Research in Ansari lab was supported by the research grant (No. MCB 1020911) from National Science Foundation.

注:在研究文章中Medler，S和安萨里，A 2日报道在使用此方法。 1.培养和收集细胞<ol><li>从新鲜划线酿酒酵母板开始在YPD培养基（10克/升酵母提取物，20g / L的蛋白胨，2％葡萄糖）的细胞的5毫升培养。 </li><li>生长细胞过夜在轨道摇床在30℃和250rpm。 </li><li>稀释过夜生长的培养的100倍，以YPD培养基100mL的最终体积的250毫升带挡板的烧瓶中。 注:共100毫升的文化，每TRO反应需要;如果需要多个反应，必须使用培养物的体积较大。 </li><li>生长细胞为0.8的光密度- 1.0在600nm（A 600〜0.8 - 1.0）。 </li><li>离心在820 xg离心在4℃下5分钟收获细胞在无菌50mL的锥形管中。 </li><li>悬浮细胞在10毫升冰冷的TMN的卜FFER（10毫米的Tris-HCl pH为7.5，5mM的MgCl 2的，100毫摩尔NaCl），并在冰上孵育5分钟。 </li><li>离心机在820×g离心5分钟，在4℃以沉淀细胞。轻轻通过抽吸去除上清液。 </li></ol> 2.透化和准备的细胞运行的分析<ol><li>通过上下吹打轻轻用截短P 1000尖悬浮在1毫升0.6％十二烷基肌氨酸钠溶液的细胞。细胞悬液转移到一个冷却的1.5毫升离心管中。 </li><li>轻轻摇动细胞在4℃下于一个平台摇床25分钟。 注:微量离心管应该被打包到50mL锥形管中在冰冷保持冷温度，从而避免在此阶段的启动子产生的任何新的起始事件的可能性。 </li><li>用台式冷冻离心机离心细胞以1200 xg离心6分钟，4℃。 </li><li>轻轻吸出上清液从每除去任何过量的液体meabilized细胞沉淀。 </li></ol> 3.转录运行的反应<ol><li>添加转录150μL的运行上缓冲液（50mM的Tris-HCl pH值7.5，100mM的氯化钾，10mM的MgCl 2的，2毫摩尔DTT（二硫苏糖醇），0.75毫每个ATP，CTP，GTP，和BrUTP，200单位核糖核酸酶抑制剂）的透化细胞沉淀。 </li><li>通过上下吹打使用截断的第200页尖轻轻拌匀。 注:如果处理多个样本，把一切都在冰上，直至所有样本都准备好，然后进行下一个步骤。 </li><li>在水浴孵育5分钟，在30℃。 注:调整为1〜5分钟之间温育的时间，以确保BrUTP的最佳掺入新生RNA。 </li><li>停止加入冷准备使用的RNA分离试剂500μL的反应。孵育在室温下5分钟。 </li></ol> 4. RNA提取<ol><li>加的酸洗的玻璃珠200微升至细胞suspensi上（步骤3.4），并在旋涡混合器剧烈摇晃20分钟，4℃。 </li><li>使用热压22克针穿刺微量离心管的底部，将其放置在一个15毫升的无菌锥形管的顶部。 </li><li>离心机在4℃下300×g离心1分钟，以收集细胞裂解物。转移细胞裂解物到1.5mL离心管中。 </li><li>添加冷的RNA分离试剂500μL，和200微升氯仿至细胞裂解物。在4℃下混合在涡旋混合器上并离心，内容物在16168×g离心20分钟。 </li><li>上层水相转移到新的离心管中。 </li><li>添加冷的酚/氯仿的等体积/异戊醇（125:24:1）的pH 4-5。剧烈摇动在涡旋混合器上并离心16168 xg离心在4℃下15分钟。 </li><li>传送上层水含有相的RNA到一个新的离心管中。重复步骤4.6两次。 </li><li>到最后含有RNA的水相，加5 M氯化钠STOCk以得到0.3 M的最终浓度添加3倍的冷乙醇的体积以沉淀RNA。 <ol><li>孵育1小时，或在-20℃过夜。 注: - 当抗BrdU珠粒孵育阻断（参见步骤5.3）4.11执行步骤4.9。 </li></ol></li><li>离心机在16168 xg离心在4℃下20分钟。去除上清，重悬在100微升DEPC（焦碳酸二乙酯）水的RNA沉淀。 </li><li>使用RNA提取试剂盒迷你的RNA从非法人BrUTP核苷酸分开。洗脱从用100微升的无RNase水列中的RNA。 </li><li>孵育在65℃水浴中的RNA的5分钟，然后转移到冰上至少2分钟。 </li></ol> 5. BrUTP标记的RNA的亲和纯化<ol><li>以25μL抗BrdU落户珠，加500 0.25X SSPE（盐水磷酸钠磷酸EDTA）结合缓冲液（20X SSPE的微升:3M氯化钠，0.2M的NaH 2 PO 4 </sub&gt;，0.02摩尔EDTA，pH值7.4，1mM EDTA中，0.05％吐温，37.5毫摩尔NaCl）。离心机在4℃下200×g离心30秒，并去除上清。 </li><li>重复步骤5.1三次。 </li><li>加入500微升封闭缓冲液（1×结合缓冲液，0.1％聚乙烯吡咯烷酮，1毫克超纯牛血清白蛋白（BSA））将珠并轻轻1摇动 - 在4℃在一个平台上摇动2小时。 </li><li>两次用结合缓冲液500微升洗珠，如在步骤5.1中描述。 </li><li>加入400微升的结合缓冲液至珠。 </li><li>转移100μLRNA（从步骤4.11）直接向珠。在4℃下2小时在一个平台摇床 - 温和振荡1孵育。 </li><li>用500微升结合缓冲液，低盐缓冲液500微升（0.2×SSPE，1毫摩尔EDTA，0.05％吐温），高盐缓冲液的500微升（0.25×SSPE，1毫摩尔EDTA，0.05％吐温洗涤珠粒顺序，的100mM NaCl）中，并两次用TET缓冲液500微升（1×TE，0.05％吐温）中，在纺200在每次洗涤之间于4℃xg离心30秒。 </li><li>用150微升洗脱从珠BrUTP标记的RNA顺序，两次和一次用200μl洗脱缓冲液（20mM DTT，150 mM氯化钠，的50mM的Tris-HCl pH为7.5，1mM EDTA中，0.1％SDS）中的，通过温育对于在42℃的水浴中，随后通过短的自旋来从上清液中分离珠4分钟。 </li></ol> 6.沉淀BrUTP标记的RNA <ol><li>加入500微升冷的酚/氯仿/异戊醇（125:24:1）pH为4 - 5到纯化BrUTP亲和性标记的RNA。在4℃下混合在涡旋混合器上并离心16168×g离心15分钟。 </li><li>上层水相转移到新的离心管中。 </li><li>加入5 M氯化钠库存得到冷乙醇0.3 M和3倍体积的最终浓度，以沉淀RNA。 </li><li>孵育在-20℃过夜。 </li><li>收集通过离心分离沉淀的RNA在16168×g离心30分钟，在4℃下&lt;/ LI&gt; <li>除去上清液，并允许沉淀风干10分钟。 </li><li>重悬DEPC水26μLRNA沉淀。 </li><li>通过使用分光光度计测定260nm处的吸光度确定最终的RNA浓度。此外，确定的260/280比例。 A比值〜2指示纯RNA样品。 </li><li>直到需要用于cDNA合成在-80存储在等分试样的RNA样品℃。 </li></ol> 7. BrUTP标记的RNA的反转录<ol><li>调整RNA浓度为100纳克/μLDEPC使用水。使用RNA的0.5微克每个cDNA合成反应。 注:RNA模板的浓度可以变化为0.5至1.0微克，以达到最佳的cDNA合成。 </li><li>扭转使用如图3A所示设计的多聚（A）位点（终止位点）下游的多个反向引转录的RNA。 </li><li>对于每个逆转录反应，加入5微升RNA templatE（0.5微克），1微升dNTP混合物（各10毫米），反向引物C，D，E，F或G的2微升（50μM各）（ 见图3A）和无核酸酶的水。在每个管中的最终的反应体积为13微升。 注意:反转录反应的数量将取决于用于cDNA合成反向引数。例如，对于ASC1被使用的五种反向引，因此5逆转录反应成立。标签用于cDNA合成的反向引物的基础上，在管为C，D，E，F和G。 </li><li>孵育含RNA模板，dNTP混合物和引物在热循环仪的管在65℃下5分钟，以除去任何二级结构构象，然后冷却至4℃至少2分钟。 </li><li>添加1μL的逆转录酶（200U /μL），4微升缓冲液和2微升的0.1M DTT的含有RNA模板和引物各管（步骤7.4）。轻轻吹打向上和向下混合。 </li><li>通过在42℃保温60分钟在热循环反应混合物中进行逆转录。 </li><li>灭活在65℃下的逆转录酶20分钟。 </li><li>存储的cDNA在-20℃或进行下一步骤。 </li></ol> 8.扩增的cDNA <ol><li>从步骤7.8进行，每一cDNA的制备（标为C，D，E，F和G为ASC1）的扩增的PCR反应如下:从步骤7.8，1.0微升正向引物B（10μM），1.0微升3.0微升cDNA模板反向引物C或D或E或F或G（10μM），2.5微升10×聚合酶PCR缓冲液，0.5微升dNTP混合物（各10毫摩尔），1.0微升MgCl 2的（2.5毫摩尔），15.5微升的PCR级水，和0.5微升热稳定聚合酶。 注:5至1::cDNA模板从1的不同稀释液100进行测试，以优化的PCR产物的产率。单启动子近端的正向引物将被用于扩增cDNA的使用不同的反向引获得。例如，位于该启动子附近的单个正向引物B组合物用于与从C到基伍反向引为ASC1。 BC，BD，BE，BF和BG中分别标为C，D，E，F和G管被用作如图3A所示。 </li><li>使用以下热循环参数运行的PCR:1个循环95℃2分钟（热启动聚合酶的活化），30 - 40个循环95℃30秒（变性），54℃15秒（退火） ，68℃1分钟（延伸），和1个循环68℃5分钟（最终延伸）。 注:退火温度将取决于具体的引物被用来变化和延长时间将取决于预期的产物大小而有所不同。 </li><li>通过电泳在0.8％，1.5％，或取决于产品的预期大小的2％琼脂糖凝胶上分离PCR产物。 </li><li>如在El Kaderi 等人所述量化PCR产物。 ，2012 11。 注:另外，采用实时定量RT-qPCR的策略，以量化新生成绩单的数量。 </li></ol>

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The authors have nothing to disclose.

这里使用的链特异性TRO协议是改编自哺乳动物细胞12用于GRO-SEQ（全球运行在测序）分析的协议。我们成功修改了协议，来研究转录新生在芽殖酵母。要具体分析转录终止缺陷，我们通过摆脱RNA水解步骤的进一步调整的协议。这使我们能够特异性检测来自子区靠近转录起始位点开始的全长新生转录物。 有迹象表明，应以最大的谨慎进行以获得有意义的结果在?...

真核转录周期包括三个主要步骤;起始，延伸和终止。转录由RNA聚合酶II终止包括两个不同的，相互依存的步骤3。第一步包括裂解，多腺苷酸化和mRNA的释放从模板，并紧接着第二个步骤，通过从所述模板的聚合酶脱开标记。适当的终止是聚合酶的转录起始/再引发在回收至关重要的，用于防止干扰的下游基因的转录，并为通过限制普遍非编码RNA 4,5转录保持异常转录检查。尽管最近的进展，终端是迄今为止RNA聚合酶II转录周期的最不被理解的一步。一个可靠的终止试验是研究机制underlyin至关重要通过体内 RNA聚合酶II转录克终止。 许多实验方法是用来检测在生理条件下可主动转录基因终止缺陷。这些措施包括Northern杂交，终止因子芯片（染色质免疫沉淀）和传统TRO检测。每种技术具有一定的优点和缺点。所使用的传统的TRO测定是在体内 1检测的转录终止缺陷的最可靠和灵敏的方法。该测定本地化于细胞内的基因的不同区域的转录活性聚合酶分子。在正常情况下，该测定显示了转录接合聚合酶分子严格分布的基因（ 图1A）的启动子和终止子区域之间。在有缺陷的终止，然而，聚合酶是无法读取终止信号并在该基因（ 图1B）的3&#39;端下游的区域进行检测。 转录终止缺陷主要表现在从启动子近侧区发起感-转录聚合酶存在，并且是无法读取终止信号，继续转录的基因的3&#39;末端的下游区， 如图2A。与真核基因组中表现出感压倒性普遍存在的转录以及反义方向和周围的基因6，7，8，9，10的实现，它很快变得清晰，传统的TRO测定无法检测，如果聚合酶信号一个基因的下游代表的启动子启动的转录物（ 图2A）或发起索姆的异常的RNAewhere在基因或终止子元件（ 图2B），或从基因（ 图2C）的3&#39;末端的反义方向的聚合酶转录的下游的中间位置。如果观测到的通读聚合酶信号表示启动子启动的扩展感的mRNA或发起检查中的基因的下游的反义转录物的链特异性TRO测定此处描述的使用BrUTP可以区分。我们已经成功地使用这种方法来证明在芽殖酵母2中转录终止Kin28激酶的作用。这里采用的办法，我们表明，Rna14需要在芽殖酵母ASC1的转录终止。

<table><tbody><tr><td>Phenol-chloroform-isoamyl alcohol mixture</td><td>Sigma-Aldrich</td><td>77619</td><td>pH 4-5</td></tr><tr><td>TRIzol reagent</td><td>Life technologies</td><td>15596-026</td><td></td></tr><tr><td>RNase Inhibitor, human placenta</td><td>New England Biolabs</td><td>M0307S</td><td></td></tr><tr><td>Anti-BrdU beads</td><td>Santa Cruz Biotechnology</td><td>sc-32323AC</td><td></td></tr><tr><td>Protoscript II</td><td>New England Biolabs</td><td>M03368L</td><td>or an equivalent enzyme</td></tr><tr><td>Advantage 2 polymerase Mix</td><td>Clontech</td><td>639201</td><td>or an equivalent enzyme</td></tr><tr><td>5-Bromouridine 5&#039; triphosphate sodium salt</td><td>Santa Cruz Biotechnology</td><td>sc-214314A</td><td></td></tr><tr><td>RNeasy Mini kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>ATP</td><td>New England Biolabs</td><td>N0451AA</td><td></td></tr><tr><td>CTP</td><td></td><td>N0454AA</td><td></td></tr><tr><td>GTP</td><td></td><td>N0452AA</td><td></td></tr><tr><td>Ultra-pure bovine serum albumin (BSA)</td><td>MC Labs</td><td>UBSA-100</td><td></td></tr><tr><td>Asc1 B</td><td></td><td></td><td>AGATTCGTCGGTCACAAGTCC</td></tr><tr><td>Asc1 C</td><td></td><td></td><td>GAACTTTATACATATTCTTAGTT&lt;br/&gt; AGCAGTC</td></tr><tr><td>Asc1 D</td><td></td><td></td><td>TGTACATATGTATTTTCGCAGCA</td></tr><tr><td>Asc1 E</td><td></td><td></td><td>GCCAAGGAGACTGAATTTAATG</td></tr><tr><td>Asc1 F</td><td></td><td></td><td>CTATGGAATGGGGGTTTTAAG</td></tr><tr><td>Asc1 G</td><td></td><td></td><td>GGTTATGGCAGACATGCCAC</td></tr><tr><td>5s cDNA</td><td></td><td></td><td>AGATTGCAGCACCTGAGT</td></tr><tr><td>5&amp;rsquo; 5s reverse</td><td></td><td></td><td>GGTTGCGGCCATATCTAC</td></tr><tr><td>3&amp;rsquo; 5s reverse</td><td></td><td></td><td>TGAGTTTCGCGTATGGTC</td></tr></tbody></table>

analysis of termination of transcription using brutp-strand-specific transcription run-on (tro) approach

这份手稿描述了在体内检测转录终止缺陷的协议。这里描述使用BrUTP的具体链-TRO协议是在生理条件下分析转录终止缺陷强大的实验方法。像传统的TRO测定法，它依赖于超出基因作为转录终止缺陷1的指示器的3&#39;端的转录活性的聚合酶的存在。它克服了与传统的TRO测定遇到的两个主要问题。首先，它可以检测是否聚合酶通过终止信号读出的是，从启动子近侧区启动转录的一个，或者如果它被简单地表示该发起非特异性从身体某处一个普遍地转录聚合酶或3&#39;该基因的末端。其次，它可以区分，如果转录活性聚合酶信号超出终止区是真正的通读义mRNA转录聚合酶或终止子启动的非编码反义RNA的信号。简单地说，该协议涉及透的指数生长的酵母细胞中，从而允许在体内启动在BrUTP核苷酸的存在下伸长，由亲和方法纯化BrUTP标记的RNA的转录，逆转录纯化的新生RNA和扩增的cDNA用链特异性的引物侧翼启动子和基因2的终止子区域。

为了证明在检测转录终止缺陷BrUTP链特异性TRO过程的适用性，我们使用RNA14的终止缺陷，温度敏感突变体称为rna14-1。 Rna14在由RNA聚合酶II转录终止的作用被使用在选择的基因1的终结者近端区域检测RNA传统TRO检测证明。超出在非允许温度下rna14-1突变体的终止子区域的RNA信号的检测被解释为终止缺陷。所观察到的信号，然而，可以归因于?...

Watch this Scientific Journal Video about Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach at JoVE.com

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

使用BrUTP链特异性转录运行的（TRO）方法终止转录分析

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

analysis-termination-transcription-using-brutp-strand-specific

Research

JoVE Journal

Genetics

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

9.8K 次观看.  Wayne State University. We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes. 

视频: Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

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

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

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5&#39;-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5&#39;-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5&#39;-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDI-TOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5&#39;-H2PO4-(8-oxoG)G CUA AAA GU], [5&#39;-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5&#39;-H2PO4-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.

MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

使用MALDI-TOF质谱法鉴定酶降解产生的RNA片段

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

生物学

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

前体mRNA底物的3&#39;末端切割：用无细胞体系中的RNA加工反应分析<em&gt;在体外</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

基于荧光引物延伸法确定转录起点和核糖核酸酶的切割位点<em&gt;在体内</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription.

This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

终结者使用PGR-蓝质粒和金门议会的快速验证

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed ...

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single strands, each requiring distinct modes of replication: continuous (leading) and discontinuous (lagging). Understanding the interactions of chromatin-associated proteins with replicating DNA strands in vivo is crucial for elucidating the mechanisms of chromatin assembly and DNA repair coupled to DNA replication. This protocol presents the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method, designed to quantify relative protein levels on nascent leading and lagging DNA strands at replication forks. The eSPAN procedure starts with chromatin immunoprecipitation (ChIP, in yeast) or cleavage under targets and tagmentation (CUT&amp;Tag; in mammalian cells) of a protein of interest, followed by enrichment of the protein-associated nascent DNA by bromodeoxyuridine (BrdU) immunoprecipitation. Strand-specific next-generation sequencing is applied to isolated ssDNA. This technique can be used to determine whether a protein is enriched at leading or lagging replication forks. The eSPAN provides genome-wide strand-preference of chromatin-associated proteins, including histones at replication forks.

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single ...

Transcription Attenuation in Prokaryotes

Transcriptional attenuation occurs when&#160;RNA transcription is prematurely terminated due to the formation of a terminator mRNA hairpin structure.&#160; Bacteria use these hairpins to regulate the transcription process and control the synthesis of several amino acids including histidine, lysine, threonine, and phenylalanine. Transcription attenuation takes place in the non-coding regions of mRNA.
There are several different mechanisms used to attenuate transcription. In ribosome mediated transcriptional attenuation, the movement of a ribosome on the transcript is stalled or proceeds forward depending upon the availability of the tRNAs charged with a specific amino acid. High amino acid concentrations allow the ribosome to move forward leading to the formation of the terminator structure; deficiency of the amino acid stalls the ribosome and causes formation of the anti-terminator structure. The trp operon in E. coli, discussed below, is a good example of this type of mechanism. tRNA mediated transcriptional attenuation, as observed in trp operon of Lactococcus lactis, depends on an RNA-RNA interaction. When uncharged tRNAs are present in sufficient numbers, they directly bind to the mRNA and stabilize the anti-terminator structure. Transcriptional attenuation is also known to be mediated by proteins as found in bgl (beta-glucoside) operon in E. coli.&#160; This involves an RNA&#8212;protein interaction where a protein binds to the transcript and regulates the formation of an anti-terminator structure. More recently, another transcriptional attenuation mechanism was discovered where small metabolites like thiamine were observed to regulate transcription by directly binding to the non-coding mRNA segments, also known as riboswitches. Riboswitches can form a terminator or an anti-terminator structure depending upon the concentration and nature of a metabolite.&#160;
Trp Operon
The trp operon in E. coli contains a 140 nucleotide leader sequence before its first structural gene. This leader sequence has four distinct segments &#8211; 1 through 4&#8211; and regulates the transcription of the downstream structural genes. Segment&#160; 1 can form a hairpin structure with segment&#160; 2. This 1-2 hairpin structure is known as a pause structure, as during transcription, it stalls RNA polymerase until the ribosome binds the newly transcribed RNA. This synchronizes transcription and translation in bacteria.&#160; When tryptophan concentrations are low, a hairpin structure forms between segments 2 and 3, known as the anti-terminator structure. This anti-terminator structure allows the continuous transcription of the downstream genes which produce enzymes for tryptophan synthesis. In contrast, when tryptophan concentrations are sufficient, a hairpin structure forms between segments 3 and 4, called the terminator structure. Along with a&#160; series of uracil bases that follow, the terminator structure causes RNA polymerase to dissociate from the RNA and template DNA strands, resulting in the termination of the transcription.

使用BrUTP链特异性转录运行的（TRO）方法终止转录分析

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