这项工作由加拿大自然科学和工程研究委员会资助-发现补助金 (RGPIN-2017-05463), 加拿大创新基金会-约翰 r. 埃文斯领袖基金 (35017), 校园艾伯塔省创新计划和艾伯塔省部经济发展和贸易。

注意: RNA 极易被 ribonucleases (RNases) 降解。采取标准的预防措施, 保持 RNA 完好无损。经常更换手套。在协议的所有步骤中使用过滤过的吸管提示、无核酸酶 plasticware 和无核酸酶化学品。使用无核酸酶或二乙基焦碳酸 (DEPC) 处理水的所有解决方案。
1. 解决方案的准备工作
<ol><li>准备 Toeprinting 缓冲器:20 毫米三盐酸 (pH 7.6), 100 毫米 KOAc, 2.5 毫米毫克 (华侨)2, 5% (瓦特/v) 蔗糖, 2 毫米 dithiothreitol (德勤), 0.5 毫米胺。<ol>
<li>在-20 摄氏度的单用途整除数中存储数码和胺, 以避免重复的冻融循环。 注: 在使用前应立即将胺和 toeprinting 缓冲器添加到该缓冲区中。缺乏胺的解决方案可以存储在-20 摄氏度。</li>
	</ol>
</li>
	<li>制备整除数85毫米 5 '-guanylyl imidodiphosphate (GMP-PNP) 和91毫米三磷酸腺苷 (ATP)。把整除数-20 摄氏度。</li>
	<li>准备450毫升 6% polyacrylamide-7M 尿素凝胶混合: 67.5 毫升40% 丙烯酰胺: 双丙烯酰胺 (19:1), 189 克尿素, 112.5 毫升 5x (三/硼酸/thylenediaminetetraacetic 酸 (EDTA)) 和120毫升水。在37摄氏度的水浴或热板上搅拌, 溶解尿素。筛选解决方案 (e. g, 0.2 微米硝化棉真空过滤器)。 注: 解决方案可存储在4摄氏度, 至少一个月。 注意: 单体丙烯酰胺是一种可以通过皮肤吸收的神经毒素。小心避免皮肤接触 (i. e), 戴上手套, 实验室大衣, 眼睛保护)。聚丙烯酰胺也应小心处理, 因为聚合可能不会进行100% 完成。</li>
	<li>制备甲酰胺负载染料: 95% 甲酰胺, 18 毫米 EDTA, 0.025% (w/v) 月桂酸钠 (SDS), 0.025% (w/v) 溴酚蓝, 0.025% (瓦特/v) 二甲苯蓝。存储在-20 摄氏度。</li>
	<li>溶解 1 nmol 底漆 (5 ' CTCGATATGTGCATCTGTA; 5 ' 末端标记以 IRDye 800) 入 100 ul 水为 10 pmol/ul 的工作集中。存储在-20 °c 在单一使用整除数 (大约10µL), 保护免受光。</li>
</ol>2. mRNA 的制备
<ol><li>从适当的模板 (i. e、基因组 dna 或质粒 dna) 放大 dna 模板, 以聚合酶链反应 (PCR) 合成 mRNA。使用高保真 DNA 聚合酶根据制造商的指示, 以以下反应条件 (35 个周期): 熔化, 98 °c, 十年代;退火, 53 °c, 二十年代;延伸, 72 °c, 三十年代。 注: 前底漆 (5 ' AAGCTTAATACGACTCACTATAG) 包含 T7 启动子序列, 允许 RNA 转录;反向底漆 (5 ' T51GAATTCGGATCCGACCGTGG) 包括51嘧啶残留物, 为转录 RNA 提供一个聚尾。请注意, RNA 也可以从质粒 DNA 中转录为体外。</li>
	<li>使用适当的转录试剂盒体外转录含有 IRES 的 rna 或 DNA 模板中的标记 rna。按照制造商的说明进行操作。在标准的 20 ul 反应量中准备 RNA 样品。治疗新合成的 RNA 与2单位的 RNase 无 DNase 30 分钟37摄氏度。</li>
	<li>将 DNase 处理的 RNA 稀释至110µL, 核酸酶无水加110µL 酸苯酚, 涡 5 s 和离心机在室温下 2万 x g 的3分钟。去除100µL 的水相到一个新的1.5 毫升离心管, 添加10µL 的3米醋酸钠, 和涡旋 2 s. 添加3容量100% 乙醇, 涡流 5 s, 并在-20 °c 一夜之间沉淀 RNA。</li>
	<li>离心机在 > 2万 x g 30 分钟4摄氏度, 并丢弃上清。用500µL 的冰冷 70% (v/v) 乙醇清洗颗粒, 重复离心。吸入尽可能多的上清, 空气干燥的颗粒为 5-10 分钟. 小心不要把球团取出来。</li>
	<li>并用重悬 RNA 在适当的容量核酸酶水中产生0.5 微克/ul 的工作浓度。这可以存储在-80 摄氏度或立即使用。</li>
</ol>3. Toeprinting 反应
<ol><li>混合 15 ul 的兔子网织裂解 (RRL, 不核酸酶处理), 1 ul (40 单位) 的 RNase 抑制剂, 1 µL 91 毫米 ATP (1.82 毫米最后) 和 1 ul GMP-PNP (85 毫米决赛) 在1.7 毫升离心管。孵育30°c 5 分钟。 注: RRL 应 aliquoted 用于单一用途, 以避免重复冻融。临界控制是缺乏 RRL 的反应, 为了阐明反向转录的自然停顿由于 RNA 的次要结构。添加 toeprinting 缓冲区以替换此控件的 RRL。</li>
	<li>添加0.5 µg (1 µL) RNA 和孵育30°c 5 分钟。</li>
	<li>添加22µL 的 Toeprinting 缓冲和孵化在30摄氏度3分钟。</li>
	<li>添加0.5 µL (5 pmol) 的 IRDye 标记底漆和孵化在冰上10分钟。</li>
	<li>添加2µL 25 毫米 dNTPs (最终浓度: 1 毫米), 2 µL 100 毫米毫克 (华侨)2, 1 µL 的禽 Myeloblastosis 病毒逆转录酶 (AMV RT) 和3.5 µL Toeprinting 缓冲。最后的音量是 50 ul。</li>
	<li>孵育反应在30°c 45 分钟。</li>
	<li>添加200µL 的核酸酶水, 并立即提取与250µL 25:24:1 phenol:chloroform:isoamyl 酒精 (pH 约 8.0)。涡流 5 s 和离心机在 2万 x g 在室温下3分钟。去除水相到一个新的1.5 毫升离心管, 增加3容量100% 乙醇, 漩涡 5 s, 并且沉淀在-20 °c 隔夜。</li>
	<li>离心机在 > 2万 x g 30 分钟4摄氏度, 并丢弃上清。用500µL 的冰冷 70% (v/v) 乙醇清洗 DNA 颗粒。离心机在 > 2万 x g 15 分钟4摄氏度。吸入尽可能多的上清, 空气干燥颗粒为 5-10 分钟. 小心不要把球团取出来。</li>
	<li>在 6 ul 的无核酸酶水中溶解颗粒, 加入3的甲酰胺负载染料 ul。</li>
</ol>4. 测序反应
<ol><li>使用 DNA 模板从2.1 和 IRDye 标记底漆从步骤3.4 的标准排序反应, 使用 dideoxynucleotides (ddNTPs) 作为链终结器。使用适当的 DNA 测序套件, 并按照制造商的指示。</li>
	<li>混合 6 ul 的每一个排序反应与 3 ul 的甲酰胺负载染料。</li>
</ol>5. 序凝胶和电泳的制备
注: 本协议使用荧光凝胶为基础的成像仪和21厘米 x 23 厘米 x 0.2 毫米凝胶, 但可以适应其他音序器或凝胶大小, 如果需要的话。
<ol><li>彻底清洁0.2 毫米垫片以及短 (23 x 25 厘米) 和长 (30 x 25 厘米) 玻璃板与100% 乙醇。空气干燥。</li>
	<li>混合30毫升 6% polyacrylamide-7M 尿素凝胶混合 200 ul 10% (瓦特/v) 过硫酸铵 (APS) 和 20 ul 的 tetramethylethylenediamine (TEMED)。倒入凝胶, 小心避免气泡, 并插入 ' 鲨鱼牙齿 ' 凝胶梳子。允许凝胶在室温下聚合1小时。</li>
	<li>装配测序仪, 用1x 填充储层。</li>
	<li>在1500伏前运行15分钟, 直到达到55摄氏度的最佳温度。</li>
	<li>将样品/负载染料混合 (从步骤3.9 或 4.2) 加热到85摄氏度, 5 分钟. 将 1 ul 加载到排序凝胶上。</li>
	<li>用1500伏的凝胶8小时。这台机器将实时读取波段。</li>
	<li>拆卸设备, 处理丙烯酰胺凝胶和运行缓冲器。</li>
</ol>

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作者没有利益冲突的披露。

Toeprinting 是一种强有力的技术, 直接测量一个 RNA 的能力, 以支持在高度控制条件下的翻译起始配合物的形成。该协议描述了一种 toeprinting 哺乳动物 rna 的简化技术。兔网织红细胞裂解剂 (RRL) 是一种方便的核糖体、eIFs、引发 tRNA 和 IRES反式作用因子 (ITAFs) 的来源。实验者提供他们的选择 RNA, 也可以补充 toeprinting 反应与他们选择的特定辅助因子。例如, 48S vs 80S 平移起始配合物可以根据 toeprints7的荧光强度分布进行区分。所观察到的起始复合物将取决于所使用的鸟嘌呤核苷酸的类型。在这里讨论的 XIAP IRES 的情况下, GMP-PNP 加 ATP 稳定48S 预启动复合体, 其特点是 toeprint 分布 + 17≥ + 18 > + 19。GMP-PNP 是一个 nonhydrolyzable GTP 模拟, 抑制核糖体亚基加入, 从而阻止翻译启动在48S 步骤13。相比之下, GTP、atp 或 atp 加 GTP 稳定80S 起始配合物, 其特征是 toeprint 分布 +17 < +18 > +198。
用户将不得不考虑他们希望 toeprint 的 RNA 类型。如果目的是研究与 cap 无关的机制, 如 IRES 元素, 则 RNA 可以是体外转录任何商业可用的工具包。然而, 任何哺乳动物 RNA 都可以接受这种 toeprinting 方法, 只要它有一个 5 ' 帽结构。必须使用适当的工具包生成上限 mRNA。无论如何, 制造商的协议应密切遵循, 因为制备高质量的 RNA 代表了过程中的一个关键步骤。另一个关键点是, 步骤3.7 中的苯酚提取必须在中性 pH 值或以上的情况下进行;如果在这个阶段使用酸苯酚, 新合成的 cDNA 将丢失。值得注意的是, 在步骤3.5 中使用的醋酸镁浓度会影响反向转录的效率, 并且可能需要对每个 mRNA 进行优化。
为了确保 toeprint 的特异性, 需要进行一些关键的控制。首先, 在没有 RRL 或核苷酸的情况下, 不应观察到强健的 toeprint。这确保观察到的反向转录暂停是由于核糖体 rna 复合体, 而不是一个稳定的 rna 二级结构或一些缺陷与反向转录反应本身。第二, 如果起始密码子被突变, 就不应观察 toeprint。这确保核糖体已经形成了一个复杂的, 特别是与起始密码子的 RNA 和用户已经正确地确定了核糖体的 3 ' 边缘在复杂与开始密码子。这对于 IRES 元素尤其重要, 它可能会将核糖体招募到另一起始密码子14。同样, 如果没有多条尾, toeprint 可能会受到损害, 就像 XIAP IRES8的情况一样。但是, 某些 IRES 元素在没有多条尾的情况下形成 toeprints, 如 CrPV IRES10。最后, 除了 toeprint 正确, 其他反向转录暂停可能会被观察到。这些都可以简单地表示由于 rna 中的二级结构稳定的停顿, 或者它们可能是由于一种称为 "核糖体跳跃" 的现象, 其中核糖体从起始密码子滑到 rna15上的其他位置。为了区分这些可能性, toeprinting 反应可以在放线菌酮8的存在下进行, 它有效地力场了起始密码子的核糖体, 并应减少由于核糖体跳跃而导致的替代 toeprints.值得注意的是, 不同的基因会有不同的次级结构, 这意味着反向转录的数量和强度 (i. e) 也会有所不同。
这项技术的一个可能的限制是, 它测量核糖体招募到一个 mRNA在体外, 但形成的翻译起始情结并不一定意味着翻译将发生在体内。因此, toeprinting 在与体内技术相结合以测量翻译 (e. g, polysome 分析16) 和由此产生的蛋白质水平 (e g, 西方分析) 时特别强大。
最近的技术是 "核糖体分析" (也称为 "核糖体足迹"), 其中高通量 RNA 测序用于测量核糖体在总细胞 mRNA 上的存在。这是一个强大的技术来衡量核糖体招募在转录范围内。核糖体分析所固有的是使用试剂, 如放线菌酮, 以稳定核糖体在基因。这可能代表一个缺点, 因为核糖体的数量不成比例, 在 5 ' 未翻译的区域 (UTR) 中可能是非特定停滞的, 这可能被误解为非规范的翻译启动17。在 toeprinting, 放线菌酮不是程序不可分割的一部分, 否定了这种误报的可能性。此外, 任何单一 mRNA 都可以通过 toeprinting 更详细地研究, 因为它是简单的进行许多控制实验和迭代 (例如, 测试几个点突变的影响, 或没有一个多的尾巴, 在核糖体招募)。在核糖体分析实验的背景下进行一组类似的控制实验, 将会耗费时间和成本高昂。因此, Toeprinting 是一种对高通量测序技术的补充方法, 而且还经常用于旨在阐明翻译规则的机制的研究8,9,10, 18。

由于翻译消耗了大部分的细胞能量, 所以翻译被严格管理的1是有意义的。相反, 失调的翻译, 以及随之而来的蛋白质输出的改变, 经常被观察到应激反应和疾病状态, 如癌症1,2。平移控制的一个主要优点是细胞能够改变蛋白质输出的速度, 以响应各种刺激3。因此, 翻译规则是一个重要的机制, 可以影响细胞生存和死亡1,2,3。在翻译步骤中, 启动是最受高度管制和复杂的3。简单地说, 大多数真核基因包含5是7G cap, 几乎总是对他们的翻译至关重要。cap 依赖启动需要真核启动因子 eIF4E, eIF4A 和 eIF4G (cap 识别复合体), 以互动的 5 ' 末端的 mRNA。43S preinitiation 核糖体复合体, 包含 eIF2-bound 引发器 tRNA 和 eIF3, 被招募到 5 ' 末端的 mRNA通过eIF4G 与 eIF3 的相互作用。preinitiation 复合体被认为扫描 mRNA, 由 eIF4A (RNA 旋) 辅助, 直到开始密码子 (AUG) 的位置。48S 起爆复合体随后形成, tRNA 被交付到核糖体的 P 位点。最后, 60S 和40S 核糖体亚基联合形成80S 起始复合体, 后跟翻译伸长1,3,4。相比之下, 内部核糖体进入站点 (IRESes) 通过将40S 核糖体亚基直接招募到起始密码子3, 绕过 5 ' cap 的要求。由于主要的真核致动因子 (eIFs) 的修饰, 生理应激条件减弱了全球 mRNA 的转化。然而, 非规范的翻译启动机制允许选择性地翻译某些基因经常编码的应力反应蛋白, 和失调的非规范化的翻译启动是牵连到疾病的状态, 如癌症1,2. 谨慎的 RNA 管理元素, 如蜂窝 IRESes, 允许翻译这样的基因2,3。
翻译控制的一个特别有趣的方面是了解给定 mRNA 的规范化与非规范化翻译的机制。Toeprinting 是一种技术, 允许详细的机械研究的翻译启动特定 rna在体外。toeprinting 的总体目标是评估一个有兴趣的 RNA 的能力, 以核化在各种条件下与核糖体形成的翻译起始复合体, 以确定哪些序列、结构元素或附件有效的翻译启动需要考虑因素。例如, 核糖体的招募可能会受到阻碍, 因为没有 5 ' 盖帽, 但刺激的存在 IRES。
该技术的原理是体外转录一个感兴趣的 RNA, 在含有翻译成分 (或纯化成分) 的细胞萃取物的存在下孵化它, 以允许起始络合物形成, 并反转转录具有特定底漆的 RNA。稳定的 RNA 核糖体复合物将导致反向转录在核糖体的 3 ' 边缘-所谓的 ' toeprint '5,6,7。
在本协议中, eIFs 的核糖体亚基、tRNAs 和 IRES反式作用因子 (ITAFs) 由兔网织红细胞裂解 (RRL) 方便地贡献。该协议的另一个优点是使用荧光标记的底漆和荧光凝胶为基础的成像仪, 而不是核素底漆。这消除了额外的步骤, 包括 radiolabeling 底漆, 以及干燥的凝胶和暴露在一个强化的屏幕。荧光带是实时记录, 因为凝胶运行, 允许更大的分辨率。名额 X 链细胞凋亡蛋白 (XIAP) IRES RNA 作为一个例子在这里, 虽然上限基因也可以分析此技术8。
与西方分析不同的是, 它测量了细胞裂解物中翻译过程的最终输出, toeprinting 是一种体外方法, 用于测量 RNA 上的翻译起始复杂形成。这种简化方法允许对调节翻译起始的基板或因素进行高度详细的研究 (e. g., 有上限或未覆盖的 mRNA, IRES 结构, 存在或没有聚尾, 提供特定的蛋白质因素,等)。因此, toeprinting 可以用来研究不同的翻译模式8或 mRNA 结构的影响, 如 IRESes, 对蛋白质合成9,10。

<table><tbody><tr><td>DEPC (Diethyl pyrocarbonate)</td><td>Sigma</td><td>D5758-100ML</td><td></td></tr><tr><td>TRIS base, Ultrapure</td><td>JT Baker</td><td>4109-01</td><td></td></tr><tr><td>KOAc (Potassium acetate)</td><td>Bio Basic</td><td>PB0438</td><td></td></tr><tr><td>Mg(OAc)&lt;sub&gt;2&lt;/sub&gt; (Magnesium acetate tetrahydrate)</td><td>Bio Basic</td><td>MB0326</td><td></td></tr><tr><td>Sucrose, molecular biology grade</td><td>Calbiochem</td><td>573113-1KG</td><td></td></tr><tr><td>Spermidine</td><td>Sigma</td><td>85558</td><td></td></tr><tr><td>GMP-PNP (Guanosine 5&amp;prime;-[&amp;beta;,&amp;gamma;-imido]triphosphate trisodium salt hydrate) 0.1 M solution</td><td>Sigma</td><td>G0635</td><td></td></tr><tr><td>ATP (Adenosine 5&amp;prime;-triphosphate) disodium salt, 100 mM solution</td><td>Sigma</td><td>A6559</td><td></td></tr><tr><td>19:1 Acrylamide:bis-acrylamide, 40%</td><td>Bio Basic</td><td>A0006</td><td></td></tr><tr><td>Urea</td><td>Bio Basic</td><td>UB0148</td><td></td></tr><tr><td>500mL bottle top filtration units, 0.2 &amp;micro;m</td><td>Sarstedt</td><td>83.1823.101</td><td></td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037-100ML</td><td></td></tr><tr><td>EDTA (disodium salt, dihydrate)</td><td>Bio Basic</td><td>EB0185</td><td></td></tr><tr><td>SDS</td><td>Bio Basic</td><td>SB0485</td><td></td></tr><tr><td>Bromophenol blue</td><td>Bio Basic</td><td>BDB0001</td><td></td></tr><tr><td>Xylene cyanol FF</td><td>Bio Basic</td><td>XB0005</td><td></td></tr><tr><td>MEGAshortscript T7 transcription kit</td><td>Ambion</td><td>AM1354</td><td></td></tr><tr><td>mMESSAGE mMACHINE T7 transcription kit</td><td>Ambion</td><td>AM1344</td><td></td></tr><tr><td>Acid Phenol:Chloroform (5:1)</td><td>Ambion</td><td>AM9722</td><td></td></tr><tr><td>25:24:1 Phenol:Chloroform:Isoamyl Alcohol</td><td>Invitrogen</td><td>15593-049</td><td></td></tr><tr><td>Rabbit Reticulocyte Lysate (RRL). Should NOT be nuclease-treated.</td><td>Green Hectares, USA</td><td></td><td>Contact Green Hectares, ask for 1:1 RRL:water</td></tr><tr><td>RiboLock RNase Inhibitor (40 U/&amp;micro;L)</td><td>Thermo Fisher</td><td>E00382</td><td></td></tr><tr><td>100 mM dNTPs</td><td>Invitrogen</td><td>56172, 56173, 56174, 56175</td><td>Mix equal parts for a stock of 25 mM each.</td></tr><tr><td>AMV-RT, 10 U/&amp;micro;L</td><td>Promega</td><td>M5101</td><td></td></tr><tr><td>Sequenase Version 2.0 DNA Sequencing Kit</td><td>Thermo Fisher</td><td>707701KT</td><td></td></tr><tr><td>Model 4200 IR2 DNA analyzer</td><td>LI-COR</td><td></td><td>Product has been discontinued</td></tr><tr><td>APS (Ammonium Persulfate)</td><td>Bio Basic</td><td>AB0072</td><td></td></tr><tr><td>TEMED</td><td>Bio Basic</td><td>TB0508</td><td></td></tr><tr><td>Phusion High Fidelity Polymerase</td><td>New England Biolabs</td><td>M0530</td><td></td></tr><tr><td>Turbo Dnase</td><td>Thermo Fisher</td><td>AM2238</td><td></td></tr></tbody></table>

toeprinting analysis of translation initiation complex formation on mammalian mrnas

翻译起始是蛋白质合成的速率限制步骤, 是细胞调节蛋白质输出的关键点。调控蛋白质合成是细胞应激反应的关键, 失调是许多疾病状态的中心, 如癌症。例如, 虽然细胞应力导致了全球翻译的抑制, 通过衰减上限依赖性的启动, 某些应力反应蛋白有选择地翻译成与盖帽无关的方式。谨慎的 RNA 调控元素, 如细胞内核糖体进入站点 (IRESes), 允许翻译这些特定的基因。这种基因的识别, 以及它们的调控机制的特性, 一直是分子生物学中的一个关键领域。Toeprinting 是一种研究 RNA 结构和功能的方法, 它与翻译起始有关。toeprinting 的目的是评估体外转录 RNA 在各种条件下与核糖体形成稳定的配合物的能力, 以确定核糖体中涉及的序列、结构元素或辅助因素绑定-一个前游标有效的翻译启动。除了其他技术, 如西方分析和 polysome 分析, toeprinting 允许一个强大的特点的机制, 调整翻译的启动。

我们以前描述了 XIAP IRES 支持与 cap 无关的翻译启动的体外8,10的能力。Toeprinting 是讯问 XIAP IRES 起爆复合体机械细节的关键技术。对包含 XIAP IRES (图 1A) 的 mRNA 进行编码的 DNA 构造是体外转录并受到 toeprinting 分析。此处使用的 XIAP IRES mRNA 的变种变体在图 1B中表示。XIAP mRNA-核糖体复合体的反向转录产生了典型的 toeprints +17 到 +19 nt 下游 AUG (图 1C, 车道 1)。这表明核糖体招募的起始密码子和形成稳定核糖体-RNA 复合物5,6,7。核糖体招聘在没有多条尾 (图 1C、车道 9) 的情况下被强烈削弱, 如先前报告的11。在缺乏 RRL 和 GMP (图 1C、车道 8) 和起始密码子突变体 (图 1C、车道 7) 的情况下, Toeprint 的形成也受到强烈的损害, 确认所观察到的 Toeprint 不是结构诱导的反向转录的停顿, 但实际上是特定于启动复杂的形成。5 ' polypyrimidine (ppt) 突变体 (图 1C、车道 2) 和 3 ' PPT 突变体 (图 1C, 车道 4) 的 Toeprint 组被削弱, 这会破坏 IRES 结构 (图 1B)8 ,10,12。当 PPT 突变体用 5 ' cap (图 1C、车道3和 5) 转录时, Toeprint 的形成恢复。对于 PPT 双突变体 (图 1C、泳道 6), 也恢复了 Toeprint 的形成, 后者恢复了辅助结构 (图 1B)10。这些数据表明, XIAP IRES 的二次结构对于非限定 RNA 的平移起始至关重要, 而 IRES 结构对于 cap 依赖的平移起始来说是可有可无的。为了进一步证明在没有 IRES 结构的情况下 5 ' cap 的具体要求, 人类β-球蛋白 (HBB) mRNA 受到 toeprinting 分析 (图 1D)。在没有 5 ' cap (图 1D, 车道 1) 的情况下没有观察到 toeprint, 但是核糖体成功地被招募到了覆盖的 HBB RNA (图 1D, 车道 2)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57519/57519fig1.jpg"> 图 1.Toeprinting 分析证实了 IRES 二次结构在名额 XIAP IRES RNA 上形成翻译起始络合物的重要性。(A)在本分析中使用的 XIAP IRES RNA 编码的 DNA 结构示意图。3 ' 末端的 toeprinting 底漆是 42 bp 下游的起始密码子。(B) XIAP IRES 的二次结构 (左上), 5 ' ppt 突变体 (右上), 3 ' ppt 突变体 (左下角) 和 ppt 双突变体 (右下角)。"PPT 突变体" 指的是临界 polypyrimidine 中的点突变 (UU 到 AA), 它扰乱了 IRES8、10、12的次级结构。开始密码子被装箱;点突变用星号 (*) 表示。(C) Toeprinting 分析 XIAP IRES 变体在使用 GMP-PNP 和 ATP 治疗 RRL 中形成翻译起始配合物的能力。开始密码子 AUG 被替换为 AAC 在开始密码子突变体。为了使 RNA 缺乏一个多的尾巴, 用于使体外转录模板的反向底漆仅仅缺乏一个多 T 道。(D)起始复合体是在人β-球蛋白 (HBB) 的上限 (但不是未覆盖) 5 ' UTR 上形成的。在面板 (C) 和 (D) 中, 最左边的4条车道代表顺序反应与在每个车道之上指示的双脱氧核苷酸;序列显示在每个面板的左侧。佛罗里达州, 全长。除另有规定外, 所有 rna 均未覆盖。此数字已从以前的出版物8、10修改。<a href="/files/ftp_upload/57519/57519fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs at JoVE.com

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Toeprinting 的目的是测量体外转录 RNA 的能力, 在各种条件下形成与核糖体的翻译起始配合物。该协议描述了一种 toeprinting 哺乳动物 RNA 的方法, 可用于研究 cap 依赖和 IRES 驱动的翻译。

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

toeprinting-analysis-translation-initiation-complex-formation-on

Nanyang Technological University

Research

JoVE Journal

Biochemistry

12.5K 次观看.  University of Lethbridge. Toeprinting 的目的是测量体外转录 RNA 的能力, 在各种条件下形成与核糖体的翻译起始配合物。该协议描述了一种 toeprinting 哺乳动物 RNA 的方法, 可用于研究 cap 依赖和 IRES 驱动的翻译。

视频: Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

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

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

遗传学

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

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

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

发育生物学

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

分离翻译含肽- tRNA的核糖体的功能和结构分析

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

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 ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

从真核细胞 中快速体内 固定和分离转化复合物

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

摘要

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定量荧光测量全球本地化翻译

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

用于分析依赖帽的翻译动力学的体外单分子成像测定

在中枢神经系统Translatome由多核糖体分馏体内拷问

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

分离翻译含肽- tRNA的核糖体的功能和结构分析

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

使用多体分析在发育中的鼠标大脑中进行翻译分析

从真核细胞中快速体内固定和分离转化复合物

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

摘要

探索更多视频

定量荧光测量全球本地化翻译

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

在中枢神经系统Translatome由多核糖体分馏体内拷问

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

分离翻译含肽- tRNA的核糖体的功能和结构分析

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

使用多体分析在发育中的鼠标大脑中进行翻译分析

从真核细胞 中快速体内 固定和分离转化复合物

用于分析依赖帽的翻译动力学的体外单分子成像测定

从真核细胞中快速体内固定和分离转化复合物