Name: saccharomyces cerevisiae metabolic labeling with 4-thiouracil and the quantification of newly synthesized mrna as a proxy for rna polymerase ii activity
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Description: Watch this Scientific Journal Video about Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity at Jo

我们感谢他的支持和诉费舍尔, k. 舒马赫和 f. El Saafin 的讨论。肺结核由 ITN (PITN-GA-2013-606806, NR) 和基金会 ARC 支持。这项工作得到了法新社谜 (ANR-15-CE11-0022 SAGA2) 的资金支持。这项研究也得到了 ANR-10-LABX-0030-INRT 的支持, 法国国家基金是由法新社谜在审批 d ' 艾文莉 ANR-10-IDEX-0002-02 框架计划下管理的。

1. 佐贺亚基细胞培养和雷帕霉素耗竭
<ol><li>对于每个S. 酵母菌株和复制, 包括野生型或控制菌株, 接种一个单一的殖民地从新鲜的盘子到5毫升的 YPD 培养基 (2% 蛋白胨, 1% 酵母提取物, 和2% 葡萄糖)。</li>
	<li>以恒定搅拌 (150 rpm) 在30摄氏度的夜间生长酵母细胞。</li>
	<li>测量 600 nm (od600) 的光学密度, 并将培养液稀释到外径600约0.1 在100毫升的 YPD 培养基中, 让其生长直到 OD<...

<ol>
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虽然基因组范围的工具来分析转录的变化仍在改善, 但通过定量的 rna 的稳态水平的定量分析对转录的分析可能无法准确地反映 rna 聚合酶 II 活性的变化。事实上, mRNA 水平不仅受 RNA 合成的调控, 而且还受其成熟和降解的制约。为了测量 mrna 合成与 mrna 降解的不耦合性, 近年来开发出了不同的实验方法, 用于分析酵母和哺乳动物中的新生转录。
对新生转录进行定量的最广泛使用?...

细胞通过基因表达程序的动态改变对内源和外源性线索作出反应。近年来, 全基因组方法的巨大发展允许在不同的条件下对转录的准确和全面的描述。在大多数转录组研究中, 微阵列杂交或高通量测序用于从总稳态 rna 分数中量化 rna 水平。在特定的摄动下转录改变可以显示出广泛的可能的结果, 与特定基因表达变化或大范围的基因要么上升或下调。基因表达是由 rna 聚合酶和影响 rna 水平的其他过程的 rna 合成之间的微调平衡或稳态的结果。RNA 聚合酶 II 转录, 包括其三不同的阶段 (起始, 伸长, 和终止), 是高度和错综复杂的与 mRNA 处理, 细胞质出口, 翻译和降解相关。
最近的一些研究表明, mRNAs 合成和衰变是耦合机制, 并表明在量化总稳态 RNA 时, 可以忽略对突变或刺激下的转录效应。首先, 通过对 mRNA 稳态水平的分析, 对转录变化的检测始终取决于 mRNAs 半衰期。一旦引入扰动, 长期半衰期的 mRNAs 的稳态水平将比短半衰期 mRNAs 的影响要小得多。因此, RNA 合成变化的可检测性强烈偏向于短寿命转录, 而较长存活的 mRNA 种类的分析可能无法揭示转录速率的动态变化。其次, 一些报告表明, 在酵母和哺乳动物中, 在分析 mRNA 的稳态水平时, 可能会忽略转录的全球变化。这可能是由于链路 mrna 合成和退化导致 mrna 缓冲的机制。这促使开发新的协议, 以量化 mrna 合成与降解分离, 通过对新转录 mrna 的分析。近年来, 提出了几种替代方案, 包括全球运行时序 (GRO)1和本机伸长率转录序列 (NET seq)2、3。在这里, 我们提出的协议最初开发的哺乳动物细胞4,5,6 , 然后适应酵母7,8,9,10, 11, 这是基于 RNA 标记与巯基核苷或基础模拟, 4-thiouridine (4sU) 或 4-硫尿嘧啶 (4tU), 分别。
这种方法专门净化新转录的 rna 从细胞的 rna 是脉冲标记与4sU 几乎没有干扰细胞稳态。因此, 一旦细胞暴露在 4sU, 分子迅速小白菜植株吸, 磷酸化到4苏三磷酸盐, 并纳入 rna 被转录。一旦脉冲标记, 它是可能提取总细胞 rna (对应于稳定状态的 rna), 并随后, 4 su 标记的 rna 分数是硫醇-特别修改, 导致生物素之间的二硫键的形成和新转录的 RNA4,5。然而, 4sU 只能由表达核苷转运体的细胞小白菜植株吸, 如人类要核苷转运 (hENT1), 防止其立即在萌芽或裂变酵母中使用。虽然可以在粟或s. 酵母中表达 hENT1, 但使用改良的碱4tU 可以实现更简便的方法, 因为酵母细胞可以占用 4tU, 而不需要核苷转运体10的表达,11,12,13. 事实上, 4tU 的新陈代谢需要酶尿嘧啶核糖 (UPRT) 的活性。在几个有机体, 包括酵母, 但不是哺乳动物, UPRT 是一个嘧啶打捞途径, 回收尿嘧啶尿苷单磷酸的关键。
转录组研究中的一个重要偏差可以通过平行分析的不同样本之间的规范化来引入。事实上, 许多偏离因素可能影响突变体和野生型菌株转录组的比较分析: 细胞裂解的效率、RNA 提取和恢复的差异, 以及用于微阵列分析的扫描器校准差异, 等等。如上所述, 当预期全球对 RNA 聚合酶 II 转录产生影响时, 这种变化会特别误导人。以远亲相关裂变酵母粟粟为内部标准, 设计了一种精确比较不同样品间 mRNA 合成速率的优雅的平均值。为此, 在细胞裂解和 RNA 提取之前, 将固定数量的标记的粟细胞添加到s. 酵母样本中, 即野生型或突变细胞.随后,粟和s 酵母的稳态和新合成的 rna 可以通过 rt-pcr 或通过微阵列芯片或高通量测序10进行量化。将这些数据与动力学建模相结合, 可以测量发芽酵母中 mRNA 合成和衰变的绝对速率。
在本手稿的框架中, 我们将展示新转录 rna 的分析如何能够揭示辅激活因子配合物佐贺和 TFIID 在萌芽酵母14,15, rna 聚合酶 II 转录中的全球作用, 16。重要的是, 过去的研究量化了S 酵母的稳态 mRNA 水平, 并建议佐贺在有限的酵母基因上发挥主导作用, 这一组受佐贺突变的强烈影响, 但相对抗 TFIID突变17,18,19。令人惊讶的是, 佐贺酶活性被证明对整个转录基因组起作用, 这表明在 RNA 聚合酶 II 转录中这一共同激活剂的作用更广泛。减少 RNA 聚合酶 II 在大多数表达基因的招募在佐贺或 TFIID 的灭活时观察到, 表明这些辅活化因子在大多数基因一起工作。因此, 新转录 mRNA 的定量显示, 佐贺和 TFIID 是需要的转录几乎所有基因的 RNA 聚合酶 II14,15,16。补偿机制的实施成为一种方法, 使细胞能够应对全球 mrna 合成减少, 这是由于 mrna 退化同时全球减少而缓冲的。佐贺添加了全球影响 rna 聚合酶 ii 转录的因素列表, 如 rna Pol ii 亚基10、调解人辅激活因子复合20、一般转录因子 TFIIH21、22和间接地, mRNA 降解机械的元素9,10,23。这种补偿性事件在佐贺突变体中普遍可见, 尽管 mrna 合成14的全球和严重减少, 但稳定状态 mRNA 水平的变化不大且有限。类似的分析也进行了BRE1删除应变, 导致完全丧失组蛋白 H2B 泛素化。有趣的是, 在缺乏 Bre1 的情况下, 可以检测到 rna 聚合酶 II 转录的温和但一致的全球影响, 这表明在酵母中新转录的 rna 的代谢标记可以检测和量化 mRNA 的广泛变化范围。合成率。

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			<td style="width:139px;">00-0079</td>
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			<td height="22" style="height:22px;width:216px;">GeneChip Scanner 3000 7G</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:139px;">00-0210</td>
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saccharomyces cerevisiae metabolic labeling with 4-thiouracil and the quantification of newly synthesized mrna as a proxy for rna polymerase ii activity

转录组研究稳态 rna 可能忽略 rna 聚合酶 II 转录的全球缺陷。事实上, mrna 合成的全球减少被证明是通过同时减少 mrna 退化来恢复正常的稳态水平而得到补偿的。因此, 全基因组定量的 mrna 合成, 独立于 mrna 衰变, 是 RNA 聚合酶 II 转录活性的最佳直接反映。在这里, 我们讨论了一种使用非扰动代谢标记的新的 rna 在酿酒酵母(酵母) 的方法。具体来说, 细胞培养6分钟与尿嘧啶模拟, 4-硫尿嘧啶, 和标记的新转录 rna 被纯化和量化, 以确定所有个体 mRNA 的合成率。此外, 使用标记粟粟细胞作为内部标准允许比较不同的酵母菌株的 mRNA 合成。利用该协议和动态动力学模型拟合数据, 可以确定相应的 mRNA 衰减率。

当对新转录的 RNA 进行代谢标记时, 需要控制几个方面: 标记的时间和效率、峰值比例、提取协议和生物素化效果 (包括信噪比),等。其他7,10,11, 这些条件已经广泛和有条不紊地显示。在这里, 我们主要关注通过 rt-pcr、微阵列或测序处理样品后可以进行的解释和即时分析。对不同突变菌株的分析表明, 该方...

Watch this Scientific Journal Video about Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity at Jo

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

这里描述的协议是基于全基因组的定量的新合成的 mRNA 纯化的酵母细胞标记为 4-硫尿嘧啶。这种方法允许测量 mrna 合成与 mrna 衰变分离, 从而提供了 RNA 聚合酶 II 转录的精确测量。

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 ...

该技术的主要优点是，它通过测量不受RNA降解影响的新合成mRNA来反映RNA聚合酶II活性。该协议最初使用微阵列杂交描述，但可以很容易地被采用到新转录的mRNA的高全序列。演示这个程序将是蒂亚戈巴普蒂斯塔，前研究生从我们的实验室。接种S.cerevisiae细胞后，在30摄氏度下过夜，在150 RPM时持续搅拌。第二天，测量600纳米的光学密度。将培养量稀释到约 0.1 的 OD600，在 100 毫升 YPD 介质中。成长的细胞，直到OD600是大约0.8。在此之后，测量S.pombe过夜文化的OD600。将这种培养剂稀释到约 500 毫升 YAS 介质中的 0.1 的 OD600，让它生长，直到 OD600 约为 0.8。首先，准备一个新的解决方案，两个摩尔四硫化。将准备好的溶液保持室温，防止光线。将四硫化溶液添加到 S.cerevisiae 和 S.pombe 培养中，使最终浓度为 5 毫摩尔。在30摄氏度和32摄氏度时，在30摄氏度和32摄氏度下培育S.cerevisiae和S.pombe培养，持续搅拌6分钟。在此之后，删除每个培养的一小部分，并使用自动细胞计数器或 Neubauer 腔室对细胞进行计数。在2，500倍g和4摄氏度的离心机5分钟收集细胞。丢弃上清液，用冰冷洗涤细胞一次PBS。再次在2，500倍的g和4摄氏度下离心5分钟。接下来，将细胞重新在五毫升的冰冷中重新暂停一次PBS。以三比一的比例混合S.cerevisiae和S.pombe细胞。将混合样品在2500倍g和4摄氏度下离心5分钟。然后取出PBS，将液氮中的细胞闪冻。将样品存放在负 80 摄氏度，直到准备好使用。当准备继续时，在冰上解冻细胞约20至30分钟。使用经过改造的酵母RNA提取试剂盒提取文本协议中概述的RNA。在此之后，将滤芯转移到最终收集管。用50微升DEPC处理的无RNase水来加热RNA，预热至100摄氏度。以16，000倍g的离心机一分钟。然后使用 50 微升预热 DEPC 处理无 RNase 水将 RNA 再次加热到同一管。以 16，000 次 g 离心一分钟，确保整个样品体积通过过滤器。如果没有，则再次离心较长时间。完成后，使用适当的设备对样品的纯度进行量化和检查。使用 DEPC 处理无核酸酶的水将以前获得的 RNA 的浓度调整为每毫升 2 毫克。200微克总RNA的等量，在60摄氏度下加热10分钟。立即在冰上冷却两分钟。添加 600 微升 DEPC 处理的无 RNase 水。添加100微升生物素化缓冲液，然后加入200微升生物素HPDP。如果生物素 HPDP 溶液沉淀出溶液，则将 DMSO 或 DMF 的体积增加至文本协议中所述的 40% 的反应量。保护样品免受光线的污染，并在室温下用温和的搅拌孵育三个小时。在此之后，在管中加入大约相等的氯仿体积，并大力混合。在13，000倍的g和4摄氏度的离心机5分钟。接下来，小心地将上相转移到新的两毫升管中。加入相当于样品体积约1/10的五摩尔氯化钠。然后混合样品。首先，在65摄氏度下加热生物素化RNA10分钟。然后在冰上冷却样品五分钟。在生物素化RNA中加入100微升的斯特雷普他丁涂层磁珠。在室温下孵育，轻微摇晃90分钟。将套件提供的柱放在磁性支架中。接下来，在柱子上加入900微升的室温洗涤缓冲液。将 200 微升珠和 RNA 混合物涂抹到柱上。收集1.5毫升管中的流经，然后再次将其应用到同一磁柱上。如有必要，保持此流通过，因为它表示未标记的 RNA 分数。要开始对不同分数进行RT-qPCR验证，请先合成文本中概述的cDNA。然后使用标准协议通过实时 qPCR 放大 cDNA。从野生细胞中纯化的份量中测得的转录量，与四硫化杆和未培养的四硫化细胞确认，这一程序特别净化了标有RNA的RNA。对spt20增量菌株的分析表明，总稳定态RNA的数量基本保持不变，或者只是被测试基因的轻微减少。对于删除的 bre1 菌株，也可以看到类似的结果。然而，对spt20增量菌株中新转录的RNA的分析表明，所有被测试基因的mRNA合成减少了3到5倍。同时，bre1 的丢失导致更谨慎但仍然可见的下降。在 spt7 的可致耗尽后，saga 复合体、新转录的 mRNA 水平的结构子单元将降低到类似于删除应变的程度。当通过全基因组分析测量总RNA水平时，只有少数基因在通过向上调节或向下调节来降低spt20的缺失时，其表达水平会有所改变。然而，对新转录的RNA的分析表明，在删除spt20时，超过4000个基因的含量显著降低至少两倍，这表明传奇对发芽酵母中RNA聚合酶II转录的全球积极影响。虽然这种方法可以提供对糖核糖精转录的见解，但它只是使用本协议，只有稍有变化，我们才能检测到mRNA合成的全局减少，而mRNA降解的全局减少可以补偿。

Research

JoVE Journal

Genetics

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

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

可诱导T7 RNA聚合酶介导的多基因表达系统，pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

科研

遗传学

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

在酵母S.酵母缺氧反应期间, 测量一段时间内的 mRNA 水平

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

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

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 ...