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Bioquímica

mRNA从植物原生质体捕获

Published: July 28, 2017
doi:
10.3791/56011
, , ,
1Department of Biology,KU Leuven

Summary

在这里，我们提出了应用于拟南芥叶肉叶肉原生质体的相互作用捕获方案。该方法严格依赖于体内紫外线交联，并允许从生理环境中分离和鉴定植物mRNA结合蛋白。

Abstract

RNA结合蛋白（RBP）决定RNA的命运。他们参与所有的RNA生物发生途径，特别有助于信使RNA（mRNA）的转录后基因调控（PTGR）。在过去几年中，已经通过使用称为“mRNA相互作用捕获”的新方法成功地分离了许多来自酵母和哺乳动物细胞系的mRNA结合蛋白质组，其允许鉴定mRNA结合蛋白（mRBP）直接从生理环境。该方法由体内紫外（UV）交联，寡核苷酸（dT）珠的信使核糖核蛋白复合物（mRNP）的下拉和纯化以及随后通过质谱（MS）鉴定交联蛋白质组成。最近，通过应用相同的方法，已经从不同的拟南芥组织来源同时报道了几种植物mRNA结合的蛋白质组：病毒幼苗，叶组织，叶肉叶原生质体和培养根细胞。在这里，我们提出了拟南芥叶肉叶片原生质体的优化的mRNA相互作用捕获方法，这是一种细胞类型，其用作包括各种细胞测定的实验的通用工具。最佳蛋白质产量的条件包括起始组织的量和UV照射的持续时间。在从中等规模实验（10 7个细胞）获得的mRNA结合蛋白质组中，发现具有RNA结合能力的RBP被超表达，并且鉴定出许多新的RBP。实验可以放大（10 9个细胞），优化的方法可以应用于其他植物细胞类型和物种，以广泛分离，编目和比较植物中mRNA结合的蛋白质组。

Introduction

真核生物使用多个RNA生物发生调控途径来维持细胞生物学过程。在已知类型的RNA，mRNA的是非常多样的并携带的蛋白质的编码能力和它们的同种型1个 .The PTGR通路引导预的mRNA 2,3的命运。来自不同基因家族的RBPs控制RNA的调节，并且在PTGR中，特异性mRBP通过直接物理相互作用指导mRNAs，形成功能性mRNP。因此，识别和表征mRBPs及其mRNP对于了解细胞mRNA代谢的调节至关重要2。在过去三十年中，各种体外方法 – 包括RNA电泳迁移率转移（REMSA）测定法，通过指数富集测定系统进化配体（SELEX），基于文库衍生构建体，RNA Bind-n-Seq（RBNS），放射性标记或定量荧光RNA结合测定法，X-射线晶体学和NMR光谱法4，5，6，7，8，9 -已被广泛应用到限制性商业惯例的研究，主要是从哺乳动物细胞。这些哺乳动物RBP研究的结果可以通过RNA结合蛋白DataBase（RBPDB）进行搜索，收集了已发表的观察10 。

尽管这些体外方法是强大的工具，但它们确定了来自给定的RNA序列库的结合RNA基序，因此在发现新的靶RNA的能力方面受到限制。计算策略预测基因组范围的RBP也是如此，它们基于蛋白质序列和结构15的保守性。为了克服这个，一个新的实验方法哈已被确定，其允许识别感兴趣的RBP相互作用的RNA图案，以及用于确定结合的精确位置。这种称为“交联和免疫沉淀”（CLIP）的方法由体内紫外线交联，然后进行免疫沉淀11 。早期研究表明，DNA和RNA核苷酸的光激活可以发生在大于245nm的激发UV波长处。通过胸苷的反应似乎是有利的（按光反应性降低的顺序排列：dT≥dC> rU> rC，dA，dG） 12 。使用波长为254nm（UV-C）的紫外光，观察到当RNA核苷酸和蛋白质残基之间的共价键仅在几埃（Å）的范围内时产生。因此，这种现象称为RNA和RBP的“零长度”交联。之后可以进行严格的净化程序很少背景13,14杜热。

与CLIP相辅相成的策略是将体内紫外线交联与蛋白质鉴定相结合，以描述RBP的景观。许多这样的全基因组基因结合的蛋白质组已经从酵母细胞，胚胎干细胞（ESC），并使用该新的实验方法的人细胞系（ 即，HEK293和HeLa）中分离，被称为“mRNA的相互作用组捕获” 18 19，20，21。该方法由体内紫外线交联，然后是mRNP纯化和基于MS的蛋白质组学。通过应用这种策略，已经发现了许多含有非典型RBD的新型“月光”RBP，并且已经清楚的是，更多的蛋白质具有比以前所想象的RNA结合能力“> 15，16，17，使用这种方法允许新的应用程序和调查限制性商业惯例时，回答新的生物学问题的能力。例如，最近的一项研究调查了mRNA的结合蛋白质（核心RBP的保护蛋白质组）酵母和人类细胞之间22 。

已经发现植物限制性商业惯例参与生长和发育（ 例如 ，在开花时间的转录后调节，昼夜节律钟，并在线粒体和叶绿体基因表达）的24，25，26，27，28，29 。此外，他们被认为在响应非生物胁迫的细胞过程中进行功能（ 例如，感冒，干旱，salinity，和脱落酸（ABA））31，32，33，34。基于RNA识别基序（RRM）和K同源性（KH）结构域序列基因， 拟南芥基因组中有超过200种预测的RBP基因;在水稻，大约250已注意到35,36。值得注意的是，许多预测限制性商业惯例似乎是独特的植物（ 例如，没有后生动物直向同源物，以含有RRM结构域预测拟南芥限制性商业惯例的约50％）35，这表明许多可以用作新的功能。大多数预测RBP的功能仍然没有表征23 。

通过使用来自拟南芥的幼苗，叶组织，培养的根细胞和叶肉叶原生质体的mRNA结合的蛋白质组分离基因相互作用组捕获近日有报道称38，39。这些研究表明在不久的将来系统地对植物功能性RBP进行编目的潜力很大。在这里，我们提出了从植物原生质体（ 即没有细胞壁的细胞）捕获mRNA相互作用的方案。 拟南芥叶片叶肉原生质体是叶细胞的主要类型。分离的原生质体允许UV光最佳地接近细胞。这种细胞类型可以在瞬时表达功能表征40,41蛋白质测定中。此外，原生质体已被应用到其它几种植物细胞类型和物种42，43，44（ 例如，彼得森等人 ，2009;和巴格曼伯恩鲍姆，2010;和Hong 等人 ，2012）。

<p class ="“jove_content”">该方法总共包含11个步骤（ 图1A ）。首先分离拟南芥叶肉叶肉原生质体（步骤1），随后UV照射形成交联的mRNP（步骤2）。当原生质体在变性条件下裂解（步骤3）时，将交联的mRNP释放在裂解/结合缓冲液中，并通过oligo-d（T） 25珠（步骤4）下拉。经过几轮严格洗涤后，将mRNPs纯化并进一步分析。在纯化交联的mRNA之前，通过蛋白酶K消化mRBPs的变性肽，并通过qRT-PCR验证RNA质量（步骤5和6）。经RNA酶处理和蛋白质浓缩（步骤7）后，蛋白质质量由SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）和银染法（步骤8）进行控制。在交联样品（CL）和非交联样品（非CL;交联样品）之间，蛋白质带图案的差异可以容易地可视化。来自不经受紫外线照射的原生质体的阴性对照样品）。蛋白质的鉴定通过基于MS的蛋白质组学获得。通过一维聚丙烯酰胺凝胶电泳（1D-PAGE）分离来自CL样品的蛋白质以除去可能的背景污染，并使用胰蛋白酶将其“凝胶内消化”成短肽，并进行纯化（步骤9）。偶联到质谱（纳米LC-MS）的纳米反相液相色谱法允许测定mRNA结合蛋白质组中的确定蛋白质的量（步骤10）。最后，使用生物信息学分析对所鉴定的mRBP进行表征和编目（步骤11）。

Protocol

拟南芥叶片叶肉原生质体分离注意：如Yoo 等人 ，2007所述， 拟南芥叶肉叶肉原生质体基本上是分离的，有几个修改40 。 植物生长 在黑暗中将大约200个拟南芥 Col-0生态型种子在4℃下在无菌水中浸泡2天以进行分层。 注意：这个种子数量足够用于一个非CL样品和一个CL样品（见步骤1.3）。 用50％?…

Representative Results

在洗涤步骤4.3中用洗涤缓冲液2（ 图1B ）观察到围绕CL样品中的珠粒的特征性晕圈。尽管尚未进行研究，但是这种现象可能通过在磁捕获过程中交联的mRNP复合物与珠聚集的干扰来解释，从而导致形成更扩散的聚集体。这表明oligo-d（T） 25珠捕获是有效的14 。 在非CL对照和CL样品中?…

Discussion

我们成功应用开发用于酵母和人类细胞的mRNA相互作用捕获，植物叶肉叶原生质体。叶肉叶细胞是植物叶片中主要类型的地面组织。该方法的主要优点是其使用体内交联从生理环境中发现蛋白质。

在这个协议中，我们主要存在许多的优化实验条件50（ 例如，原生质体的数量以作为起始原料和UV辐射的持续时间使用）。当使用至少10 7个原?…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们承认Joris Winderickx教授的实验室，该实验室提供配有常规紫外灯的紫外线交联设备。 KG由KU Leuven研究基金支持，并承认FWO授予G065713N的支持。

Materials

REAGENTS
0.8 M Mannitol Sigma M1902-500G Primary isotonic Enzyme solution &
MMg solution
2M KCl MERCK Art. 4935 Primary isotonic Enzyme solution &
W5 buffer
0.2 M MES (pH 5.7)
(4-morpholineethanesulfonic acid)		 Sigma-aldrich M2933 Primary isotonic Enzyme solution,
W5 buffer &
MMg solution,
Filtration sterilization
Cellulase R10 Yakult Pharmaceutical Industry Co., Ltd. CELLULASE
“ONOZUKA”
R-10, 10 g		 Final isotonic enzyme solution
Macerozyme R10 Yakult Pharmaceutical Industry Co., Ltd. MACEROZYME R-10, 10g Final isotonic enzyme solution
10% (w/v) BSA
(Bovine Serum Albumin)		 Sigma-aldrich A7906-100G Final isotonic enzyme solution &
Filtration sterilization
1M CaCl2 Chem-Lab NV CL00.0317.1000 Final isotonic enzyme solution,
W5 buffer &
Digestion buffer
1M NaCl Fisher Chemical S/3160/60 W5 buffer
2M MgCl2 Sigma M8266-100G MMg solution
1M LiCl
(Lithium Chloride)		 Acros 199885000 Lysis/binding buffer,
Wash buffer 1,
Wash buffer 2 &
Low salt buffer
5% (w/v) LiDS
(Lithium Dodecyl Sulphate)		 Sigma-aldrich L4632-25G Lysis/binding buffer,
Wash buffer 1 &
Filtration sterilization
1M DTT
(Dithiothreitol)		 Thermo Fisher Scientific
Wash buffer 1 &
Wash buffer 2		 307866 Lysis/binding buffer,
1M Tris-HCl (pH 7.5) (Tris(hydroxymethyl)aminomethane, Hydrochloric acid S.G. (HCl)) Acros &
Fisher Chemical		 167620010 &
H/1200/PB15		 Lysis/binding buffer,
Wash buffer 1,
Wash buffer 2,
Low salt buffer &
Elution buffer
0.5 M EDTA (pH 8.0) (Ethylenediaminetetraacetic acid) Sigma-aldrich ED-500G Lysis/binding buffer,
Wash buffer 1,
Wash buffer 2,
Low salt buffer &
Elution buffer
Tween 20 MERCK 8.22184.0500 Regeneration of oligo-d(T)25 beads
0.1 M NaOH VWR PROLABO CHEMICALS 28244.295 Regeneration of oligo-d(T)25 beads
1X PBS (pH 7.4)
(Phosphate Buffered Saline)
containing
(NaCl; KCl; Na2HPO4; KH2PO4)		 Fisher Chemical, MERCK,
Sigma-aldrich & SAFC		 S/3160/60,
Art. 4935,
71640-250G &
60230		 Regeneration of oligo-d(T)25 beads
Proteinase K solution (2 μg/μL) Thermo Fisher Scientific 11789020 Protein digestion
Loading dye Invitrogen LC5925 SDS-PAGE
qPCR master mix Promega A6001 qRT-PCR assay
RNase Cocktail Thermo Fisher Scientific AM2286 RNA digestion
Methanol Sigma-aldrich 322415 Gel fixation and gel destaining
Acetic acid Sigma-aldrich 537020 Gel fixation and gel destaining
Coomassie Brilliant Blue R-250 Thermo Fisher Scientific 20278 Gel staining
1M NH4HCO3
(Ammonium bicarbonate)
 		 Sigma-aldrich 09830-500G Gel hydration &
Digestion buffer
CH3CN
(Acetonitrile)		 Sigma-aldrich 34851-100ML Gel dehydration &
Peptide dissolving solution
IAA
(Iodoacetic acid)		 Sigma-aldrich I4386-10G Alkylating agent
TFA
(Trifluoroacetic acid)		 Sigma-aldrich 302031-10X1ML Peptide dissolving solution
FA
(Formic acid)		 Sigma-aldrich 06554-5G Peptide extraction
Trypsin solution (6 ng/μL) Promega V5280 Digestion buffer
Name Company Catalog Number Comments
EQUIPMENT
Soil Peltracom LP2D Plant growth
Vermiculite 3 Sibli AS 05VERMICULIET Plant growth
Petri dish (150 x 20 mm) Sarstedt 82.1184.500 Carrier for protoplast suspension
0.22 μm filter Millipore SE2M229104 Homogenization of final isotonic enzyme solution
Razorblade Agar Scientific T585 Rosette leaf strips
35-75 μm nylon mesh SEFAR NITEX 74010 Protoplast suspension filtration
50 mL round bottom tubes Sigma-aldrich T1918-10EA Carrier for protoplast suspension
Hemocytometer
(Bürker hemocytometer)		 MARIENFELD 650030 Protoplast cell counting
UV crosslinking apparatus
(HL-2000 HybriLinker)		 UVP, LLC UVP95003101 in vivo UV crosslinking
UV lamp
(Sankyo-Denki G8T5)		 SANKYO
DENKI		 SD G8T5 in vivo UV crosslinking
50 mL glass syringe FORTUNA Optima Z314560 Homogenization of protoplast lysate
Narrow needle (0.9 x 25 mm) Becton Dickinson microlance 3 2021-04 Homogenization of protoplast lysate
Rotator Model L26 Labinco BV 26110912 Sample incubation by rotating
Oligo-d(T)25 magnetic beads
(5 mg/mL)		 New England BioLabs S1419S mRNPs and mRNAs binding and pull-down
Magnetic rack Invitrogen CS15000 mRNPs and mRNAs binding and pull-down
Centrifugal filter units
(Amicon Ultra-4 centrifugal filter units)		 EMD Millipore UFC800308 mRBP concentration
Pierce Silver Stain Kit Thermo Fisher Scientific 24612 Silver-staining assay
RNA purification kit
(InviTrap Spin Plant RNA Mini Kit)		 STRATEC Molecular 1064100300 RNA purification
Spectrophotometer device (NanoDrop 1000 Spectrophotometer) Thermo Fisher Scientific ND-1000 RNA quality and quantity
Real-Time PCR cycler
(StepOne Real-Time PCR cycler)		 Thermo Fisher Scientific 4376600 cDNA quantification
µ-C18 columns
(Millipore Zip Tip µ-C18 columns)		 Sigma-aldrich 720046-960EA Peptide purification
Mass spectrometer
(Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer)		 Thermo Fisher Scientific IQLAAEGAAPFALGMAZR Mass spectrometry-based proteomics
Liquid chromatography instrument (Ultimate 3000 ultra-high performance liquid chromatography (UHPLC) instrument) Thermo Fisher Scientific ULTIM3000RSLCNANO Mass spectrometry-based proteomics
C18 column
(Easy Spray Pepmap RSLC C18 column)		 Thermo Fisher Scientific ES800 Mass spectrometry-based proteomics
C18 precolumn
(Acclaim Pepmap 100 C18 precolumn)		 Thermo Fisher Scientific 160321 Mass spectrometry-based proteomics
Name Company Catalog Number Comments
Primers for qRT-PCR assay Sequences
UBQ10 mRNA
(Li et al., 2014)		 Fw: AACTTTGGTGGTTTGTGTTTTGG
Rv: TCGACTTGTCATTAGAAAGAAAGAGATAA
18S rRNA
(Durut et al., 2014)		 Fw: CGTAGTTGAACCTTGGGATG
Rv: CACGACCCGGCCAATTA
DOWNLOAD MATERIALS LIST

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Tags

Keywords: MRNA InteractomeMRNA-bound ProteomePlant ProtoplastsPost-transcriptional Gene RegulationUV CrosslinkingArabidopsis ThalianaLeaf Mesophyll ProtoplastsMMG SolutionCentrifugation
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Zhang, Z., Boonen, K., Li, M., Geuten, K. mRNA Interactome Capture from Plant Protoplasts. J. Vis. Exp. (125), e56011, doi:10.3791/56011 (2017).
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