MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合
Summary
MAPS 技术已开发出来，以仔细审查体内特定监管 RNA 的目标。感兴趣的 sRNA 标有 MS2 贴录器，通过 RNA 测序实现其 RNA 合作伙伴的共同纯化及其标识。此修改后的协议特别适合革兰氏阳性细菌。
Abstract
虽然小的调节RNA（SRNA）在生命的细菌领域很普遍，但由于难以确定其mRNA目标，其中许多RNA的功能仍然很差。在这里，我们描述了MS2亲和力纯化与RNA测序（MAPS）技术的修改协议，旨在揭示特定sRNA在体内的所有RNA合作伙伴。从广义上讲，MS2 的贴合器与感兴趣的 sRNA 的 5′ 四肢融合在一起。然后，此构造以体内表示，允许 MS2-sRNA 与其细胞伙伴进行交互。细菌收获后，细胞被机械地解化。粗提取物被加载到以前涂有MS2蛋白的淀粉样色谱柱中，该色谱柱与麦芽糖结合蛋白融合。这能够对 MS2-sRNA 进行特定捕获并交互 RNA。洗净后，通过高通量RNA测序和随后的生物信息分析确定共同纯化RNA。以下协议已在Gram阳性人类病原体 金黄色葡萄球菌 中实施，原则上可转为任何革兰氏阳性细菌。综上所述，MAPS 技术是深入探索特定 sRNA 监管网络的有效方法，可提供其整个目标图的快照。然而，必须记住，MAPS 确定的假定目标仍需要通过补充实验方法进行验证。
Introduction
在大多数细菌基因组中，已经发现了数百种，甚至数千种小型调节RNA（SRNA），但其中绝大多数的功能仍然不寻常。总的来说，sRNA是短的非编码分子，在细菌生理学和适应波动环境1，2，3中起着主要作用。事实上，这些大分子是众多复杂监管网络的中心，影响代谢途径、应激反应，但也影响毒性和抗生素耐药性。从逻辑上讲，它们的合成是由特定的环境刺激（例如营养饥饿、氧化或膜应力）触发的。大多数 sRNA 通过短和非连续基配对在转录后级别调节多个目标 mRNA。他们通常通过与核糖体竞争翻译启动区域4来阻止翻译的启动。sRNA：mRNA复式的形成也常常导致目标mRNA通过招募特定的RNA而主动退化。
sRNA 靶点（即其目标 RNA 的整组）的定性允许识别其干预的代谢通路及其回答的潜在信号。因此，特定 sRNA 的功能通常可以从其目标中推断出。为此，已经开发了几个西里科预测工具，如因塔纳和科普拉纳5，6，7。它们特别依赖于序列互补性、配对能量和潜在交互站点的可访问性，以确定假定 sRNA 合作伙伴。但是，预测算法并不集成影响体内基础配对的所有因素，例如 RNA 陪护8的参与，这些因素有利于次优交互或双方的共同表达。由于其固有的局限性，预测工具的误报率仍然很高。大多数实验性的大规模方法是基于sRNA的共纯化：mRNA夫妇与标记RNA结合蛋白（RBP）6，9相互作用。例如，通过利格化和测序（RIL-seq）方法识别出RNA复式与RNA伴郎（如大肠杆菌10、11中的Hfq和ProQ）共同纯化。一种类似的技术称为紫外线交叉链接，利化和混合测序（CLASH）被应用到大肠杆菌12，13的RNA和Hfq相关sRNA。尽管Hfq和ProQ在多种细菌8、14、15的sRNA介介调节中的作用十分突出，但在像S.Aureus16、17、18这样的几个生物体中，基于sRNA的调节似乎与RNA无关。即使像沃特斯和同事13所证明的那样，与RNA关联的RNA复式净化是可行的，但这种情况仍然很棘手，因为RNA会引发它们的快速退化。因此，MS2-亲和力纯化加上RNA测序（MAPS）方法19，20构成了这种生物体的坚实替代品。
与上述方法不同，MAPS 使用特定的 sRNA 作为诱饵来捕获所有相互作用的RNA，因此不依赖于 RBP 的参与。整个过程在 图1中描述。简言之，sRNA 标记为 5′ 与 MS2 RNA 贴机，该贴子由 MS2 涂层蛋白特别识别。这种蛋白质与麦芽糖结合蛋白（MBP）融合，固定在淀粉酶树脂上。因此，MS2-sRNA 及其 RNA 合作伙伴保留在亲和力色谱列中。与麦芽糖分离后，使用高通量RNA测序进行联合纯化RNA识别，然后进行生物信息分析（图2）。MAPS 技术最终绘制了一张内部发生的所有潜在交互的交互映射图。
MAPS技术最初是在非致病性革兰阴性大肠杆菌21中实施的。值得注意的是，MAPS 帮助识别了一个 tRNA 衍生片段，该片段与 RyhB 和 Rybb sRNA 特别交互，并防止任何 sRNA 转录噪声在非诱导条件下调节 mRNA 目标。此后，MAPS 已成功应用于其他大肠杆菌sRNA，如 DsrA22、RprA23、CyaR23和 GcvB24（表 1）。除了确认以前已知的目标外，MAPS 还扩展了这些众所周知的 sRNA 的目标范围。最近，MAPS已经在沙门氏菌蒂菲穆里姆进行，并透露，SraL sRNA绑定到rho mRNA，编码转录终止因子25。通过这种配对，SraL 保护rho mRNA 免受由 Rho 本身触发的过早转录终止。有趣的是，该技术不限于 sRNA，可以应用于任何类型的蜂窝RNA，例如使用 tRNA 衍生的片段26和 mRNA22的 5’未翻译区域 （表 1）。
MAPS方法也已适应致病性革兰阳性细菌S.金黄色葡萄球菌19。具体来说，裂解协议已被广泛修改，以有效地打破细胞，由于比Gram阴性细菌更厚的细胞壁，并保持RNA完整性。这个经过调整的协议已经解开了RSA27、RsaI28和RSAC29的相互作用。这种方法深入了解了这些 sRNA 在细胞表面特性、葡萄糖吸收和氧化应激反应的调控机制中的关键作用。
2015年在 大肠杆菌 中制定和实施的协议最近被详细描述了30。在这里，我们提供修改后的 MAPS 协议，该协议特别适合研究革兰阳性（较厚的细胞壁）细菌中的 sRNA 监管网络，无论是非致病细菌还是致病菌（安全预防措施）。
Protocol
Representative Results
Discussion
革兰氏阳性细菌的修改协议
MAPS的初始协议是为了研究大肠杆菌20、30模型中的sRNA相互作用而开发的。在这里，我们描述了一个修改后的协议，它适用于机会性人类病原体金黄色葡萄球菌中依赖sRNA的监管网络的特征，并且肯定可转化为其他Gram阳性细菌，病原学与否。
特别注意细胞裂解步骤。法国的印刷?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了”国家重建联盟”（ANR，授予ANR-16-CE11-0007-01，RIBOSTAPH，和ANR-18-CE12-0025-04，科诺科，公关）的支持。它还在实验室交易所 NetRNA ANR-10-LABX-0036 和 ANR-17-EURE-0023（对 PR）的框架下发布，作为未来项目投资的一部分，由 ANR 管理的国家提供资金。DL得到了欧盟Horizna 2020研究和创新计划的支持，该计划是根据玛丽·斯考多斯卡-库里赠款协议第753137-萨纳雷格号。E. Massé实验室的工作得到了加拿大卫生研究所（CIHR）、加拿大自然科学和工程研究理事会（NSERC）和国家卫生研究院NIH团队赠款R01 GM092830-06A1的运营赠款的支持。
Materials
| 1.5 mL microcentrifuge tube | Sarstedt | 72.690.001 | |
| 15 mL centrifuge tubes | Falcon | 352070 | |
| 2 mL microcentrifuge tube | Starstedt | 72.691 | |
| 2100 Bioanalyzer Instrument | Agilent | G2939BA | RNA quantity and quality |
| 250 mL culture flask | Dominique Dutscher | 2515074 | Bacterial cultures |
| 50 mL centrifuge tubes | Falcon | 352051 | Culture centrifugation |
| Absolute ethanol | VWR Chemicals | 20821.321 | RNA extraction and purification |
| Allegra X-12R Centrifuge | Beckman Coulter | Bacterial pelleting | |
| Ampicilin (amp) | Sigma-Aldrich | A9518-5G | Growth medium |
| Amylose resin | New England BioLabs | E8021S | MS2-affinity purification |
| Anti-dioxigenin AP Fab fragment | Sigma Aldrich | 11093274910 | Northern blot assays |
| Autoradiography cassette | ThermoFisher Scientific | 50-212-726 | Northern blot assays |
| BamHI | ThermoFisher Scientific | ER0051 | Plasmid construction |
| BHI (Brain Heart Infusion) Broth | Sigma-Aldrich | 53286 | Growth medium |
| Blocking reagent | Sigma Aldrich | 11096176001 | Northern blot assays |
| CDP-Star | Sigma Aldrich | 11759051001 | Northern blot assays (substrate) |
| Centrifuge 5415 R | Eppendorf | RNA extraction and purification | |
| Chloroform | Dominique Dutscher | 508320-CER | RNA extraction and purification |
| DIG-RNA labelling mix | Sigma-Aldrich | 11277073910 | Northern blot assays |
| DNase I | Roche | 4716728001 | DNase treatment |
| Erythromycin (ery) | Sigma-Aldrich | Fluka 45673 | Growth medium |
| FastPrep device | MP Biomedicals | 116004500 | Mechanical lysis |
| Guanidium Thiocyanate | Sigma-Aldrich | G9277-250G | Northern blot assays |
| Hybridization Hoven Hybrigene | Techne | FHB4DD | Northern blot assays |
| Hybridization tubes | Techne | FHB16 | Northern blot assays |
| Isoamyl alcohol | Fisher Scientific | A/6960/08 | RNA extraction and purification |
| LB (Lysogeny Broth) | Sigma-Aldrich | L3022 | Growth medium |
| Lysing Matrix B Bulk | MP Biomedicals | 6540-428 | Mechanical lysis |
| MicroPulser Electroporator | BioRad | 1652100 | Plasmid construction |
| Milli-Q water device | Millipore | Z00QSV0WW | Ultrapure water |
| NanoDrop spectrophotometer | ThermoFisher Scientific | RNA/DNA quantity and quality | |
| Nitrocellulose membrane | Dominique Dutsher | 10600002 | Northern blot assays |
| Phembact Neutre | PHEM Technologies | BAC03-5-11205 | Cleaning and decontamination |
| Phenol | Carl Roth | 38.2 | RNA extraction and purification |
| Phusion High-Fidelity DNA Polymerase | New England Biolabs | M0530 | Plasmid construction |
| pMBP-MS2 | Addgene | 65104 | MS2-MBP production |
| Poly-Prep chromatography column | BioRad | 7311550 | MS2-affinity purification |
| PstI | ThermoFisher Scientific | ER0615 | Plasmid construction |
| Qubit 3 Fluorometer | Invitrogen | 15387293 | RNA quantity |
| RNAPro Solution | MP Biomedicals | 6055050 | Mechanical lysis |
| ScriptSeq Complete Kit | Illumina | BB1224 | Preparation of cDNA librairies |
| Spectrophotometer Genesys 20 | ThermoFisher Scientific | 11972278 | Bacterial cultures |
| SpeedVac Savant vacuum device | ThermoFisher Scientific | DNA120 | RNA extraction and purification |
| Stratalinker UV Crosslinker 1800 | Stratagene | 400672 | Northern blot assays |
| T4 DNA ligase | ThermoFisher Scientific | EL0014 | Plasmid construction |
| TBE (Tris-Borate-EDTA) | Euromedex | ET020-C | Northern blot assays |
| ThermalCycler T100 | BioRad | 1861096 | Plasmid construction |
| Tween 20 | Sigma Aldrich | P9416-100ML | Northern blot assays |
| X-ray film processor | hu.q | HQ-350XT | Northern blot assays |
| X-ray films Super RX-N | FujiFilm | 4741019318 | Northern blot assays |
Referências
- Carrier, M. C., Lalaouna, D., Masse, E. Broadening the Definition of Bacterial Small RNAs: Characteristics and Mechanisms of Action. Annual Review of Microbiology. 72, 141-161 (2018).
- Hör, J., Matera, G., Vogel, J., Gottesman, S., Storz, G. Trans-Acting Small RNAs and Their Effects on Gene Expression in Escherichia coli and Salmonella enterica. EcoSal Plus. 9 (1), (2020).
- Desgranges, E., Marzi, S., Moreau, K., Romby, P., Caldelari, I. Noncoding RNA. Microbiology Spectrum. 7 (2), (2019).
- Adams, P. P., Storz, G. Prevalence of small base-pairing RNAs derived from diverse genomic loci. Biochimica et Biophysica Acta – Gene Regulatory Mechanisms. 1863 (7), 194524 (2020).
- Pain, A., et al. An assessment of bacterial small RNA target prediction programs. RNA Biology. 12 (5), 509-513 (2015).
- Desgranges, E., Caldelari, I., Marzi, S., Lalaouna, D. Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs. Biochimica et Biophysica Acta – Gene Regulatory Mechanisms. , 194506 (2020).
- Wright, P. R., et al. CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains. Nucleic Acids Research. 42, 119-123 (2014).
- Smirnov, A., Schneider, C., Hor, J., Vogel, J. Discovery of new RNA classes and global RNA-binding proteins. Current Opinion in Microbiology. 39, 152-160 (2017).
- Saliba, A. E., Santos, S., Vogel, J. New RNA-seq approaches for the study of bacterial pathogens. Current Opinion in Microbiology. 35, 78-87 (2017).
- Melamed, S., Adams, P. P., Zhang, A., Zhang, H., Storz, G. RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles. Molecular Cell. 77 (2), 411-425 (2020).
- Melamed, S., et al. Global Mapping of Small RNA-Target Interactions in Bacteria. Molecular Cell. 63 (5), 884-897 (2016).
- Iosub, I. A., et al. Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation. Elife. 9, (2020).
- Waters, S. A., et al. Small RNA interactome of pathogenic E. revealed through crosslinking of RNase E. The EMBO Journal. 36 (3), 374-387 (2017).
- Dos Santos, R. F., Arraiano, C. M., Andrade, J. M. New molecular interactions broaden the functions of the RNA chaperone Hfq. Current Genetics. , (2019).
- Kavita, K., de Mets, F., Gottesman, S. New aspects of RNA-based regulation by Hfq and its partner sRNAs. Current Opinion in Microbiology. 42, 53-61 (2018).
- Bohn, C., Rigoulay, C., Bouloc, P. No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus. BMC Microbiology. 7, 10 (2007).
- Jousselin, A., Metzinger, L., Felden, B. On the facultative requirement of the bacterial RNA chaperone, Hfq. Trends in Microbiology. 17 (9), 399-405 (2009).
- Olejniczak, M., Storz, G. ProQ/FinO-domain proteins: another ubiquitous family of RNA matchmakers. Molecular Microbiology. 104 (6), 905-915 (2017).
- Lalaouna, D., Desgranges, E., Caldelari, I., Marzi, S. Chapter Sixteen – MS2-Affinity Purification Coupled With RNA Sequencing Approach in the Human Pathogen Staphylococcus aureus. Methods in Enzymology. 612, 393-411 (2018).
- Lalaouna, D., Prevost, K., Eyraud, A., Masse, E. Identification of unknown RNA partners using MAPS. Methods. 117, 28-34 (2017).
- Lalaouna, D., et al. A 3′ external transcribed spacer in a tRNA transcript acts as a sponge for small RNAs to prevent transcriptional noise. Molecular Cell. 58 (3), 393-405 (2015).
- Lalaouna, D., Morissette, A., Carrier, M. C., Masse, E. DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli. Molecular Microbiology. 98 (2), 357-369 (2015).
- Lalaouna, D., Prevost, K., Laliberte, G., Houe, V., Masse, E. Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli. Nucleic Acids Research. 46 (5), 2600-2612 (2018).
- Lalaouna, D., Eyraud, A., Devinck, A., Prevost, K., Masse, E. GcvB small RNA uses two distinct seed regions to regulate an extensive targetome. Molecular Microbiology. 111 (2), 473-486 (2019).
- Silva, I. J., et al. SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA. Proceedings of the National Academy of Sciences. 116 (8), 3042-3051 (2019).
- Lalaouna, D., Masse, E. Identification of sRNA interacting with a transcript of interest using MS2-affinity purification coupled with RNA sequencing (MAPS) technology. Genomics Data. 5, 136-138 (2015).
- Tomasini, A., et al. The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms. Nucleic Acids Research. 45 (11), 6746-6760 (2017).
- Bronesky, D., et al. A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus. The EMBO Journal. 38 (6), (2019).
- Lalaouna, D., et al. RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation. Nucleic Acids Research. 47 (1), 9871-9887 (2019).
- Carrier, M. C., Laliberte, G., Masse, E. Identification of New Bacterial Small RNA Targets Using MS2 Affinity Purification Coupled to RNA Sequencing. Methods in Molecular Biology. 1737, 77-88 (2018).
- Garibyan, L., Avashia, N. Polymerase chain reaction. Journal of Investigative Dermatology. 133 (3), 1-4 (2013).
- Revie, D., Smith, D. W., Yee, T. W. Kinetic analysis for optimization of DNA ligation reactions. Nucleic Acids Research. 16 (21), 10301-10321 (1988).
- Seidman, C. E., Struhl, K., Sheen, J., Jessen, T. Introduction of plasmid DNA into cells. Current Protocols in Molecular Biology. , (2001).
- Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., Roe, B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. Journal of Molecular Biology. 143 (2), 161-178 (1980).
- Grosser, M. R., Richardson, A. R. Method for Preparation and Electroporation of S. aureus and S. epidermidis. Methods in Molecular Biology. 1373, 51-57 (2016).
- Krumlauf, R. Northern blot analysis. Methods in Molecular Biology. 58, 113-128 (1996).
- Koontz, L. Agarose gel electrophoresis. Methods in Enzymology. 529, 35-45 (2013).
- Afgan, E., et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Reseaerch. 44, 3-10 (2016).
- Jagodnik, J., Brosse, A., Le Lam, T. N., Chiaruttini, C., Guillier, M. Mechanistic study of base-pairing small regulatory RNAs in bacteria. Methods. 117, 67-76 (2017).
- Mann, M., Wright, P. R., Backofen, R. IntaRNA 2.0: enhanced and customizable prediction of RNA-RNA interactions. Nucleic Acids Res. 45, 435-439 (2017).
- Georg, J., et al. The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs. Molecular Microbiology. 113 (3), 603-612 (2020).
