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Peer-reviewed

Immunoprecipitation for Protein-Protein Interactions and for RNA Enrichment in Drosophila melanogaster

黑腹果蝇中蛋白质-蛋白质相互作用和 RNA 富集的免疫沉淀

GR Giulia Romano
RK Raffaella Klima
FF Fabian Feiguin

发布: 2021年12月05日第11卷第23期 DOI: 10.21769/BioProtoc.4250 浏览次数: 2143

评审: Alexandros AlexandratosRajesh RanjanMario Ruiz

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Abstract

To determine the molecular and functional interactions between RNA-binding proteins (RBPs) and their targets RNAs, is of fundamental importance to understand the dynamic organization of the nervous system in health and disease. Nevertheless, this task has remained elusive due to the lack of specific protocols and experimental systems that would allow the combination of biochemical analysis with in vivo functional genetics. In this manuscript, we describe a trustworthy and detailed methodology to establish the molecular organization and intracellular function of RBPs/RNA multimeric complexes in a cell type-defined manner by using the powerful GAL4/UAS system for gene expression in Drosophila melanogaster.



Graphic abstract:



Immunoprecipitation for protein-RNA interaction in Drosophila.


Keywords: RNA-binding proteins (RNA结合蛋白) search TDP-43 (TDP-43) search Immunoprecipitation (免疫沉淀) search Drosophila melanogaster (黑腹果蝇) search

Background

Understanding the molecular and physical interactions between RNA-binding proteins (RBPs) and their RNA targets, especially in in vivo models, can provide important information regarding the functional organization of the eukaryotic nucleus, including RNA metabolism. In this manuscript, we concentrate our analysis on the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) due to its role in the pathogenesis of different neurodegenerative diseases (Prasad et al., 2019; Boer et al., 2021), and describe a protocol to isolate TDP-43-binding mRNA and protein complexes in a cell type (Godena et al., 2011; Romano et al., 2014 and 2020). This methodology is reproducible, relatively costless, and details the passages needed to immunoprecipitate TDP-43 from Drosophila melanogaster neurons and detect the messenger RNA molecules (mRNA), which are able to interact with its RNA-binding domain in vivo. A complementary procedure is also described to separate TDP-43/RBPs protein complexes and identify additional interactors. In addition, we indicate how the powerful genetic tools available in D. melanogaster enable the possibility to study the assembly of RBP complexes in different genetic backgrounds and/or alternative developmental stages, including adulthood and aging. This scope is achieved by utilizing the amenable GAL4/UAS system, which allows the expression of any gene of interest in D. melanogaster, making our approach distinctive and particularly efficient to isolate RBP/mRNA protein combinations in previously inaccessible compartments. In addition, this protocol can be adapted to different experimental models by simply adjusting reagents, concentrations, or proportions accordingly to the ratios described. Finally, we indicate how the material can be safely stored and successively analyzed at whatever time.

Materials and Reagents

  1. RNase-free disposable pellet pestle (Fisher Scientific, catalog number: 12-141-368)

  2. 1.5 ml tubes (Eppendorf, catalog number: 0030120086)

  3. 2.0 ml tubes (Eppendorf, catalog number: 0030120094)

  4. Glass Petri Dish 100 × 22 mm (Duroplan, catalog number: 217554804)

  5. Drosophila melanogaster strains for research are maintained and distributed from several stock centers, listed here below:

    Bloomington Drosophila Stock Center (BDSC) (https://bdsc.indiana.edu/)

    Kyoto Stock Center (DGRC) (https://kyotofly.kit.jp/cgi-bin/stocks/index.cgi)

    Vienna Drosophila Resource Centre (VDRC) (https://stockcenter.vdrc.at/control/main)

    FlyORF (https://www.flyorf.ch/)

    Fly Stocks of National Institute of Genetics (NIG-FLY) (https://shigen.nig.ac.jp/fly/nigfly/)

    Note: In Figures 1 and 2, the following strains have been used: w1118 (#3605, BDSC) – elav-GAL4/CyO (#8765, BDSC) – UAS-mCD8::GFP/CyO (#30002, BDSC) – UAS-TBPHF/L/TM3, Sb (Godena et al., 2011) – UAS-TBPH/CyO (Feiguin et al., 2009).

  6. qPCR primers [see Romano et al. (2020)]:

    Rpl11: forward 5’-ccatcggtatctatggtctgga-3’ reverse 5’-catcgtatttctgctggaacca-3’

    Dcr-2: forward 5’gcttttatgtgggtgaacaggg-3’ reverse 5’-ggctgtgccaacaagaactt-3’

    Syntaxin: forward 5’-tgttcacgcagggcatcatc-3’ reverse 5’-gccgtctgcacatagtccatag-3’

  7. Liquid nitrogen

  8. Anti-Flag M2 antibody (Sigma-Aldrich, catalog number: F3165)

  9. DynabeadsTM Protein G (Thermo Fisher, catalog number: 10003D)

  10. Complete free protease inhibitors (Roche, catalog number: 11836170001)

  11. Trizol (Invitrogen, catalog number: 15596026)

  12. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888)

  13. Potassium chloride (KCl) (Merck, catalog number: P3911)

  14. Magnesium chloride solution (Sigma-Aldrich, catalog number: M1028)

  15. Na2HPO4 (Thermo Fisher, catalog number: 10182863)

  16. KH2PO4 (Thermo Fisher, catalog number: P285-500)

  17. Tris (Thermo Fisher, catalog number: 17926)

  18. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 436143)

  19. Sodium deoxycholate (DOC) (ThermoFisher, catalog number: 89904)

  20. Urea (Sigma-Aldrich, catalog number: U5128)

  21. 1,4-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 10197777001)

  22. Bromophenol Blue (Sigma-Aldrich, catalog number: B0126)

  23. (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES (Sigma-Aldrich, catalog number: H3375)

  24. Ethylenediaminetetraacetic acid (EDTA) (Merck, catalog number: 200-449-4)

  25. Heparin (Sigma-Aldrich, catalog number: H3149)

  26. Glycerol (Sigma-Aldrich, catalog number: G5516)

  27. RNase OUT, RNAse inhibitor (Thermo Fisher, catalog number: 10777019)

  28. cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (Roche, catalog number: 11873580001)

  29. Tween 20 (Merck, catalog number: 9005-64-5)

  30. Triton X-100 (Merck, catalog number: 9002-93-1)

  31. Chloroform (CHCl3) (Merck, catalog number: 67-66-3)

  32. 2-Propanol (Merck, catalog number: 67-63-0)

  33. Ethanol (Merck, catalog number: 64-17-5)

  34. SuperScriptTM IV VILOTM Master Mix (ThermoFisher, catalog number: 11756050)

  35. iQTM-SYBR® Green Supermix (Bio-Rad, catalog number: 1708880)

  36. Ultrapure Glycogen (ThermoFisher, catalog number: 10814010)

  37. 1× PBS (see Recipes)

  38. 1× Protein-mRNA-Lysis Buffer (see Recipes)

  39. 1× Protein-Protein-Lysis Buffer (see Recipes)

  40. Bead Binding Buffer (see Recipes)

  41. 1× Washing Buffer (see Recipes)

  42. 5× Laemmli Buffer (see Recipes)

  43. 50 mg/ml Heparin stock solution (see Recipes)

Equipment

  1. Stereomicroscope (Leica, model: M1028)

  2. Glass Dounce Style Tissue Grinder 1 ml (Wheaton, model: 357538)

  3. DynaMag Spin Magnet Stand (ThermoFisher, catalog number: 12320D)

  4. Multi Rotator PTR-35 (Grant-Bio)

  5. Centrifuge 5424 R (Eppendorf)

  6. CFX Connect Real-Time PCR detection system (Bio-Rad)

  7. Tweezers Dumont n.°5 student standard tip 0.1 × 0.06 (FST, catalog number: 91150-20)

Software

  1. ImageJ (Wayne Rasband, NIH, https://imagej.nih.gov/)

  2. Prism (GraphPad, https://www.graphpad.com/scientific-software/prism/)

Procedure

English
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文章信息

版权信息

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

如何引用

Romano, G., Klima, R. and Feiguin, F. (2021). Immunoprecipitation for Protein-Protein Interactions and for RNA Enrichment in Drosophila melanogaster. Bio-protocol 11(23): e4250. DOI: 10.21769/BioProtoc.4250.

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分类

神经科学 > 神经系统疾病 > 神经退行性病变

发育生物学 > 细胞信号传导 > 能量平衡

生物化学 > RNA > RNA-蛋白质相互作用

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