Summary

MS2亲和力纯化与革兰阳性细菌中的RNA测序相结合

Published: February 23, 2021
doi:

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

1. 缓冲区和媒体 对于 MAPS 实验,请准备以下缓冲区和媒体:- 缓冲区A(150毫克,20米三轮车-HCl pH 8,1毫米M毫克2 和1米DTT)- 缓冲E(250毫克,20米三轮车-HCl pH 8,12米麦芽糖,0.1%特里顿,1毫克2 和1毫克DTT)- RNA加载缓冲区(0.025%二甲苯氰醇和0.025%溴酚蓝色在8M尿素)- 脑心输液 (BHI) 介质 (12.5 g 小牛脑, 10 克肽, 5 克牛肉心脏, 5 克 NaCl, 2.5 克纳…

Representative Results

具有代表性的结果源于对金黄色葡萄球菌29的RSAC靶点的研究。RsaC 是一种非常规的 1,116 nt 长 sRNA。其 5′ 端包含多个重复区域,而其 3′ 端 (544 nt) 在结构上是独立的,并包含与其 mRNA 目标的所有预测交互站点。当锰 (Mn) 稀缺时,就会诱导这种 sRNA 的表达,这通常在宿主免疫反应的背景下遇到。使用 MAPS 技术,我们识别了几个 mRNA 直接与 RsaC 相互作用,揭示了它在氧化?…

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。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?…

Disclosures

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

References

  1. 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).
  2. 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).
  3. Desgranges, E., Marzi, S., Moreau, K., Romby, P., Caldelari, I. Noncoding RNA. Microbiology Spectrum. 7 (2), (2019).
  4. 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).
  5. Pain, A., et al. An assessment of bacterial small RNA target prediction programs. RNA Biology. 12 (5), 509-513 (2015).
  6. 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).
  7. Wright, P. R., et al. CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains. Nucleic Acids Research. 42, 119-123 (2014).
  8. 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).
  9. 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).
  10. 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).
  11. Melamed, S., et al. Global Mapping of Small RNA-Target Interactions in Bacteria. Molecular Cell. 63 (5), 884-897 (2016).
  12. Iosub, I. A., et al. Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation. Elife. 9, (2020).
  13. 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).
  14. Dos Santos, R. F., Arraiano, C. M., Andrade, J. M. New molecular interactions broaden the functions of the RNA chaperone Hfq. Current Genetics. , (2019).
  15. 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).
  16. Bohn, C., Rigoulay, C., Bouloc, P. No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus. BMC Microbiology. 7, 10 (2007).
  17. Jousselin, A., Metzinger, L., Felden, B. On the facultative requirement of the bacterial RNA chaperone, Hfq. Trends in Microbiology. 17 (9), 399-405 (2009).
  18. Olejniczak, M., Storz, G. ProQ/FinO-domain proteins: another ubiquitous family of RNA matchmakers. Molecular Microbiology. 104 (6), 905-915 (2017).
  19. 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).
  20. Lalaouna, D., Prevost, K., Eyraud, A., Masse, E. Identification of unknown RNA partners using MAPS. Methods. 117, 28-34 (2017).
  21. 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).
  22. 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).
  23. 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).
  24. 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).
  25. 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).
  26. 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).
  27. 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).
  28. Bronesky, D., et al. A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus. The EMBO Journal. 38 (6), (2019).
  29. 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).
  30. 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).
  31. Garibyan, L., Avashia, N. Polymerase chain reaction. Journal of Investigative Dermatology. 133 (3), 1-4 (2013).
  32. Revie, D., Smith, D. W., Yee, T. W. Kinetic analysis for optimization of DNA ligation reactions. Nucleic Acids Research. 16 (21), 10301-10321 (1988).
  33. Seidman, C. E., Struhl, K., Sheen, J., Jessen, T. Introduction of plasmid DNA into cells. Current Protocols in Molecular Biology. , (2001).
  34. 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).
  35. 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).
  36. Krumlauf, R. Northern blot analysis. Methods in Molecular Biology. 58, 113-128 (1996).
  37. Koontz, L. Agarose gel electrophoresis. Methods in Enzymology. 529, 35-45 (2013).
  38. Afgan, E., et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Reseaerch. 44, 3-10 (2016).
  39. 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).
  40. 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).
  41. 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).

Play Video

Cite This Article
Mercier, N., Prévost, K., Massé, E., Romby, P., Caldelari, I., Lalaouna, D. MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (168), e61731, doi:10.3791/61731 (2021).

View Video