。 不像蛋白质印迹和其测量翻译过程的最终输出35的S-蛋氨酸标记技术，多核糖纹具有测量与积极转译的mRNA核糖体相关联的优点，因而允许的翻译机制在不同条件下的更详细的研究。因此，该技术可以很容易地适应具体研究不同翻译4-6的方式，参与翻译调控7-9，影响蛋白质的合成1,10,11或mRNA的结构，例如IRES或上游的影响胁迫条件下的蛋白因子对蛋白质合成的开放阅读框ESIS 12-14。如今，多核糖体分析应用更广泛的使用DNA微阵列15或下一代测序16,17的分析多聚核糖体相关的mRNA。

<table><tbody><tr><td>Gradient Master 108 automated gradient maker</td><td>BIOCOMP</td><td>107-201M</td><td></td></tr><tr><td>Auto Densi-Flow two-chamber gradient maker</td><td>LABCONCO</td><td>4517000</td><td></td></tr><tr><td>Beckman Ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>Model # L-100-XP</td><td></td></tr><tr><td>Density gradient fractionator</td><td>TELEDYNE ISCO</td><td>69-3873-246</td><td>Composed of: tube piercing system,&amp;nbsp; peristaltic pump, UA-6 Detector with 254 and 280 nm filters and Foxy R1 Fraction Collector</td></tr><tr><td>WinDaq data acquisition software</td><td>DATAQ INSTRUMENTS</td><td>WINDAQ/LITE</td><td>http://www.dataq.com/products/software/acquisition.htm</td></tr><tr><td>Pollyallomer ultracentrifuge tubes (for SW41, 14 x 89 mm)</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>Nuclease-free 2-ml centrifuge tubes (dolphin nose)</td><td>DiaMed</td><td>PRE1900-N</td><td></td></tr><tr><td>Nuclease-free 2-ml centrifuge tubes (flat bottom)</td><td>SafeSeal</td><td>12000</td><td></td></tr><tr><td>Sucrose (nucleases-free)</td><td>Calbiochem</td><td>573113</td><td>MW = 342.3 g/mol</td></tr><tr><td>Cycloheximide</td><td>SIGMA</td><td>C7698</td><td>MW = 281.4 g/mol. Resuspend in DMSO and keep at -80C for long term sotrage. Caution: TOXIC! Can cause repiratory tracts and skin irritation. Wear mask when weighing.</td></tr><tr><td>Sodium chloride (nucleases-free)</td><td>OmniPur</td><td>7710</td><td>MW = 58.44 g/mol</td></tr><tr><td>MgCl2.6H2O (nucleases-free)</td><td>OmniPur</td><td>5980</td><td>MW = 203.3 g/mol</td></tr><tr><td>Tris Base (nucleases-free)</td><td>J.T. Baker</td><td>4109-01</td><td>MW = 121.14 g/mol</td></tr><tr><td>Triton X-100</td><td>OmniPur</td><td>9400</td><td>Caution: TOXIC! Can cause repiratory tracts and skin irritation</td></tr><tr><td>Recombinant RNasin Ribonuclease Inhibitor</td><td>Promega</td><td>N2515</td><td></td></tr><tr><td>RNaseZap Rnase inactivation solution</td><td>Ambion, Life Technologies</td><td>AM9780</td><td></td></tr><tr><td>GlycoBlue Coprecipitant (15 mg/ml)</td><td>Invitrogen, Life Technologies</td><td>AM9516</td><td></td></tr><tr><td>Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td><td>Ambion, Life Technologies</td><td>AM9722</td><td>Caution: TOXIC ! Can cause repiratory tracts irritation as well as skin irritation and corrosion</td></tr></tbody></table>

assessment of selective mrna translation in mammalian cells by polysome profiling

蛋白质合成的调控表示强调的细胞反应的关键控制点。特别的，谨慎的RNA调控元件均表现为允许对特定的mRNA，其通常所需的特定应激反应的蛋白质进行编码的选择性转换。这些mRNA，以及负责选择性翻译调控机制的表征鉴定已在分子生物学的前沿一段时间。多核糖体分析是在这些研究的基础方法。多核糖体剖面的目的是捕捉mRNA的翻译通过对不同的转录物固定化积极地翻译的核糖体和通过超速离心法分离出来的蔗糖梯度所产生的多核糖体，因此允许高度翻译的转录和翻译的不好的人之间的区别。这些然后可以进一步特征在于，传统的生化和分子生物学方法。重要的是，结合Polysom​​e剖析与高通量基因组学方法允许翻译调控的大尺度分析。

我们最近已描述为IRES-携带的mRNA编码凋亡抑制剂XIAP和Bcl-xL 12选择性翻译抑制剂对肿瘤抑制PDCD4的一种新的角色。多核糖体分析是一个关键的技术展示PDCD4的IRES介导的翻译的具体参与。 HEK293细胞中瞬时转染到耗尽PDCD4的（ 图1A）的内源性水平，然后进行多核糖体信息分析。我们观察到，PDCD4的减少水平没有影响全局蜂窝翻译，作为判断缺乏比较siCTRL和siPDCD4处理的细胞（ 图1B）时的变化，在多核糖体轮廓。同样，XIAP蛋白19的非IRES变体的多核糖体分布处理和未处理的细胞中（ 图1C）之间变化。与此相反，两个IRES-窝藏mRNA的多核糖体分布，XIAP和Bcl-xL，被显著改变。在不存在P的DCD4这两种mRNA的分布转移到重ribopolysomes（ 图1D，E）。因为这不伴随变化的XIAP和Bcl-xL的表达（ 图1F），这些结果稳态水平演示了增强XIAP和Bcl-xL的翻译在细胞中具有减少PDCD4的水平，这进一步通过Western印迹（ 图证实1G）。 <img alt="图1" src="/files/ftp_upload/52295/52295fig1highres.jpg" /> 图1:多核糖纹识别PDCD4如XIAP和Bcl-xL的翻译的选择性抑制剂（A）的 HEK293细胞与PDCD4的siRNA或对照（CTRL）进行了治疗，非靶向siRNA，裂解并进行Western印迹分析用抗PDCD4和抗核仁素抗体来验证PDCD4的敲低的程度。（B）中 PDCD4的被撞倒如（A）和细胞裂解物进行多核糖纹。代表性的多核糖体信息显示在顶部面板;在XIAP蛋白非IRES（C）的分布，Bcl-xL的（D）和XIAP的IRES（E）的 mRNA表达相对于GAPDH的被示为总的mRNA（％的mRNA）中的每个级分的百分比（F）稳定-state mRNA水平通过RT-qPCR的中PDCD4的siRNA或对照（CTRL）的siRNA处理的细胞中测得的。（G），从裂解物（A）中也进行免疫印迹分析用抗XIAP蛋白，抗Bcl-xL的，并抗核仁抗体。 （特别注意:数据显示在图1中最初发表于12）， <a href="https://www.jove.com/files/ftp_upload/52295/52295fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling at JoVE.com

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

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 ...

assessment-selective-mrna-translation-mammalian-cells-polysome

Research

JoVE Journal

Biology

28.1K 次观看.  University of Ottawa. 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. 

视频: Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

科研

生物化学

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 ...

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 ...

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 ...

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, characterizing differences in the translatome (the whole of translated mRNAs) provides an additional layer of information particularly useful when trying to understand the function of RNA regulating or binding proteins. A number of methods for accomplishing this have been developed, including ribosome profiling and polysome analysis. However, both methods carry significant technical challenges and cannot be applied to specific cell populations within a tissue unless combined with additional sorting methods. In contrast, the RiboTag method is a genetic-based, efficient, and technically straightforward alternative that allows the identification of ribosome associated RNAs from specific cell populations without added sorting steps, provided an applicable cell-specific Cre driver is available. This method consists of breeding to generate the genetic models, sample collection, immunoprecipitation, and downstream RNA analyses. Here, we outline this process in adult male mouse germ cells mutant for an RNA binding protein required for male fertility. Special attention is paid to considerations for breeding with a focus on efficient colony management and the generation of correct genetic backgrounds and immunoprecipitation in order to reduce background and optimize output. Discussion of troubleshooting options, additional confirmatory experiments, and potential downstream applications is also included. The presented genetic tools and molecular protocols represent a powerful way to describe the ribosome-associated RNAs of specific cell populations in complex tissues or in systems with aberrant mRNA storage and translation with the goal of informing on the molecular drivers of mutant phenotypes.

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

雄性小鼠生殖细胞中的核糖核菌免疫沉淀

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, ...

发育生物学

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.

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 ...

生物学

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. ...

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. ...

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.

使用多体分析在发育中的鼠标大脑中进行翻译分析

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

神经科学

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

TRAP-RC，翻译核糖体亲和纯化的稀有细胞群<em&gt;果蝇</em&gt;胚胎

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...