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In vitro Reconstitution Assay of miRNA Biogenesis by Arabidopsis DCL1

拟南芥DCL1催化miRNA合成的体外实验

Tian Wang Tian Wang
Claudia Castillo-González Claudia Castillo-González
LY Lan You
RL Rui Li
LW Liwei Wen
Hongliang Zhu Hongliang Zhu
Xiuren Zhang Xiuren Zhang

发布: 2015年04月20日第5卷第8期 DOI: 10.21769/BioProtoc.1454 浏览次数: 11052

评审: Feng LiAnonymous reviewer(s)

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Sep 2013

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Abstract

microRNAs (miRNAs) are small non-coding RNAs, regulating most if not all, biological processes in eukaryotic organisms. miRNAs are initially processed from primary transcripts (pri-miRNAs) to produce miRNA precursors (pre-miRNAs), that are further processed into miRNA and its complementary strands (miRNA/*). In Arabidopsis, and possibly other plants, the processing from pri-miRNAs to pre-miRNAs and from pre-miRNAs to miRNA/* are both implemented through Dicer-like 1 (DCL1) complexes. Recently, we demonstrated isolation of DCL1 complexes of unprecedented quality from in planta. We further successfully reconstituted DCL1 cleavage assays in vitro that were able to fully recapitulate in vivo miRNA biogenesis. Here we provide a detailed protocol of DCL1 reconstitution assays. The protocol comprises three major parts (Figure 1): 1) Preparation of pri- and pre-miRNA transcripts (Procedures A-C); 2) Purification of the recombinant Arabidopsis DCL1 machinery from Nicotiana benthamiana (N. benthamiana) through immunoprecipitation (IP) (Procedures D and E); and 3) in vitro processing of radioisotope-labeled pri- or pre-miRNAs using the isolated DCL1 complexes (Procedure F). It is our desire that the protocol be a powerful tool for the RNAi community to study mechanistic issues or to develop RNA silencing technologies.

Keywords: Microprocessor (微处理器) search Pri-miRNA (miRNA初级转录产物) search MiRNA (miRNA) search Dicer (Dicer) search Arabidopsis (拟南芥) search

Materials and Reagents

  1. Five to six-week old plants of N. benthamiana
  2. Agrobacterium strain: ABI (Zhang et al., 2006)
  3. Plasmid [pBA-2Flag-4Myc-DCL1, pBA-6Myc-HYL1, pBA-6Myc-SE. Detailed approach could refer to the Online Methods from Zhu et al. (2013). Negative control plasmid: pBA]
  4. G-Tube® snap cap siliconized microcentrifuge tubes (VWR International, catalog number: 22179-004 )
  5. Anti-FLAG® M2 magnetic beads (Sigma-Aldrich, catalog number: M8823 )
  6. 3x Flag peptide (NH2-MDYKDHDGDYKDHDIDYKDDDDK-COOH) (Sigma-Aldrich, catalog number: F4799 )
  7. DNase (Promega Corporation, catalog number: M6101 )
  8. 3,000 Ci/mmol 10 mCi/ml [γ-32P]-ATP (PerkinElmer, catalog number: BLU502A100UC )
  9. SuperaseIn RNase inhibitor (Ambion, catalog number: N8080119 )
  10. T7 RNA polymerase (Ambion, catalog number: 18033019 )
  11. Calf intestine alkaline phosphatase (NEB, catalog number: M0290S )
  12. Phenol: chloroform: isoamyl alcohol (25:24:1) (Life Technologies, InvitrogenTM, catalog number: 15593049 )
  13. NaAc (Sigma-Aldrich, catalog number: S7670 )
  14. 5 M ammonium acetate (Ambion, catalog: AM9071 )
  15. GlycoBlue (Ambion, catalog number: AM9516 )
  16. Decade marker (Ambion, catalog number: AM7778 )
  17. Anti-c-Myc Agarose Affinity Gel (Sigma-Aldrich, catalog number: A7470 )
  18. EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 05892953001 )
  19. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P2714 )
  20. HEPES (Sigma-Aldrich, catalog number: H3375 )
  21. Spermidine (Sigma-Aldrich, catalog number: S2626 )
  22. DTT (DL-Dithiothreitol) (Sigma-Aldrich, catalog number: 43817 )
  23. MgCl2 (Sigma-Aldrich, catalog number: M8266 )
  24. NTP (Thermo scientific, catalog number: R0481 )
  25. Deionized formamide (Ambion, catalog number: AM9342 )
  26. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  27. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
  28. EDTA (Sigma-Aldrich, catalog number: V900106 )
  29. SDS (Sigma-Aldrich, catalog number: L3771 )
  30. DEPC (Sigma-Aldrich, catalog number: V900882 )
  31. KCl (Sigma-Aldrich, catalog number: V900068 )
  32. Tris-HCl (Sigma-Aldrich, catalog number: V900312 )
  33. Triton X-100 (Sigma-Aldrich, catalog number: V900502 )
  34. PMSF (Sigma-Aldrich, catalog number: 78830 )
  35. NaCl (Sigma-Aldrich, catalog number: V900058 )
  36. ATP (Thermo scientific, catalog number: R1441 )
  37. GTP (Thermo scientific, catalog number: R1461 )
  38. Glycerol (Sigma-Aldrich, catalog number: V900122 )
  39. 0.1 M Glycine-HCl (Sigma-Aldrich, catalog number: 55097-5ML-F )
  40. 5x transcription buffer (see Recipes)
  41. RNA loading buffer (see Recipes)
  42. RNA elution buffer (see Recipes)
  43. DEPC H2O (see Recipes)
  44. RNA dissolve buffer (see Recipes)
  45. Chloroform: isoamyl alcohol (24:1) (see Recipes)
  46. IP buffer (see Recipes)
  47. Protease inhibitor cocktail stock (see Recipes)
  48. TBS buffer (see Recipes)
  49. 3x Flag elution buffer stock (4 mg/ml) (see Recipes)
  50. Washing buffer (see Recipes)
  51. Assay buffer (see Recipes)
  52. Fixing buffer (see Recipes)

Equipment

  1. Compact UV lamp (UVP, model: UVGL-25 )
  2. Fluor-Coated TLC Plate (10 x 10 cm) (Ambion, catalog number: AM10110 )
  3. Geiger counter (Medcom, model: CRM-100 )
  4. Vertical electrophoresis system (Whatman, model: V15.17 )
  5. Thermomixer C (Eppendorf, model: 5382000023 )
  6. Gel dryer (Bio-Rad Laboratories, model: 583 )
  7. Gel analyzer Quantity One (Bio-Rad Laboratories, version: 4.6.9 )
  8. DynaMagTM-2 (Life technologies, model: 12321D )
  9. Rugged Rotator (Glas-Col, model: 099A MR1512 )
  10. PolyATtract® System 1000 Magnetic Separation Stand (Promega Corporation, model: Z541A )
  11. Phospho imager (PharosFXTM plus system)

Procedure

English
中文翻译

文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Wang, T., Castillo-González, C., You, L., Li, R., Wen, L., Zhu, H. and Zhang, X. (2015). In vitro Reconstitution Assay of miRNA Biogenesis by Arabidopsis DCL1. Bio-protocol 5(8): e1454. DOI: 10.21769/BioProtoc.1454.

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分类

植物科学 > 植物分子生物学 > RNA > RNA 干扰

植物科学 > 植物生物化学 > 蛋白质 > 分离和纯化

分子生物学 > RNA > RNA 干扰

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