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A New Method for Studying RNA-binding Proteins on Specific RNAs

一种研究特异性RNA结合蛋白的新方法

WS Weiping Sun *
ZZ Ziheng Zhang *
JL Ji-Long Liu
MZ Min Zhuang

 (*contributed equally to this work) 发布: 2021年05月20日第11卷第10期 DOI: 10.21769/BioProtoc.4022 浏览次数: 10806

评审: Anonymous reviewer(s)

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Abstract

Proximity-based protein labeling has been developed to identify protein-nucleic acid interactions. We have reported a novel method termed CRUIS (CRISPR-based RNA-United Interacting System), which captures RNA-protein interactions in living cells by combining the RNA-binding capacity of CRISPR/Cas13 and the proximity-tagging activity of PUP-IT. Enzymatically deactivated Cas13a (dCas13a) is fused to the proximity labeling enzyme PafA. In the presence of a guide RNA, dCas13a binds specific target RNA region, while the fused PafA mediates the labeling of biotin-tagged Pup on proximal proteins. The labeled proteins can be enriched by streptavidin pull-down and identified by mass spectrometry. Here we describe the general procedure for capturing RNA-protein interactions using this method.

Keywords: Proximity-based protein labeling (临近蛋白标记) search CRUIS (基于CRISPR的RNA联合相互作用系统) search RNA-binding protein (RNA结合蛋白) search PUP-IT (PUP-IT) search CRISPR (CRISPR) search

Background

RNA-binding proteins (RBPs) play important roles in regulating cellular processes and function. Mapping RNA-protein interactions is a key endeavor to address how proteins regulate the location and function of target RNAs. The traditional methods are mostly protein-centric methods, including RNA immunoprecipitation (RIP) and crosslinking and immunoprecipitation (CLIP). They rely on specific antibodies to immunoprecipitate RNA-protein complexes and identify the RNAs by sequencing. Although some RNA-centric methods have been developed, they focused on systematic labeling of all RNAs, rather than one specific RNA, for the purification of RNA-protein complexes (Chu et al., 2015; McHugh et al., 2015). Therefore, there is an urgent need to develop a method that can be widely used to study RNA-protein interactions of the specific RNA in living cells. Since the CRISPR-based RNA-targeting Cas nucleases have been reported, it provides us a new powerful tool to bind and cleave specific RNAs to regulate RNA function (Abudayyeh et al., 2016; Cox et al., 2017). In addition, the enzymatically deactivated Cas13a provides a new strategy for tracking the specific RNA. The proximity labeling system PUP-IT has been widely used to identify protein-protein interactions. It employs the proximity ligase PafA to mediate the ligation of a small protein (PupE) to lysines on the surrounding proteins (Liu et al., 2018). Combining PUP-IT with the RNA-targeting Cas nucleases (Figure 1), we have developed a new class of RNA-centric methods for studying RNA-protein interactions in living cells (Zhang et al., 2020).



Figure 1. The workflow of capturing RNA-protein interactions. A. The workflow for isolating HEK293T cell line stably expressing CRUIS. B. The workflow of mass spectrometry sample preparation.


Materials and Reagents

  1. Pipette tips (Filtered) (Thermo, catalog numbers: TF112-1000-Q, T104RS-Q, TF140-200-Q)

  2. 0.2 ml PCR tubes (Thermo, catalog number: 431-MIXED-Q)

  3. 1.5 ml Microtubes (AXYGEN, catalog number: MCT-150-L-C)

  4. 50 ml Tubes (Falcon, catalog number: 352098)

  5. 96-well plates (Corning, catalog number: 3599)

  6. 24-well plates (Corning, catalog number: 3524-ND)

  7. 6-well plates (Corning, catalog number: 3516)

  8. 10 cm dishes (Corning, catalog number: 430167)

  9. pC0040-LwaCas13a crRNA backbone (Addgene, catalog number: 103851)

  10. pB-CAGGS-dCas9 (Addgene, catalog number: 110823)

  11. Super PiggyBac Transposase (SBI, catalog number: PB210PA-1)

  12. DMEM (Life, catalog number: C11995500CP)

  13. FBS (Gemini, catalog number: 100-307)

  14. Penicillin-Streptomycin (Life, catalog number: 15140122)

  15. Opti-MEM (Gibco, catalog number: 31985062)

  16. PEI (Polysciences, catalog number: 23966)

  17. PBS (Life, catalog number: C20012500CP)

  18. 0.25% Trypsin-EDTA (Life, catalog number: 25200072)

  19. Biotin (SCRC, catalog number: 67000260)

  20. Triton® X-100 (Sangon Biotech, catalog number: T0694-100 ml)

  21. 1 M Tris pH 7.5, sterile (Sangon Biotech, catalog number: B548127-0500)

  22. 100× protease inhibitor cocktail (Biotool, catalog number: B14001)

  23. Urea (Sangon Biotech, catalog number: UT0907)

  24. DTT (MDBio, catalog number: D023-5G)

  25. Iodoacetamide (SIGMA, catalog number: I1149-5G)

  26. Streptavidin magnetic beads (PIERCE, catalog number: 88816)

  27. 1 M Tris pH 8.0, sterile (Sangon Biotech, catalog number: B548127-0500)

  28. NaCl (Sangon Biotech, catalog number: A501218-0001)

  29. SDS (MDBio, catalog number: S001-100)

  30. EDTA (Sangon Biotech, catalog number: E0105-500g)

  31. Ammonium bicarbonate reagent plus(R) (Sigma, catalog number: A6141-500G)

  32. RapiGest SF (Waters, catalog number: 186008090)

  33. Sequencing Grade Modified Trypsin (Promega, catalog number: V5113)

  34. Formic acid (Sigma, catalog number: 94318-250ML)

  35. Acetonitrile (Merck Chemicals, catalog number: 1.00030.4008)

  36. Trifluoroacetic acid LC-MC Ultra (Sigma, catalog number: 14264-50ML)

  37. Amino acid sequence information of CRUIS and Bio-PupE are available at https://doi.org/10.1093/nar/gkaa143

  38. ZIPTIP C18 (Millipore,catalog number: ZTC18S960)

  39. Anti-myc antibody (Cell Signaling, catalog number: 2276s)

  40. Goat anti-mouse IgG Antibody (H&L) [HRP] (GenScript, catalog number: A00160)

  41. Streptavidin-HRP (Cell Signaling, catalog number: 3999s)

  42. BSA (AMRESCO, catalog number: 0903-5G)

  43. SurePAGE (GenScript, catalog number: M00657)

  44. PVDF Western blotting membranes (Roche, catalog number: 30100400)

  45. BbsI-HF (NEB, catalog number: R3539)

  46. Lysis buffer (see Recipes)

  47. Buffer 1 (see Recipes)

  48. Buffer 2 (see Recipes)

  49. Buffer 3 (see Recipes)

  50. Buffer 4 (see Recipes)

Equipment

  1. Standard molecular biology lab equipment

  2. Eppendorf mixer (Eppendorf, catalog number: 5382000074)

  3. Magnetic separator(Bimake, catalog number: B23803)

  4. -80°C freezer (Therom, catalog number: 905)

  5. FACSAriaTM III (BD)

  6. Trans-blot turbo (Biorad, catalog number: 1704150)

  7. Chemiluminescence imaging system (GE, Amersham Imager 600)

  8. Vacuum concentrator (Thermo, SpeedVac)

  9. Spectrometer (Thermo, Orbitrap Fusion)

Procedure

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文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Sun, W., Zhang, Z., Liu, J. and Zhuang, M. (2021). A New Method for Studying RNA-binding Proteins on Specific RNAs. Bio-protocol 11(10): e4022. DOI: 10.21769/BioProtoc.4022.

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分类

分子生物学 > RNA > RNA-蛋白质相互作用

神经科学 > 细胞机理

生物化学 > 蛋白质 > 标记

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