游泳池的设计和寡复苏<ol><li>第一步是要研究设计的前体mRNA池。这是可以做到使用UCSC基因组浏览器和下载的特定基因，拼接路口，或其他感兴趣的领域。 </li><li>一旦利益的窗户已被选中，在它们之间的瓷砖计算使用下列条件：读取的长度应为30与10核苷酸重叠，因此​​每个寡核苷酸转移从之前的20个核苷酸核苷酸。 *寡重叠，应增加在接近的剪接位点，以确保足够的覆盖范围;增加10至20个核苷酸的重叠，可以做到这一点。 </li><li>侧翼每个与通用引物序列，这将是用来放大池下游30个核苷酸的寡核苷酸。应提交安捷伦，将一个自定义的寡核苷酸芯片印刷序列平铺寡订单。 </li><li>要恢复从芯片表面的DNA寡核苷酸开始放置在阵列杂交室阵列封面幻灯片和幻灯片上轻轻移液500μL的dh 2 </li><li>夹心数组封面幻灯片上，照顾，以避免气泡，可以破坏的过程。可以肯定的数组正面朝下放置，以便与寡核苷酸的一边是接触水面。一个好的经验法则是“安捷伦触及安捷伦”，意思是阵列上的安捷伦标签面临着封面上的幻灯片安捷伦标签</li><li>关闭杂交室和旋转在99℃过夜，在杂交炉</li><li>翌日，小心地取出数组，并从会议厅制定的水，它现在包含寡核苷酸阵列的表面解放。这是寡核苷酸池</li><li>在50％幅度将在1.5 mL Eppendorf管的游泳池和超声池3-5个脉冲</li><li>接下来，由低循环PCR扩增池，使用的末尾加上一个T7号标记的通用引物。对于第一轮，变性94 ° C 1分钟，55℃退火20秒，1分钟拉长为72 ° C。随后的几轮，做10秒，20秒，10秒，在每个相应的温度，5分钟，最后延伸72 ° C。由于寡恢复的效率取决于放大池所需的周期数，可能需要尝试多个周期的数字。要小心不要过度放大的游泳池，这是由丙烯酰胺凝胶涂乐队标志着。 PCR扩增产物可存放在4℃直至需要。 </li><li>在寡准备的最后一步是利用Ambion公司MEGAshortscript™套件转录RNA。 *以下协议改编自Ambion公司<ol class="lalpha"><li>解冻的T7 10X反应缓冲液，在室温下4个核苷酸的解决方案和水，同时保持冰的T7酶组合</li><li>组装反应混合物在室温下的无RNase的离心管。反应缓冲液的组成部分可以使模板DNA沉淀，如果反应是在冰上混合。 </li><li>按照以下顺序，吸管3μL无核酸水，10X反应缓冲液，每75毫米NTP解决方案2μL，5μL模板DNA（PCR扩增寡核苷酸池）和2μL的T7酶，一个终体积20μL </li><li>管轻轻一抖混合反应，然后简要离心收集管底部的混合物。 </li><li>在热循环反应在37℃2小时。孵化时间将依赖模板，因此它可能是必要的使用时间课程的实验，以确定获得最大的收益率孵育时间</li><li> 2小时后，取出一个额外的15分钟的模板DNA加入1μL涡轮DNA酶反应和孵化，在37 ° C </li><li>终止反应和恢复酚/氯仿抽提和乙醇沉淀的RNA。 <ol class="lalpha"><li>为20μL的反应体积，加115μL的dh 2 O和15μL3 M醋酸钠的反应。 </li><li>调匀再加入75μL酸性苯酚和75μL氯仿。由涡旋混合溶液，然后在室温下13000转一分钟的离心。水层转移到一个新的管和重复提取</li><li>转移到一个新的管水层，并添加150μL氯仿。涡旋和以前一样的混合物离心机。水层转移到一个新的管。 </li></ol></li><li>两卷的100％的冷乙醇添加水层和混合以及收回的RNA。冰寒的混合物，最少为15分钟，在-20 ° C，然后离心机在4 ° C时最高速度在15分钟沉淀的RNA。小心取出上清液，和105的0.1倍的Tris - EDTA（TE）缓冲液重悬沉淀.. </li></ol></li><li>这是很难删除所有从T7套件反应的游离核苷酸，最准确的结果，量化的RNA使用RiboGreen的。 <ol class="lalpha"><li>要开始定量，先稀足够的1X TE，2300μL+50μL/sample。 </li><li>接下来，RiboGreen。您将需要1000μL，50μL/样本。对于大范围的样品（1微克/毫升至20毫微克/毫升）混合在1x TE RiboGreen 1:200;低范围样本（50毫微克/毫升为1毫微克/毫升）混合在1x TE RiboGreen 1:2000 ;移液器到每一个不透明板以及50μLRiboGreen解决方案，开始与A1和向下移动，而不是跨</li><li>稀释到2微克/毫升高范围的样品或为100 ng /μL低范围的样品RNA原液</li><li>利用股票的解决方案，创建标准曲线由六个随后1:2稀释原来的2微克/毫升或100毫微克/毫升股票;移液器的股票240到μL的PCR条管。在余下的7管，吸液管120μL的1X TE。取120μLRNA的股票，混合管2。从管2 120μL和混合管3。重复，直到管7。管8只会有TE和将您的空白。吸取50μL的标准曲线的解决方案不透明板，A1：H2（两次运行曲线） </li><li>在一个新的管中，45μL的TE 5μL的寡核苷酸池混合，并添加所有50μL到包含50μLRiboGreen良好。 </li><li>使用酶标仪设置本机读取从顶部的荧光，后一分钟的组合激发波长485 nm，发射波长为530 nm读板</li><li>使用标准曲线生成量化回收的RNA，应储存在-20 ° C </li></ol></li></ol>免疫共沉淀的寡核苷酸池与利益的RNP <ol><li>免疫共沉淀开始之前，准备A和蛋白G磁珠从Invitrogen公司以1:1的比例混合，最终成交量的50％μL反应的蛋白质。例如，对于两种反应中，您将添加50μL的每个珠子。 </li><li>使用一个像这样一个自Novagen磁分离架举行到位的珠子，而你去除上清，洗珠与冷的1X PBS等体积的两倍</li><li>第二次洗涤后，在同等体积的冷1X PBS重悬珠，并添加2％反应的抗体吴。这一数额可能会有所不同，根据抗体，因此试验是必要的，以找到最佳的条件。 </li><li>抗体/珠的混合物过夜孵育4 ° C的一个旋转平台</li><li>上午，添加2微克每超声酵母总RNA的反应，以阻止非特异性结合，和一个额外的30分钟旋转4 ° C。 </li><li>使用磁机架举行的珠子，去除上清，洗珠两次用冷的1X PBS，留在管内的第二次洗的PBS。 </li><li>取出新的管子，每个反应和每管分装50μL珠混合。让珠坐，同时准备一个主结构</li><li>每个反应中添加200毫微克的寡核苷酸池，保护引物摩尔比，超声酵母总RNA 2微克，20μLHeLa细胞的核提取物（这是RNP源），和缓冲区发送带来的音量高达120微升。保护引物的通用引物RNA的版本。订购转录模板在末尾加上一个T7号标记的通用引物，然后抄写使用MEGAshortscript™套件模板的RNA。 </li><li>从珠分装和主结构在120μL重悬珠取出上清液。在4 ° C为1-2小时，岩石的反应。 </li><li>孵化后，每个反应，一个“总”的RNA样品中删除一个小等份（包括珠） </li><li>将其余磁选立场的反应，去除上清。虽然是没有必要的分析样品下游流的，它可能是有用的保存的。 </li><li>洗磁珠冷1X PBS 50μL的1-2倍。绑定珠抗体的RNA是“知识产权”的样本。 </li><li>在1％的0.1倍TE缓冲液中的SDS 50μL重悬的“总”和“IP”珠和样品的温度在65℃，15分钟洗脱RNA。 </li><li>去除上清，它现在包含以前绑定的珠子和干净的酚/氯仿抽提乙醇沉淀的RNA。干颗粒，并在50μL的0.1倍TE缓冲液重悬。 </li></ol>合作IP样本的逆转录和扩增准备aminoallyl标签<ol><li>接着，RNA样品，必须反转录和PCR扩增。逆转录反应，使用反向通用引物。 </li><li>对于每个样本，混合1μL开始反向通用引物，1μL模板的RNA（免疫反应）和10μLDH 2 O。热的反应，在70 ° C 5分钟，然后寒意在冰上或保持在4 ° C。 </li><li>当样品冷却，使以下主结构。对于每一个反应，结合10毫米dNTPs 4μL，2μL10X Arrayscript缓冲区，1μLRNase抑制剂，和1μL反转录酶。调匀，加入8μL主结构每个逆转录反应。 </li><li>使用PCR仪，样品在42 ° C孵育2小时，95 ° C 5分钟，然后4 ° C，直到样品需要放大。 </li><li>反向转录后的RNA样品，PCR扩增使用的通用引物，含有T7启动标记的引物之一。放大池所需的周期数将取决于合作IP的效率，因此它可能是需要尝试多个周期。每个反应需要5μL10倍的Taq反应缓冲液，0.2μL每个引物（100毫米），cDNA模板1μL（逆转录反应），10毫米dNTPs 1μL，42.2μLDH 2 O， 0.4μLTaq酶。 开始3分钟，94℃孵育样品的PCR。接下来，每个周期内，变性94 ° C 45秒，退火55 ° 30秒，在72 ° C和拉长最终伸长率在72℃10分钟，一分钟。 </li><li>下一步，抄写样品​​再次使用MEGAshortscript™套件，然而，这一次加2μLaminoallyl双绞线，而不是T7双绞线。 aminoallyl双绞线将用于标签的Cy染料的RNA。 </li><li>酚/氯仿和乙醇沉淀的样品。不要使用Glycoblue或醋酸铵，这两者都可能干扰样品的杂交到阵列。干颗粒和重悬在4.5μL0.1 M的碳酸钠。 </li></ol>氨基烯丙基标记的RNA用Cy染料<ol><li>要开始的标签协议的荧光染料，Cy3和Cy5溶解于45μL二甲基亚砜，。为了避免光线，在黑暗中管包裹在铝箔和储存在-20 ° C </li><li>添加2.5μL无核酸酶水RNA样品调匀</li><li>接下来，添加3μL的“总”的样本，Cy3标记，与3μL的Cy5的“知识产权”样本。在黑暗中孵育样品在室温下1小时，CY - 3样品会变成红色或粉红色，而CY - 5会变成蓝色。 </li><li>终止反应，加入6 4米羟胺盐酸UL，每个样品，调匀，自旋简要。 15分钟在室温下孵育。 </li><li>净化酚/氯仿抽提，乙醇沉淀和重新悬浮颗粒50μL的0.1倍TE标记的样品</li></ol>杂交样品芯片使用安捷伦阵列杂交试剂盒<ol><li>首先，轻轻混匀50μL封闭液的10倍，140核酸无H2 O，Cy3标记的“总”池，25μL的Cy5的标记为“知识产权”池25μL，10μL的微升杂交25X碎片缓冲区和250μL的2倍的杂交缓冲液，同时注意避免引入气泡。离心样品简单地收集在试管底部的解决方案，同时也减少气泡。 </li><li>将O形圈在杂交室的封面幻灯片，并仔细移液器到幻灯片的解决方案。 </li><li>将“三明治”的解决方案的封面幻灯片上的探测阵列。请记住，东方的阵列幻灯片，使阵列上的安捷伦标签面临着封面上的幻灯片安捷伦标签。 </li><li>夹在杂交室幻灯片/阵列三明治和旋转50 ° C时为3小时。 </li><li>而阵列旋转，请在50 mL锥形管50毫升的解决方案：一个圆锥形充满50毫升生署2 O两个1X SCC的缓冲溶液中，每一个单独的管，和一个2倍SCC的缓冲区。 </li><li>三个小时的潜伏期后，小心地松开阵列/幻灯片夹层，放置于50 ml含有2倍的SCC的缓冲区锥形。使用镊子，轻轻分开的封面幻灯片阵列拉出封面幻灯片。要小心，不要触摸幻灯片拉出阵列。关闭锥形，轻轻地反转60秒。 </li><li>使用镊子，转移到1倍SCC解决方案的阵列，帽筒，轻轻倒置1分钟，然后转移到阵列中的第二1X SCC解决方案，并再次轻轻地反转一分钟。 </li><li>最后，转移到阵列中的DH 2 O管，轻轻地反转30秒。 </li><li>干的幻灯片的边缘上放置一个Kimwipe数组。继续旋转的阵列擦拭确保不会破坏含有寡核苷酸的一面。 </li><li>将干阵列一个空的50毫升锥形管和微阵列扫描仪扫描</li></ol>解读基因芯片<ol><li>芯片扫描仪将创建一个数组的TIF图像。使用网格文件（由安捷伦提供的），这是您的特定池独特的形象，通过安捷伦的特征提取软件运行。 </li><li>安捷伦软件将解释每个功能的信号，创建一个文件，这是一个Perl脚本解析。在每个功能的信号是归在该通道的总强度。例如在一个特定功能的绿色通道，个别功能的绿色通道的强度除以总强度以上所有功能的绿色通道。最后的输出是在阵列上的每个功能的绿色通道，红色通道的日志比例。 </li><li>从芯片的寡核苷酸池中的数据可以被重新映射到基因组得到一个图片在哪里，以及如何强烈，利益的RNP约束。 </li></ol> 图1。实验原理图 <img src="/files/ftp_upload/1622/1622fig1a.jpg" alt="图1a" /> A.切片方案。选择和下载利益的前体mRNA序列后，通过选择面积平铺在30 10个核苷酸的增量转向核苷酸窗口。通用引物结合位点侧翼每边的窗口。 <img src="/files/ftp_upload/1622/1622fig1b.jpg" alt="图1b" /> B.合成阵列。应提交安捷伦，将一个自定义的寡核苷酸芯片印刷序列平铺寡订单。 <img src="/files/ftp_upload/1622/1622fig1c.jpg" alt="图1c" /> C.免疫共沉淀。煮沸后关闭阵列的寡核苷酸，在HeLa细胞的核提取物孵育，然后加入准备对利益的RNP抗体的磁珠提取。标签用Cy3寡核苷酸池，并用Cy5绑定的IP地址池，适用于探测阵列和扫描。 图2。注释基因组和浓缩数据分析。 <img src="/files/ftp_upload/1622/1622fig2.jpg" alt="图2" /> A.平均得分从数组中的数据，在每个基因组的协调基地，十个日志和地图转换得分给定的坐标。这个平均步的一个例证是3个重叠的30 nt的寡核苷酸2分，4，0.5，平均每10 NT窗口浓缩得分作图以上。可以可视化USCS基因组浏览器中使用自定义跟踪选定的基因组区域。例如浏览器窗口是在CLTA基因，基因的功能（外显子/内含子/选择性剪接等），沿底部，并从客运大楼的约束寡核苷酸日志平均富集分数由暗红色的竖线代表。客运大楼的强烈结合在polypyrimidine道外显子2上游。

执行此过程时，重要的是记住，池创建灵活和开放的修改，在RNA或蛋白质水平。在RNA水平同源地区，疾病基因突变，多态性，或随机突变可以被引入到寡核苷酸序列。在蛋白质水平的RNA结合因子可以修改的磷酸化或其他翻译后修饰。此外，具有约束力的环境可以被操纵，以增加或减少交互或与RNA结合蛋白的利益竞争的其他因素的水平。

a rapid high-throughput method for mapping ribonucleoproteins (rnps) on human pre-mrna

测序合作与RNA结合蛋白免疫沉淀（CO - IP）增加表明装订位置的RNA拼接的认识，往往影响一个剪接因子的功能。然而，与任何抽样策略确定绑定到剪接因子一种RNA的机会是其细胞丰度成正比。我们已经开发出一种在体外培养的方法的前体mRNA上，否则瞬态测量结合的特异性的小说。这种方法采用了专门设计的寡核苷酸池，跨内含子，外显子剪接路口，或其他前体mRNA的瓷砖。池是受到某种分子选择。在这里，我们展示了通过分离成一个绑定和非绑定小数寡核苷酸的方法，利用两种颜色的数组战略，在绑定的分数记录每一个寡核苷酸的富集。阵列中的数据能够识别特定序列和结构determinates核蛋白（RNP）的前体mRNA结合生成高分辨率地图。这种方法具有独特的优势是它的能力，以避免对当前的IP和SELEX技术与相关的基因的抽样偏差，池是专门设计和合成的前体mRNA序列。寡核苷酸池的灵活性是另外一大优势，因为实验者选择哪个地区的研究和瓷砖跨他们的个性化需求，剪裁池。使用这种技术，可以检测约束力的大规模成一个功能的剪接记者的克隆库，并确定是在所包含的部分丰富的寡核苷酸多态性或突变的影响。这小说在体外高分辨率测绘计划提供了一个独特的方式来研究瞬态mRNA前体的物种，其丰度低，使得他们难以电流在体内的技术研究与RNP的相互作用。

Watch this Scientific Journal Video about A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA at JoVE.com

A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

由于mRNA前体的瞬态的性质，它可以很难分离和研究<em&gt;在体内</em&gt;。在这里，我们提出了一种新<em&gt;在体外</em&gt;调查使用一种人工合成的寡核苷酸池的RNA -蛋白质相互作用的方法，整个选定区域的前体mRNA的瓷砖。

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

a-rapid-high-throughput-method-for-mapping-ribonucleoproteins-rnps-on

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

11.7K 次观看.  Brown University. 由于mRNA前体的瞬态的性质，它可以很难分离和研究在体内。在这里，我们提出了一种新在体外调查使用一种人工合成的寡核苷酸池的RNA -蛋白质相互作用的方法，整个选定区域的前体mRNA的瓷砖。

视频: A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

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

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

生物化学

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 ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

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

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

生物学

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

人线粒体 DNA 中核苷酸的同时定位与定量

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

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

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

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

通过选择性核糖体分析对共翻译相互作用网络进行全局鉴定

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