高分辨率基因表达谱RNA的合成，加工和衰变的新转录RNA在细胞培养的代谢标记
Summary
细胞总RNA提供了一种用于研究的短期变化，以及在RNA合成和衰变动力学的RNA加工模板差。这里，我们描述遵循与4 – thiouridine的新转录的RNA的代谢标记通过硫醇特异性的生物素化和纯化的新转录的RNA，来克服这些限制。
Abstract
全转录组的芯片和新一代测序的发展已经彻底改变了我们的细胞基因表达的复杂性的理解。随着更好地理解所涉及的分子机制，底层动力学的精确测量变得越来越重要。在这里，这些有力的方法面临的主要限制，因为他们学习的模板样本， 即细胞总RNA的内在属性。在许多情况下，细胞总RNA发生变化，要么过慢或过快，代表潜在的分子事件和动力学有足够的分辨率。此外，RNA的合成，加工，和衰减的贡献改变不容易区分。
我们最近开发高分辨率基因表达谱来克服这些限制。我们的做法是根据新转录RNA代谢标记4 thiouri用餐（因此也称为4SU标记），然后通过严格的纯化的新转录的RNA，使用硫醇特异性的生物素和链霉亲和素包被的磁珠。它是适用于范围广泛的生物体包括脊椎动物， 果蝇 ，酵母或细菌。我们成功地应用于4SU标记实时动力学研究转录因子的活动，提供精确的测量RNA半衰期，到RNA加工的动力学，并取得了新的见解。最后，计算模型可以被用来产生一个综合的，全面的分析，潜在的分子机制。
Introduction
基因表达谱是一个关键的工具，用来研究细胞过程和相关的复杂的相互作用网络。 mRNA丰度的研究，通常的首选方法，以获得基本的洞察潜在的分子机制。全转录组微阵列1和，最近，新一代的RNA测序（RNA-SEQ）2-4的发展助长了这种方法。虽然这些技术已经彻底改变了我们的细胞基因表达的复杂性的理解，他们面临着由于细胞总RNA模板样本， 即内在性能的主要限制。首先，短期变动的总RNA水平不相匹配的转录率的变化，但本质上是依赖于各自的转录的RNA的半衰期。虽然五倍一个短命的成绩单， 如编码转录因子的诱导，将随时检测总RNA中在一个小时之内，同样的一项长期的成绩单， 例如编码一种代谢酶的诱导，仍将几乎看不见。另外，即使是一个完整的关机的转录率的平均基因的RNA的5个小时的半衰期（> 1000倍的下调）将简单地需要5个小时，它的总的RNA水平降低仅由两方面。因此，总RNA的分析有利于短命的转录调节的检测，其中有许多编码的转录因子和基因监管的功能5。此外，调节掩盖真正的动能级联和主要信号事件的不能从二级分化的。 ，反过来，可能会导致大量的偏见在下游的生物信息学分析。其次，总RNA水平的改变不能被改变RNA的合成或腐烂。后者需要测量细胞侵袭的方法， 例如阻断transcripti使用6，放线菌素D和扩展监控正在进行的RNA随着时间的推移衰减。平均mRNA的半衰期在哺乳动物细胞中的5 – 10小时5,7，大多数基因的mRNA水平的减少小于双重转录停止在几个小时后。在这些相当小的差异，导致在大多数由于基本的数学方程的指数性质的细胞基因的mRNA的半衰期非常不精确的测量。最后，当细胞总RNA的RNA-seq中透露，大约有一半是我们的基因选择性剪接事件，底层动力学以及动力机制仍然知之甚少，指导组织和上下文特定的调控RNA加工。此外，贡献差异表达基因的RNA加工，特别是对于非编码RNA，仍有待确定。总而言之，这些限制主要障碍生物信息学动力学建模相关的分子机制。
我们最近开发出一种方法，称为高分辨率基因表达谱，以克服这些问题5,7,9。它是基于使用4 thiouridine（4SU标记），一种自然发生的尿嘧啶核苷衍生物的新转录RNA代谢标记，并提供直接访问新转录转录以最小的干扰细胞生长和基因表达（ 见图1）5， 10-12。曝光的真核细胞快速吸收4SU磷酸，磷酸化，并纳入新转录RNA 4SU结果。细胞总RNA的分离，4SU标记的RNA部分是巯基的特异性生物素化的生物素和新转录的RNA之间产生一个二硫键。 “细胞总RNA'然后可以进行定量分离成标记（”新转录“）和未标记的（'预existin的克）RNA具有高纯度，使用链霉亲和素包被的磁珠。最后，标记的RNA回收从有孔玻璃珠，通过简单地增加裂解二硫键的还原剂（ 如二硫苏糖醇）和释放从有孔玻璃珠的新转录的RNA。
新转录的RNA描绘每一个基因的转录活性4SU曝光的时段内。 4SU 标记分钟的时间表，从而提供了一个快照图片真核生物基因表达和理想的模板，下游的生物信息学分析（ 如启动子分析）。在稳态条件下可以假设的情况下，新转录/总的比例，新转录/未标记的未标记/，总RNA提供非侵入性的访问精确RNA半衰期7,13。此外，重要的是要注意4SU标记只需5分钟（5分钟4SU RNA）纯化后，新转录的RNA是年龄小于15分钟和60分钟4SU-RNA。当进行超短逐步不再4SU标记的RNA-seq的结合在一个单一的实验设置，RNA加工的动力学透露核苷酸分辨率9。最后，时间过程分析与计算模型相结合的新的转录总RNA允许RNA合成的综合分析和衰减14。
总之，该方法可以直接分析RNA的合成，处理，并在真核细胞中的降解动力学。这是适用于所有主要模式生物，包括哺乳动物，昆虫（ 果蝇 ），两栖类（ 爪蟾 ）和酵母5,15,16。这是直接兼容微阵列分析5,17，9,13,14 RNA定序，适用于体内 12,15。在这里，我们详细的方法，在培养的哺乳动物细胞中的标签，隔离和净化新转录RNA。此外，电位人的问题和缺陷进行了讨论。
Protocol
Representative Results
Discussion
新转录RNA代谢标记大幅提高电源的高通量技术，如芯片和RNA-SEQ提供更合适的模板来解决生物学问题的兴趣。本议定书进行了广泛的优化。它允许新转录RNA> 1000倍浓缩，并提供高度可重复性的结果。
4SU标记实验的实验设计是至关重要的新转录的RNA，将描绘仅在4SU细胞的曝光时间的实时的转录活性。如果刺激后转录率的实际变化已经消退，这些都将错过新转录RNA进行分析…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们想感谢阿米里根仔细阅读的手稿。支持这项工作是由NGFN加授＃01GS0801，MRC奖学金授予G1002523 NHSBT授予WP11-05，LD和DFG授予FR2938/1-1 CCF
Materials
| Name | Company | Catalog Number | Comments |
| 4-thiouridine | Carbosynth | T4509 | Prepare 50 mM stock in sterile H2O, store at -20 °C in aliquots of 50-500 μl, discard unused reagent, do not refreeze. |
| Trizol | Invitrogen | 15596026 (100 ml), 15596018 (200 ml) | WARNING – CORROSIVE and HAZARDOUS TO HEALTH! Ensure immediate access to Phenol antidote (PEG-Methanol); Store at 4 °C. |
| Chloroform | Sigma | 372978 | WARNING – HAZARDOUS TO HEALTH |
| Isopropanol | Sigma | 650447 | |
| Sodium citrate, nuclease-free | Sigma | C8532 | Prepare 1.6 M stock solution using nuclease-free water. |
| 5M nuclease-free NaCl | Sigma | 71386 | Stock solution |
| Nuclease-free H2O | Sigma | W4502 | Make 1 ml aliquots in nuclease-free tubes. |
| RNA precipitation buffer | 1.2 M NaCl, 0.8 M sodium citrate in nuclease free water. Prepare in advance under strictly nuclease-free conditions. Store at room temperature in 50 ml falcon tubes. | ||
| Ethanol | Sigma | 459844 | Use with nuclease-free water to prepare 80% ethanol, store at -20 °C. |
| 1 M nuclease-free Tris Cl, pH 7.5 | Lonza | 51237 | Stock solution |
| 500 mM nuclease-free EDTA, pH 8.0 | Invitrogen | 15575-020 | Stock solution |
| 10x Biotinylation Buffer (BB) | 100 mM Tris pH 7.4, 10 mM EDTA in nuclease-free water, make aliquots of 1 ml. | ||
| Dimethylformamide (DMF) | Sigma | D4551 | |
| EZ-Link biotin-HPDP | Pierce | 21341 | Prepare 1 mg/ml stock solution by dissolving 50 mg biotin-HPDP in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 °C in aliquots of 1 ml. |
| Phase Lock Gel Heavy tubes 2.0 ml | Eppendorf | 0032 005.152 | Optional for the chloroform extraction step. |
| Zeta membrane | BIORAD | 162-0153 | |
| 10x Dot blot binding buffer | 100 mM NaOH, 10 mM EDTA | ||
| Biotin-oligo | 5′-biotin, 25 nucleotides, any sequence | ||
| Sodium dodecyl sulphate | Fisher | BPE9738 | For 100 ml 20% stock solution, add 20 g SDS to 80 ml PBS pH 7-8 and adjust volume to 100 ml. Keep all high-percentage SDS solutions above 20 °C. Warm the solutions slightly should SDS precipitate. |
| EZ-Link Iodoacetyl-LC-Biotin | Pierce | 21333 | Prepare 1 mg/ml stock solution by dissolving 50 mg iodoacetyl-biotin in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 °C in aliquots of 1 ml. Generates irreversible, thiol-specific biotinylation. |
| Phosphate buffer saline | Gibco | 10010-015 | |
| Dot blot blocking buffer | Mix 20 ml 20% SDS with 20 ml 1 x PBS pH 7-8 and add EDTA to the final concentration of 1 mM. | ||
| Streptavidin-horseradish peroxidase | Vector Laboratories | SA5004 | Store at -20 °C. Mix 10 ml 20% SDS with 10 ml 1 x PBS. Add 20 μl Streptavidin-HRP before use. |
| ECL reagent | GE Healthcare | RNP2109 | Use following the manufacturer’s instructions. |
| Super RX, X-RA Film, 18×24 cm | Fujifilm | 47410 19236 | |
| μMacs Streptavidin Kit | Miltenyi | 130-074-101 | Store the beads at 4 °C. |
| Tween 20 | Sigma | P1379 | |
| Washing buffer | 100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20 in nuclease-free H2O. | ||
| Dithiothreitol (DTT) | Sigma | 43817 | Prepare as 100 mM DTT in nuclease-free H2O, always prepare fresh before use. |
| RNeasy MinElute Kit | Qiagen | 74204 | Store columns at 4 °C, remaining components of the kit at room temperature. |
| 1.5 ml screw-top polypropylene tubes | Sarstedt | 72.692.005 | Compatible with Dimethylformamide |
| 2.0 ml screw-top polypropylene tubes | Sarstedt | 72.694.005 | Compatible with Dimethylformamide |
| 15 ml tubes | BD Falcon | 352096 | Compatible with Dimethylformamide |
| 50 ml tubes | BD Falcon | 352070 | Compatible with Dimethylformamide |
| All solutions/reagents should be stored at room temperature unless otherwise specified. | |||
| Equipment | |||
| UV/VIS spectrophotometer | Thermo Scientific | NanoDrop 1000 | Or equivalent. Use low volume (1-2 μl) for measurements of low RNA concentrations to avoid excessive sample loss. |
| Polypropylene 15 ml centrifuge tubes | VWR International | 525-0153 | In contrast to standard 15 ml tubes, these tolerate up to 15,000 × g |
| High-speed centrifuge | Beckman Coulter | Avanti J-25 | Or equivalent equipment capable of reaching 13,000×g |
| High-speed rotor | Beckman Coulter | JLA-16250 | Or equivalent equipment capable of reaching 13,000×g |
| Adaptors for 15 ml tubes | Laborgeräte Beranek | 356964 | Or equivalent equipment capable of reaching 13,000×g |
| Refrigerated table-top centrifuge | Eppendorf | 5430 R | Or equivalent. |
| Thermomixer | Eppendorf | Thermomixer compact | Or equivalent. |
| Magnetic stand | Miltenyi Biotec | 130-042-109 | One stand holds 8 μMacs columns. |
| Waterbath | Grant | SUB Aqua 5 | Or equivalent. |
| Ultra-fine scale | A&D | GR-202 | Or equivalent. |
| E-Gel iBase Power System | Invitrogen | G6400UK | For RNA gels; or equivalent. |
| E-Gel EX 1% agarose precast gels | Invitrogen | G4020-01 | For RNA gels; or equivalent. |
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