Summary

mRNA从植物原生质体捕获

Published: July 28, 2017
doi:

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( 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?…

Divulgations

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

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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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