这项工作得到了瑞士国家科学基金会 Sinergia 赠款 CRSII3 147697, 基金会《应用 Krebsforschung 和苏黎世 Krebsliga 的支持。

1. 胞浆和核蛋白分数的制备
<ol><li>板 4 x 106 ACC-MESO-4 细胞在 T150 细胞培养瓶中生长 RPMI 1640 培养基补充10% 血清, 1x 青霉素/链霉素 (100x), 2 毫米 l-谷氨酰胺 (200 毫米)。当细胞达到 80-90% (~ 5-5. 5 x 106细胞) 的汇合处时, 继续进行蛋白质提取核和胞浆分数。 注: ACC-MESO-4 细胞线是从山本理 BioResource 中心11获得的。</li>
	<li>吸入培养基, 通过加入15毫升的 1x PBS 来洗涤细胞, 轻轻地将盘子倾斜几次, 吸入 1x PBS。添加3毫升0.25% 胰蛋白酶-EDTA 和孵化培养瓶在37°c 3 分钟。 注: 观察显微镜下的细胞脱离;如果仍然附有水龙头对手手掌3次。</li>
	<li>添加7毫升的 RPMI-1640 培养基, 辅以10% 的血清, 1x 青霉素/链霉素 (100x), 2 毫米 l-谷氨酰胺 (200 毫米), 收集的细胞在15毫升管, 并在 200 x g 向下旋转5分钟。</li>
	<li>吸入培养基和并用重悬细胞颗粒, 1.5 毫升 1x PBS, 然后将细胞转化为1.5 毫升离心管。向下旋转 500 x g 和4°c 为5分钟, 丢弃上清。 注意: 从这个步骤开始, 执行所有离心步骤在4摄氏度, 并保持所有试剂在冰上。</li>
	<li>并用重悬的细胞颗粒与冰冷1.5 毫升 1x PBS 和旋转向下的细胞在 500 x g 和4°c 3 分钟. 丢弃上清。 注: 所需量的 cer I、cer II 和净净试剂分别为10、0.55 和5卷的估计细胞颗粒包装量;下面的协议假定容量为20µL。我们建议只准备所需量的试剂 CER i。</li>
	<li>添加200µL 的冰冷细胞质萃取试剂 (CER I) 补充2µL 的1x 蛋白酶抑制剂,例如, 稀释1片蛋白酶抑制剂 (无 EDTA) 在500µL 的水获得100x 的库存。这一量的试剂 CER 我足够溶解20µL 细胞颗粒包装体积。<ol>
<li>要估计细胞颗粒的包装体积, 比较的管含有细胞颗粒与1.5 毫升管填充 10, 20, 50 或100µL 1x PBS。</li>
	</ol>
</li>
	<li>并用重悬细胞颗粒通过大力涡流管十五年代和孵化的管在冰上10分钟. 加入11µL 的冰冷细胞质萃取试剂 ii (CER ii) 到管, 大力漩涡 5 s, 并孵化1分钟冰。</li>
	<li>漩涡大力为 5 s 和离心机管在 1.6万 x g 和4°c 5 分钟. 将上清液转移到冰上干净的预冷管上。上清是在 RNA 下拉实验中进一步使用的细胞质分数。</li>
	<li>并用重悬100µL 冷核萃取试剂中剩余的颗粒, 补充1µL 蛋白酶抑制剂 (100x) 和大力涡十五年代。我们建议只准备所需量的净净试剂补充蛋白酶抑制剂。</li>
	<li>在冰上孵化样品40分钟, 偶尔涡流十五年代 (每10分钟)。</li>
	<li>离心管在 1.6万 x g 10 分钟转移上清 (这是核蛋白分数) 到一个干净的预冷管。</li>
	<li>立即通过使用二辛可宁 (评估) 方法测量蛋白质浓度 (参见材料表) 来进行。为了控制每个分数的蛋白质纯度, 执行免疫印迹法(图 1) 对α蛋白 (仅在胞浆分数中的阳性检测) 和聚 ADP-核糖聚合酶-PARP (仅在核分数中进行阳性检测)。对于蛋白质可视化, 请参阅第4节。</li>
	<li>为了保持蛋白质的活性和其自身的状态, 准备25µL 的整除数胞浆和核萃取物。把这些整除数放在液氮中5秒, 然后直接储存在-80 摄氏度。</li>
</ol>2. 用 Desthiobiotin 标记 RNA
<ol><li>有商业合成的 RNA 探针和高效液相色谱纯化。并用重悬与核酸酶水在10µM。 注意: 探测序列列在表 1中。</li>
	<li>每个 rna 下拉反应使用 50 pmol rna。解冻并保持所有的试剂在冰上, 除了 PEG 30%, 用来标记 3 ' 的 RNA 寡核苷酸的终点。</li>
	<li>转移5µL 10 µM RNA 探针, 标记为 CALB2 3 ' UTR (是), CALB2 3 ' UTR (mtARE) 和不相关的 RNA (愤怒), 入0.5 毫升薄壁离心管并且孵化在 PCR 机在85°c 为5分钟. 立即将管子放在冰上。 注意: 这一步很重要, 因为它促进了 RNA 探针的松弛和可访问性以进行标记。</li>
	<li>对于单一的30µL 反应, 添加到含 RNA 管10µL 混合: 3 µL 核酸酶游离水, 3 µL 10x RNA 连接酶反应缓冲器, 1 µL RNase 抑制剂 (40 U/µL), 1 µL Desthiobiotinylated 胞苷 Bisphosphate (1 毫米)和2µL T4 RNA 连接酶 (20 U/µL)。 注: 要准备3个样本超过 (3.2) 的主混合 A, 混合9.6 µL 的核酸酶游离水, 9.6 µL 的 10x RNA 连接酶反应缓冲器, 3.2 µL RNase 抑制剂 (40 U/µL), 3.2 µL 的 Desthiobiotinylated 胞苷 Bisphosphate (1 毫米), 6.4 µL T4 RNA 连接酶 (20 U/µL)。</li>
	<li>仔细添加15µL 的 PEG 30% 的反应。使用另一个提示混合反应。在16摄氏度一夜之间孵化反应。</li>
	<li>第二天, 准备以下内容: 5 米氯化钠 (新鲜/核酸酶), 氯仿: 异戊醇在24:1 比率 (例如, 每反应-96 µL 氯仿和4µL 异戊醇), 冰冷100% 乙醇和冰冷70% 乙醇.</li>
	<li>添加70µL 的核酸酶水到 RNA 标记反应管。添加100µL 氯仿: 异戊醇和涡旋简要。在 1.3万 x g 的旋转向下3分钟小心地移除上部阶段和转移到一个新的无核酸酶 1.5 mL 管;避免接触较低的相位。</li>
	<li>添加10µL 5 米氯化钠, 1 µL 糖原和300µL 冰冷100% 乙醇。把管子放在-20 摄氏度, 2 小时。 注: 在这一步, 实验可以继续在第二天。</li>
	<li>离心机在 1.3万 x g 和4°c 为15分钟, 小心地丢弃上清, 不干扰颗粒。300µL 70% 乙醇和离心机再次在 1.3万 x g 和4°c 为5分钟。</li>
	<li>完全丢弃上清液, 并风干颗粒 (15 分钟)。并用重悬20µL 的核酸酶水中的颗粒。 注意: 在同一天进行 RNA 下拉。</li>
	<li>孵育 RNA 在90°c 2 分钟和地方在冰。在链亲和素磁性微珠预洗涤的以下步骤中, 将标记 RNA 放在冰上。 注意: 长时间潜伏期可能会损害 RNA 探针。</li>
</ol>3. RNA 蛋白质下拉
<ol><li>
预洗链亲和素磁性微珠与 desthiobiotin RNA 孵化
	<ol>
<li>每 50 pmol RNA 使用50µL 链亲和素磁性珠子。但是, 这应该根据实验进行优化。</li>
		<li>涡流管与链亲和素磁珠十五年代, 并迅速删除200µL (主混合; 足够的 3 + 1 反应) 成一个干净的1.5 毫升安全锁管使用切割吸管提示。把管子放在磁性支架上, 这样珠子就会在管子的侧面收集, 等待1分钟. 取出泥沙液体。</li>
		<li>要清洗珠子, 从磁支架上取下管子, 加入400µL 0.1 米氢氧化钠, 0.05 米氯化钠溶液, 轻轻吸管上下几次。把管子放回磁性支架上。等1分钟, 收集上清。再次重复此步骤。</li>
		<li>用200µL 100 毫米氯化钠冲洗珠子。移除上清。</li>
		<li>添加200µL 20 毫米三 (pH 7.5), 并用重悬珠吹打, 放置在磁性立场的管, 等待1分钟, 并删除上清。重复步骤。</li>
		<li>将管子从磁支架上取下, 加入200µL 1x RNA 捕获缓冲器, 并用重悬链亲和素磁性微珠涡流。去除50µL 的链亲和素磁性珠子, 并添加到每个标记 RNA 管使用切割吸管尖端。在滚筒上室温下孵化管30分钟。</li>
	</ol>
</li>
	<li>
蛋白质与 RNA 的结合
	<ol>
<li>将管子放进磁性支架, 等待1分钟, 取出上清。</li>
		<li>添加50µL 20 毫米三 (pH 7.5) 的珠子和并用重悬由吹打。把管子放到磁性支架上, 等待1分钟, 取出上清。重复此步骤。</li>
		<li>添加100µL 1x 蛋白-RNA 结合缓冲剂的珠子和并用重悬由吹打。</li>
		<li>同时, 准备以下组合:10 µL 10x 蛋白-RNA 结合缓冲液, 30 µL 50% 甘油, 胞浆蛋白50µg, 无核酸酶水可达100µL。 注: 准备主混合 B 为3个样品过剩 (3.3) 如下:33 µL 10x 蛋白-RNA 结合缓冲, 99 µL 50% 甘油, 165 µg 细胞质蛋白和核酸酶水多达330µL. 把管子的主混合 B 放在冰上。</li>
		<li>把管子放到磁性支架上, 等待1分钟, 然后收集上清。添加100µL 的主混合 B, 并用重悬轻轻吹打上下, 并避免产生气泡。孵化反应管为60分钟在4°c 在滚筒上。</li>
	</ol>
</li>
	<li>
洗涤和洗脱
	<ol>
<li>把管子放到一个磁性的支架上, 收集上清液 (流经 FT), 然后转移到新的管子里。 注意: 将此 FT 放在冰上以供以后分析。</li>
		<li>吸管100µL 的1x 洗涤缓冲器上的珠子, 轻轻并用重悬, 放回磁性支架, 等待1分钟, 并丢弃上清。重复此步骤2次。</li>
		<li>在磁珠上添加40µL 洗脱缓冲器, 混合吹打上下, 并在37摄氏度上孵化在管振动筛 (950 rpm) 30 分钟。</li>
		<li>将管子放入磁性支架, 等待1分钟, 收集洗脱样品 (洗脱液)。放置在冰上, 用于下游分析。 注意: 在这个步骤中, 每个经过测试的 RNA 探针都由一个流经 (FT) 和洗脱液 (E) 样本组成。</li>
	</ol>
</li>
</ol>4. 聚丙烯酰胺电泳与印迹分析
注: 有关本节中使用的缓冲区的食谱, 请参见表2。根据 Laemmli 方法12执行西方污点。
<ol><li>Handcast 12% SDS 页凝胶 (10 井, 1.5 毫米间隔, 66 µL)。准备一个运行的凝胶混合: 4 毫升30% 丙烯酰胺-bisacrylamide 库存溶液, 2.4 毫升下三缓冲器, 3.2 毫升的 dH2O, 9.6 µL TEMED, 96 µL 10% 硫酸铵溶液 (APS)。准备堆叠凝胶混合: 3.25 毫升 dH2O, 0.5 毫升30% 丙烯酰胺-bisacrylamide 库存溶液, 上三个缓冲器1.3 毫升, 10 µL TEMED, 20 µL 10% APS。</li>
	<li>添加16.6 µL 6x Laemmli 缓冲区 (例如, 14 毫升的三/HCl/sds pH 值 6.8 (上部三缓冲器), 2 克的 sds, 1.86 克的德勤, 6 毫升甘油和0.012% 溴酚蓝色; 存储在-20 °c) 到 FT 样本, 6.6 µL 6x Laemmli 缓冲区进入 e 样品。孵育样品5分钟, 在95摄氏度。</li>
	<li>装载样品 (20 µL 的 FT 和整个洗脱 ~ 40 µL) 和运行他们30分钟在 60 v, 继续与 20 V 隔夜。</li>
	<li>使用半干 electroblotting 系统, 将蛋白质转移到 PVDF 膜上80分钟, 在 15 v。 注: 在执行蛋白质转移之前, 必须激活 PVDF 膜。将膜在100% 甲醇中孵化1分钟, 然后在超纯水中孵化2分钟。</li>
	<li>蛋白质转移后, 用考马斯溶液孵化 SDS 页凝胶3小时, 然后用超纯水洗涤三步, 每30分钟。</li>
	<li>在蛋白质转移结束时, 让 PVDF 膜干燥1小时, 如步骤4.5 所示, 重新激活膜。在 1x TTBS 中用 5% BSA 阻挡膜1小时。</li>
	<li>用第一个抗体孵化膜: 虚宾或间皮素, 在室温下或一夜4摄氏度稀释1:1000 在 5% BSA 在 1x TTBS 4h。</li>
	<li>在 1x TTBS 中冲洗三次, 用兔抗小鼠辣根过氧化物酶 (HRP)-共轭次生抗体稀释 1:10,000 5% BSA, 1x TTBS。</li>
	<li>使用增强化学发光和数字成像仪可视化蛋白质。</li>
</ol>

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作者声明他们没有竞争的财政利益。

3 ' UTRs 属于非编码基因组3, 所有非编码 rna 可以与蛋白质相互作用, 以发挥其功能7。当哺乳动物基因组被发现被普遍转录并产生长非编码 rna 16 的很大一部分时, 新的证据表明这些长非编码 rna 在调节基因表达时的作用与染色质重塑复合体17。这种知识是利用 RNA 下拉法得到的。因此, 要开始破译特定 RNA 的 interactors, 第一步可能?...

基因表达和基因产物的最终水平可以通过影响 mrna 稳定性和 mrna 翻译率1来严格调节。这些转录后调控机制是通过非编码 rna 和/或 rna 结合蛋白 (限制性商业惯例) 与靶向 mRNA 的相互作用而施加的。它通常是 3 ' 未翻译的 mRNA 区域 (3 ' UTR-属于基因组2) 的非编码部分, 其中包含特定的cis-管理元素 (), 由反演作用因素 (如 miRNA 或限制性商业惯例) 识别。3. 最好研究的cis元素在 3 ' UTR, 是腺嘌呤丰富的元素 (是) 的主题, 这是公认的特定的 AU 结合蛋白 (AUBP), 反过来, 诱导 mRNA 退化/deadenylation (是介导的衰变) 或mRNA 稳定化4。
3 ' UTR 的 calretinin mRNA (CALB2) 的大小是 573 bp 长, 包含一个假定的 AUUUA pentamer, 如 AREsite2 的预测, 生物信息学工具 5.与假定的存在相一致的是主题, pmirGLO 向量-报告分析显示了这个元素在 CALB2 mRNA6中的稳定作用。最后, 用体外RNA 下拉法确定了通过 AUBP 稳定 calretinin mRNA 的方法。
由于所有非编码 rna 与蛋白质7相互作用,体外rna 下拉是确定 rna interactors 以帮助破译分子机制的一种很好的方法和首选的检测手段。在这个 rna 下拉法, 商业上被合成的 rna 探针, 被标记以被改进的生物素 (desthiobiotin) 的形式在 RNA 链的 3 ' 末端被使用了。rna desthiobiotin 是固定的通过与链亲和素的相互作用的磁性珠子, 这是用来拉下来的蛋白质, 特别是与绑定 RNA 的利益。非变性和活性蛋白从间皮瘤细胞胞浆的比例被用作蛋白质的来源。这种 RNA 束缚的蛋白质是洗脱从磁性珠子, 运行通过 12% SDS 页凝胶, 转移到膜, 并探讨了不同的抗体。
在标准的链亲和素-生物素亲和纯化过程中, 需要苛刻的变性条件来破坏强不可逆转的生物素-链亲和素键洗脱绑定蛋白 8, 这可能导致离解蛋白质复合物。与生物素不同的是, desthiobiotin 可逆地结合到链亲和素, 并以缓冲的生物素溶液进行竞争性置换, 允许蛋白质的温和洗脱, 避免自然生物素化分子的分离 9,表明该技术是理想的分离本地蛋白质复合体在当地条件下。
体外约束条件和严格性是由盐浓度、还原剂和洗涤剂百分比决定的, 应与细胞上下文中存在的情况相近, 以便识别真正的体内交互作用。在此之前所实现的绑定条件已被证明是适当的, 以确定虚宾作为一种金结合蛋白10。这种方法可以节省时间, 因为优化适当的绑定条件可能会耗费时间和挑战性。此外, 该方法可作为任何 RNA 下拉实验的启动协议, 通过改变盐和洗涤剂的浓度, 改变甘油的百分比, 添加其他盐, 可以逐步优化。此外, 我们证明, 即使是一个短的25核苷酸 RNA 探针窝藏 pentamer 是-主题可以用来证明与特定 AUBP 的互动。

<table><tbody><tr><td>NE-PER Nuclear and cytoplasmic extraction kit</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>78833</td><td></td></tr><tr><td>Pierce Protease Inhibitor Tablets, EDTA-free</td><td>Thermo Fisher Scientific</td><td>A32965</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>Pierce Magnetic RNA-protein Pull-Down Kit&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>20164</td><td>Includes magnetic beads and therefore the kit need to be stored at 4 &amp;deg;C</td></tr><tr><td>Pierce RNA 3&amp;rsquo; End Desthiobiotinylation Kit&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>20163</td><td>The kit is a part of the &quot;Pierce Magnetic RNA-protein Pull-Down Kit&quot;, and needs to be stored at -20 &amp;deg;C unlike the pull-down kit (4 &amp;deg;C).</td></tr><tr><td>DynaMag-2,&amp;nbsp; magnetic stand</td><td>Thermo Fisher Scientific</td><td>12321D</td><td></td></tr><tr><td>Anti - HuR (MOUSE) monoclonal antibody</td><td>Thermo Fisher Scientific</td><td>-</td><td>The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit&amp;nbsp; - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td></tr><tr><td>Anti - &amp;alpha; - tubulin (MOUSE) monoclonal antibody</td><td>Santa Cruz</td><td>8035</td><td>1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td></tr><tr><td>Anti - PARP (RABBIT) Polyclonal antibody</td><td>Cell Signaling</td><td>9542</td><td>1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS</td></tr><tr><td>Anti - Mesothelin (MOUSE) Monoclonal Antibody&amp;nbsp;</td><td>Rockland</td><td>200-301-A87</td><td></td></tr><tr><td>Secondary goat anti-rabbit antibody</td><td>Cell Signaling</td><td>7074</td><td></td></tr><tr><td>Secondary rabbit anti-mouse antibody</td><td>Sigma-Aldrich</td><td>A-9044</td><td></td></tr><tr><td>RPMI - 1640 medium</td><td>Sigma-Aldrich</td><td>R8758</td><td></td></tr><tr><td>FBS - Filtrated Bovine Serum</td><td>Pan Biotech&amp;nbsp;</td><td>P40-37500</td><td></td></tr><tr><td>Penicillin - streptomycin (100x)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P4333</td><td></td></tr><tr><td>L-Glutamine solution (200 mM)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G7513</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Gibco by Life technologies&amp;nbsp;</td><td>25200-056</td><td></td></tr><tr><td>Mini-PROTEAN 3 Cell</td><td>Bio-Rad</td><td>1653301</td><td></td></tr><tr><td>Mini Protean System Glass Plates&amp;nbsp;</td><td>Bio-Rad</td><td>1653308</td><td></td></tr><tr><td>Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer</td><td>Bio-Rad</td><td>1653312</td><td></td></tr><tr><td>Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 &amp;micro;L</td><td>Bio-Rad</td><td>1653365</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell Casting Modules</td><td>Bio-Rad</td><td>1658050</td><td></td></tr><tr><td>RNaseZap RNase Decontamination Solution</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1</td><td>Roth</td><td>3029</td><td></td></tr><tr><td>20% SDS&amp;nbsp;</td><td>PanReac AppliChem</td><td>A3942</td><td></td></tr><tr><td>PVDF transfer membrane&amp;nbsp;</td><td>Perkin Elmer</td><td>NEF1002</td><td>Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min&amp;nbsp;</td></tr><tr><td>Trans-Blot SD semi-dry transfer cell&amp;nbsp;</td><td>Bio-Rad</td><td>1703940</td><td></td></tr><tr><td>Clarity Western ECL Substrate</td><td>Bio-Rad</td><td>1705061</td><td></td></tr><tr><td>FusionFX&amp;nbsp; Digital Imager</td><td>Vilber</td><td>-</td><td></td></tr><tr><td>RNA oligo synthesis</td><td>Mycrosynth AG, Switzerland</td><td>-</td><td>the synthesis scale - 0.04 &amp;micro;mol - HPLC purified</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7030</td><td></td></tr><tr><td>Hydrochloric Acid (HCl) 6 M</td><td>PanReac AppliChem</td><td>182883.1211</td><td></td></tr><tr><td>Ultra Pure 1 M Tris, pH 7.5&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>15567027</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>17904</td><td>50% sterile aliquots to be stored at -20&amp;deg;C</td></tr><tr><td>Pierce Streptavidin magnetic beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>88817</td><td>Please note that these magnetic beads have an average diameter of 1 &amp;micro;m (range from 0.5 - 1.5 &amp;micro;m). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 &amp;micro;m, are also used in RNA-pulldown but be aware that this may affect purification process.</td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Ammonium persulfate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>Coomassie G-250</td><td>Applichem</td><td>A3480</td><td></td></tr><tr><td>Aluminiumsulfate-(14-18)-hydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>368458</td><td></td></tr><tr><td>ortho-phosphoric acid&amp;nbsp;</td><td>Applichem</td><td>A0637</td><td></td></tr></tbody></table>

desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells

体外RNA 下拉仍然主要用于确定 rna 结合蛋白识别特定 rna 结构和图案的协议的第一步。在这个 rna 下拉协议, 商业上合成的 rna 探针被标记以修改的形式生物素, desthiobiotin 在 RNA 链的 3 ' 末端, 可逆地束缚到链亲和素并且因而允许蛋白质洗脱在更加生理上条件。rna desthiobiotin 是固定的, 通过与链亲和素的相互作用, 磁性珠子, 这是用来拉下来的蛋白质, 特别是与 RNA 的利益。非变性和活性蛋白从间皮瘤细胞胞浆的比例被用作蛋白质的来源。此处描述的方法可用于检测已知的 rna 结合蛋白与含有一系列兴趣序列的25核苷酸 (nt) 长 RNA 探针之间的相互作用。这对于完成使用报告向量法实现的 RNA 分子中稳定或不稳定元素的功能表征是有用的。

在这个实验中, 一个 25 nt 长片段的 calretinin 3 ' UTR 窝藏是主题 (CALB2 3 ' UTR (是) 25 nt) 被用来测试它是否结合了人类抗原 R (虚) 蛋白, 一个已知的 mRNA 稳定剂。为了测试这些元素的特异性, 25-nt RNA 探针 CALB2 3 ' UTR (mtARE) 包含了一个有主题的突变, 以前被证明是为了取消 "是" 的主题的稳定效果, 使用了6。第三个 RNA 探针代表负控制, 这是一个 28 nt 无关的 rna, 它包...

Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin-链亲和素亲和介导纯化间皮瘤细胞 RNA 相互作用蛋白的研究

Desthiobiotin 标记的合成 25-核苷酸 RNA 寡核苷酸, 其中含有腺嘌呤丰富的元素 (是) 的主题, 允许特定的结合胞浆是结合蛋白。

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

desthiobiotin-streptavidin-affinity-mediated-purification-rna

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

8.0K 次观看.  University Hospital Zurich. Desthiobiotin 标记的合成 25-核苷酸 RNA 寡核苷酸, 其中含有腺嘌呤丰富的元素 (是) 的主题, 允许特定的结合胞浆是结合蛋白。

视频: Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

科研

遗传学

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

酿酒酵母4-硫尿嘧啶代谢标记及新合成 mRNA 作为 RNA 聚合酶 II. 活性的一种代理

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

生物化学

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

生物素和光裂解链接器所附 rna 对高分子配合物的单步纯化

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

通过旗-生物素串联纯化通过交叉链接和免疫沉淀对蛋白质-RNA相互作用进行转录组-全过程分析

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

使用短生物素化RNA对RNA结合蛋白进行优化的定量下拉分析

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

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

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...