核糖体剖面对芽殖酵母中翻译的全基因组定量
Summary
翻译调控在蛋白质丰度控制中起着重要的作用。在这里, 我们描述一个高通量的方法, 定量分析翻译在萌芽酵母酿酒酵母。
Abstract
mRNA 转化为蛋白质是一个复杂的过程, 涉及多个层次的调节。通常认为, mRNA 转录的变化反映了蛋白质合成的变化, 但许多例外情况已经观察到。最近, 一种叫做核糖体剖面 (或 Ribo-Seq) 的技术已经成为一种强有力的方法, 它允许识别, 精确度高, 将 mRNA 的区域转化为蛋白质, 并在全基因组水平上量化翻译。在这里, 我们提出了一个广义的协议的基因组全定量翻译使用 Ribo-Seq 在萌芽酵母。此外, 结合 Ribo 序列数据与 mrna 丰度测量, 使我们能够同时量化的翻译效率数以千计的 mrna 转录在同一样本, 并比较这些参数的变化, 以响应实验操纵或在不同的生理状态。我们描述了一个详细的协议, 生成核糖体足迹使用核酸消化, 分离完整的核糖体足迹复合物通过蔗糖梯度分馏, 并准备 DNA 库的深度测序, 以及适当的确保准确分析体内翻译所需的质量控制。
Introduction
mRNA 翻译是细胞中的基本过程之一, 在蛋白质表达调控中起着重要的作用。因此, mRNA 翻译受到严格控制, 以响应不同的内外生理刺激1,2。然而, 翻译规则的机制仍然理解。在这里, 我们描述了协议的基因组全定量翻译在萌芽酵母核糖体剖面。核糖体剖面技术的总体目标是研究和量化不同细胞条件下的特定基因的翻译。这项技术使用下一代测序来定量分析核糖体在整个基因组中的占有情况, 并允许监测蛋白质合成的速率在体内在单一密码子解析3,4。目前, 这种方法提供了最先进的测量蛋白质翻译水平的手段, 并已证明是一个有用的发现工具提供的信息, 不能透露其他现有的技术, 如微阵列或转换状态数组分析 (TSAA) 5。由于核糖体分析报告的转录水平和翻译输出的组合变化, 它也提供了比其他方法更大的灵敏度。
这种方法是基于核糖体保护的 mRNA 片段3的深度测序。在蛋白质翻译期间, 核糖体保护 ~ 28 nt 部分的 mRNA (称为脚印) 6。通过确定核糖体保护片段的序列, Ribo-Seq 可以映射核糖体在翻译的 mrna 上的位置, 并确定哪些区域的 mrna 可能被积极转化为蛋白质3,7。此外, 我们可以定量测量 mrna 的翻译, 通过计算与给定的 mrna 转录值对齐的脚印数。
为了分离核糖体保护的碎片, 细胞裂解最初处理的翻译抑制剂, 以拖住核糖体后, 核糖核酸消化。虽然游离的 mrna 和部分的翻译基因不受核糖体的保护, 退化的核糖核酸, 核糖体保护的 mrna 片段可以恢复纯化完整的核糖体足迹复合物。然后将这些 mRNA 的足迹转化为 cDNA 文库, 并通过深度测序 (图 1) 进行分析。在核糖体剖面的同时, 完整的 mRNA 从相同的样本中提取并测序。通过比较 Ribo-Seq 和 mrna 丰度测量的翻译水平, 我们可以确定在翻译水平上具体上调或下调的基因, 并在全基因组水平上计算 mrna 的翻译效率。虽然本文中描述的协议是针对酵母的, 但对于那些试图在其他系统中建立 Ribo 的研究人员来说, 它也应该是有用的。
Protocol
1. 提取制剂 YPD 板上的单一菌落的冷冻储存的条纹酵母菌株 (1% 酵母提取物、2% 蛋白胨、2% 葡萄糖和2% 琼脂)。在30° c 下孵育2天。 接种酵母从 YPD 板 (使用一个单一的殖民地) 到15毫升的 YPD 培养基 (1% 酵母提取物, 2% 蛋白胨, 2% 葡萄糖) 在50毫升圆锥离心管和成长过夜与震动 (200-250 rpm) 在30° c。 在2个无菌烧瓶中稀释培养成500毫升的 YPD 培养基, 使 OD600和 #60; 0.1。将酵母?…
Representative Results
详细的管道的生物分析核糖体的剖析数据已经描述了以前的8,9。此外, 几个研究小组已经开发了生物信息学工具的差异基因表达分析和处理的测序数据, 这是特定的核糖体分析方法10,11,12 ,13,14,<sup cla…
Discussion
Ribo-Seq 方法已成为一个强大的技术, 分析 mRNA 翻译在体内在全基因组的水平3。使用这种方法进行的研究, 允许 single-codon 的分辨率进行监测, 有助于我们理解翻译规则。尽管有其优点, Ribo 还是有几个局限性。核糖体 RNA (rRNA) 片段总是 co-purified 在核糖体保护的足迹的分离, 减少了有用的测序读数, 可以获得在 Ribo-Seq 实验9,25的产量。…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院的资助, AG040191 和 AG054566 向 VML 提供了支持。这项研究是进行的, 而 VML 是一个遥远的研究赠款接受来自美国老龄研究联合会。
Materials
| 0.45 μM membrane filters | Millipore | HVLP04700 | |
| 0.5 M EDTA | Invitrogen | AM9261 | |
| 0.5 mL centrifugal filters (100 kDa MWCO) | Millipore | UFC510024 | |
| 1 M Tris-HCl, pH 7.0 | Invitrogen | AM9850G | |
| 1 M Tris-HCl, pH 7.5 (pH 8.0 at 4°C) | Invitrogen | 15567-027 | |
| 10X TBE buffer | Invitrogen | AM9863 | |
| 10% TBE-urea gel | Invitrogen | EC6875BOX | |
| 15% TBE-urea gel | Invitrogen | EC6885BOX | |
| 2 M MgCl2 | RPI | M24500-10.0 | |
| 2X TBE-urea sample buffer | Invitrogen | LC6876 | |
| 3M NaOAc, pH 5.5 | Invitrogen | AM9740 | |
| 5' Deadenylase (10 U/μL) | Epicentre | DA11101K | |
| 5X Nucleic acid sample loading buffer | Bio-Rad | 161-0767 | |
| 8% TBE gel | Invitrogen | EC6215BOX | |
| Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1) | Invitrogen | AM9722 | |
| Blue light transilluminator | Clare Chemical Research | DR-46B | |
| Chrome-steel beads, 3.2 mm | BioSpec Products | 11079132c | |
| Cryogrinder | Biospec product | 3110BX | |
| Cycloheximide | RPI | C81040-5.0 | |
| Data Acquisition System | DATAQ Instruments | DI-245 | |
| Deoxynucleotide (dNTP) solution mix (10 mM) | NEB | N0447L | |
| Glycogen | Invitrogen | AM9510 | |
| Gradient fractionation system | Brandel | BR-184X | |
| High-fidelity DNA polymerase (2,000 U/mL) | NEB | M0530S | Supplied with 5X Phusion HF Buffer |
| Next-generation sequencing library quantification kit | Kapa Biosystems | KK4824 | |
| Nucleic acid gel stain | Invitrogen | S11494 | |
| Optima XE-90 ultracentrifuge | Beckman Coulter | A94471 | |
| Poly(A) mRNA isolation kit | Invitrogen | 61011 | |
| Rec J exonuclease (10 U/μL) | Epicentre | RJ411250 | |
| Reverse transcriptase (200 U/μL) | Invitrogen | 18080093 | Supplied with 5X first-strand buffer and 0.1 M DTT |
| RNA fragmentation buffer | NEB | E6186A | |
| RNase I (100 U/μL) | Invitrogen | AM2295 | |
| RNase inhibitor (20 U/μL) | Invitrogen | AM2696 | |
| Silicone rubber caps | BioSpec Products | 2008 | |
| ssDNA ligase (100 U/μL) | Epicentre | CL9021K | Supplied with 10X CircLigase II buffer and 50 mM MnCl2 |
| Stainless steel microvials, 1.8 mL | BioSpec Products | 2007 | |
| Sucrose | RPI | S24060-5000.0 | |
| SW-41 Ti rotor | Beckman Coulter | 331362 | |
| Syringe pump | New Era Pump Systems | NE-300 | |
| T4 polynucleotide kinase (10,000 U/mL) | NEB | M0201S | Supplied with 10X T4 polynucleotide kinase buffer |
| T4 RNA ligase 2 truncated KQ (200,000 U/mL) | NEB | M0373S | Supplied with 10X T4 RNA ligase buffer and 50% PEG8000 |
| Thermal cycler | Bio-Rad | 1851148 | |
| Thinwall polyallomer tubes, 13.2 mL | Beckman Coulter | 331372 | |
| Triton X-100 | Sigma Aldrich | X100-100ML | |
| UV monitor | Bio-Rad | 7318160 | |
| Saccharomyces cerevisiae strain BY4741 | Open Biosystems | YSC1048 |
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Citer Cet Article
Beaupere, C., Chen, R. B., Pelosi, W., Labunskyy, V. M. Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling. J. Vis. Exp. (130), e56820, doi:10.3791/56820 (2017).