RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)
Summary
在这里,我们提出一个协议,以丰富内源RNA结合位点或”足迹”的RNA:蛋白质(RNP)复合物从哺乳动物细胞。这种方法涉及RNP亚单位的两个免疫沉淀,因此被称为RNA免疫沉淀串联(RIPiT)。
Abstract
RNA免疫沉淀串联(RIPiT)是一种在RNA:蛋白(RNP)复合物内丰富一对蛋白质的RNA足迹的方法。RIPiT 采用两个纯化步骤。首先,标记的RNP亚单位的免疫沉淀后是轻度RNase消化和随后的非变性亲和洗脱。另一个RNP亚单位的第二次免疫沉淀允许扩充定义的复合物。在RNA和蛋白质变性后,RNA足迹被转换成高通量的DNA测序库。与更流行的紫外线 (UV) 交联后免疫沉淀 (CLIP) 方法以丰富 RBP 结合位点不同,RIPiT 是紫外线交联独立。因此,RIPiT可应用于RNA相互作用中存在的众多蛋白质,这些蛋白质对RNA调控至关重要,但不直接接触RNA或紫外线交联,与RNA接触不良。RIPiT 中的两个纯化步骤提供了另一个优势,用于识别感兴趣的蛋白质与另一个共同因子合作作用的结合位点。双重纯化策略也有助于通过限制背景来增强信号。在这里,我们提供了一个逐步的过程来执行RIPiT,并从分离的RNA足迹生成高通量测序库。我们还概述了 RIPiT 的优势和应用,并讨论了它的一些局限性。
Introduction
在细胞内,RNA与蛋白质复合体存在,形成RNA:蛋白质复合物(RNPs)。RNPs围绕RNA结合蛋白(RBPs,那些直接结合RNA的蛋白质)进行组装,但也包括非限制性核糖核酸(那些结合RPS但非RNA的),并且在性质上通常是动态的。RP 及其辅助因素在 RnP 中共同发挥作用,以执行监管职能。例如,在无意义介导的mRNA衰变(NMD)通路中,UPF蛋白(UPF1、UPF2和UPF3b)识别过早终止的核糖体。每个UPF蛋白都可以与RNA结合,但只有当它们聚集在一起时,活性NMD复合物才会开始形成。在这个复合体中,UPF1通过非RBP SMG1进一步通过磷酸化激活,这种UPF1激活最终导致mRNA衰变诱导因子1的招募,2。在此示例中,RBP 需要非 RBP 辅助因素来招募和激活触发 NMD 的 RNP 复合物。然而,RnPs的另一个属性是它们的组成异质性。考虑存在于不同E、A、B或C复合物中的拼接体。不同的拼接体复合物具有重叠和不同的蛋白质3。为了研究RNP功能,必须阐明哪些RNA被RBP及其相关蛋白质所束缚。有许多方法可以实现它,每种方法都有其明显的优点和缺点4,5,6,7。
广泛流行的方法来识别RBP结合位点 – 交联后免疫沉淀(CLIP)及其各种变异 – 依靠紫外线(UV)光将RBP与RNA8交联。然而,对于RNP中不直接接触RNA的非RRNA,这不是一种有效的方法。在这里,我们描述了一种适用于RSP和非限制性商业惯例的替代方法,用于分离和识别其RNA结合位点。这种方法称为RNA免疫沉淀串联(RIPiT)由两个免疫沉淀步骤组成,与单个纯化相比,这有助于实现更高的特异性(图1)9,10。与 CLIP 相比,单个免疫沉淀 (IP) 步骤可以以较低的严格性执行,因此 RIPiT 不依赖于在免疫沉淀期间能够承受强洗涤剂存在的抗体的可用性。RIPiT最独特的优势是能够以两个不同的纯化步骤靶向两种不同的蛋白质;这提供了一个强大的方法来丰富一个成分独特的RNP复合物从其他类似的复合物11。
对 RIPiT 程序的微小变化可以进一步增强 RNP 富集性。例如,RNA-蛋白质或蛋白质-蛋白质-蛋白质在RNPs内的一些相互作用是暂时的,可能难以有效地纯化这种复合物的足迹。为了稳定这种相互作用,在细胞裂解和RIPiT之前,RNPs可以在细胞内与甲醛交联。例如,我们观察到,外子结复合体 (EJC) 核心因子 EIF4AIII 和 EJC 拆解因子之间的弱相互作用,PYM12可以通过甲醛处理来稳定,以便更多 RNA 足迹得到丰富(数据不如图所示)。在细胞收获和RIPiT之前,细胞也可以用药物治疗,以稳定或丰富特定状态的RNP。例如,在研究在翻译过程中从mRNA中去除的蛋白质(例如,EJC 13,UPF114)时,使用翻译抑制剂(如紫霉素、环霉素或哈林托宁)进行治疗可导致增加的占用率。RNA上的蛋白质。
从RIPiT中恢复的RNA量通常较低(0.5-10 pmoles,即10-250 ng RNA,考虑到平均RNA长度为75 nt)。主要原因是,只有一小部分特定蛋白质与RNPs内的其他蛋白质复合存在(第一步中任何”自由”蛋白IP在第二个IP期间丢失)。为了从这个RNA生成RNA-Seq库,我们在此还概述了以前发布的协议,适用于这种低RNA输入15,16(图2),在3中产生高通量测序就绪样品。天。
Protocol
Representative Results
Discussion
在这里,我们将讨论一些成功执行 RIPiT 的关键注意事项。首先,必须优化单个 IP,以便在每个步骤中实现尽可能高的效率。此处描述的输入细胞的FLAG agarose 珠子的量已被证明对于我们测试过的各种蛋白质是可靠的。由于只有一小部分伙伴蛋白与 FLAG 蛋白共同免疫,因此高效第二 IP 所需的抗体量通常较低(小于 10 μg)。小规模的RIPiT(从一个10厘米的板)然后西方印版验证蛋白质在每个部分在两个免疫沉淀步…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH赠款GM120209(GS)的支持。作者感谢OSUCCC基因组共享资源核心的服务(CCC支持赠款NCI P30 CA16058)。
Materials
| Anti-FLAG Affinity Gel | Sigma | A2220 | |
| ATP, [γ-32P]- 3000Ci/mmol 10mCi/ml EasyTide, 250µCi | PerkinElmer | BLU502A250UC | |
| BD Disposable Syringes with Luer-Lok Tips (200) | Fisher | 14-823-435 | |
| Betaine 5M | Sigma | B0300 | |
| biotin-dATP | TriLink | N-5002 | |
| biotin-dCTP | Perkin Elmer | NEL540001EA | |
| Branson Sonifier, Model SSE-1 | Branson | ||
| CircLigase I | VWR | 76081-606 | ssDNA ligase I |
| DMEM, High Glucose | ThermoFisher | 11995-065 | |
| DNA load buffer NEB | NEB | ||
| Dynabeads Protein A | LifeTech | 10002D | |
| Flp-In-T-REx 293 Cell Line | ThermoFisher | R78007 | |
| GeneRuler Low Range DNA Ladder | ThermoScientific | FERSM1203 | |
| Hygromycin B | ThermoFisher | 10687010 | |
| Mini-PROTEAN TBE Gel 10 well | Bio-Rad | 4565013 | |
| Mini-PROTEAN TBE-Urea Gel | Bio-Rad | 4566033 | |
| miRCAT-33 adapter 5′-TGGAATTCTCGGGTGCCAAGGddC-3′ | Any | this protocol is only compatible with the Illumina sequencing platform | |
| Mirus transIT-X2 transfection reagent | Mirus | MIR 6004 | |
| Mth RNA ligase | NEB | E2610S | |
| PE1.0 5′-AATGATACGGCGACCACCGAGATCTACACT CTTTCCCTACACGACGCTCTTCCGATC*T-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| PE2.0 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTC GGCATTCCTGCTGAACCGCTCTTCCGATC*T-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| Phenol/Chloroform/Isoamyl Alcohol (25:24:1, pH 6.7, 100ml) | Fisher | BP1752I-100 | |
| Purple Gel Loading Dye (6x) | NEB | NEB #7025 | |
| Q5 DNA Polymerase | NEB | M0491S/L | |
| RNase I, E. coli, 1000 units | Eppicenter | N6901K | |
| SPIN-X column | Corning | CLS8160-24EA | |
| Streptavidin beads | ThermoFisher | 60210 | |
| Superscript III (SSIII) | ThermoScientific | 18080044 | reverse transcriptase enzyme |
| SybrGold | ThermoFisher | S11494 | gold nucleic acid gel stain |
| T4 Polynucleotide Kinase-2500U | NEB | M0201L | |
| T4RNL2 Tr. K227Q | NEB | M0351S | |
| Tetracycline | Sigma | 87128 | |
| Thermostable 5´ App DNA/RNA Ligase | NEB | M0319S | |
| TruSeq_SE1 5′-pGGCACTANNNNNAGATCGGAAGA GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE10 5′-pGGTGTTCNNNNNAGATCGGAAG AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE11 5′-pGGTAAGTNNNNNAGATCGGAA GAGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE12 5′-pGGAGATGNNNNNAGATCGGAAGA GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE2 5′-pGGGTAGCNNNNNAGATCGGAAGAG CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE35′-pGGTCGATNNNNNAGATCGGAAG AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCT CTTCCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE4 5′-pGGCCTCGNNNNNAGATCGGAAGA GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE5 5′-pGGTGACANNNNNAGATCGGAAGA GCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTC TTCCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE6 5′-pGGTAGACNNNNNAGATCGGAAGAG CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTTC CGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE7 5′-pGGGCCCTNNNNNAGATCGGAAG AGCGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCT TCCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE8 5′-pGGATCGGNNNNNAGATCGGAAGAG CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTT CCGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| TruSeq_SE9 5′-pGGACTGANNNNNAGATCGGAAGAG CGTCGTGTAGGGAAAGAGTGT-SPACER 18-CTCGGCATTCCTGCTGAACCGCTCTTC CGATCTCCTTGGCACCCGAGAATTCCA-3′ |
Any | this protocol is only compatible with the Illumina sequencing platform | |
| Typhoon 5 Bimolecular Imager | GE Healthcare Life Science | 29187191 |
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