JoVE Logo
Faculty Resource Center

Sign In





Representative Results






Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Published: September 30th, 2016



1Faculty of Biology, Technion - Israel Institute of Technology, 2Department of Biomolecular Sciences, Weizmann Institute of Science

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-binding proteins (RBPs) represent about 10% of S. cerevisiae proteins1,2 and about 15% of mammalian proteins3-5. They are implicated in many cellular processes such as mRNA post-transcriptional processing and regulation, translation, ribosome biogenesis, tRNA aminoacylation and modification, chromatin remodeling, and more. An important subgroup of RBPs is the mRNA-binding proteins (mRNPs)6,7. In the course of mRNA maturation, different RBPs bind the transcript and mediate its nuclear processing, export out of the nucleus, cellular localization, translation and degradation6-8. Thus, the distinct set of RBPs bound....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Note: Insert a sequence consisting of 12 MS2-binding sites (MS2 loops; MS2L) into the desired genomic locus, usually between the open reading frame (ORF) and the 3' UTR. A detailed protocol for this integration is provided elsewhere22. Verify proper insertion and expression by PCR, northern analysis or RT-PCR20,23. It is important to verify that the integration did not intervene with the synthesis of the 3'UTR. In addition, a plasmid-expressing MS2-CP fused to SBP under the expression of an .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

RaPID enables the isolation of a specific target RNA with its associated proteins. Critical for its success is keeping the RNA intact as much as possible, thereby obtaining a sufficient amount of proteins. To determine the isolation efficiency and quality of RNA, northern analysis is performed (Figure 1A). Northern analysis has the advantage of directly reporting the efficiency and quality of RaPID. Thus, the relative amounts of full length and degradation products can be.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Various methods use the isolation of specific mRNAs to identify their associated proteins11,34 35. These methods apply in vitro and in vivo strategies to probe RNA-protein interactions. In vitro methods incubate exogenously transcribed RNA with cell lysate to capture RBPs and isolate RNP complexes36,37. An effective approach of this type was presented recently, which enabled the identification of novel proteins that bind a regulatory RNA motif18. A dr.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We thank Prof. Jeff Gerst and Boris Slobodin for their helpful advice in setting up the RaPID protocol and providing the necessary plasmids. We also thank Dr. Avigail Atir-Lande for her help in establishing this protocol and Dr. Tamar Ziv from the Smoler Proteomics Center for her help with the LC-MS/MS analysis. We thank Prof. T.G. Kinzy (Rutgers) for the YEF3 antibody. This work was supported by grant 2011013 from the Binational Science Foundation.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Tris sigma T1503
SDS bio-lab 1981232300
DTT sigma D9779
Acidic Phenol (pH 4.3) sigma P4682
Acidic Phenol: Chloroform (5:1, pH 4.3) sigma P1944
Chloroform bio-lab 3080521
Formaldehyde Frutarom 5551820
Glycine sigma G7126
NP-40 Calbiochem 492016
Heparin Sigma H3393
Phenylmethylsulfonyl Flouride (PMSF) Sigma P7626
Leupeptin Sigma L2884
Aprotinin Sigma A1153
Soybean Trypsin Inhibitor Sigma T9003
Pepstatin Sigma P5318
DNase I Promega M610A
Ribonuclease  Inhibitor Takara 2313A
Glass Beads Sartorius BBI-8541701 0.4-0.6mm diameter 
Mini BeadBeater BioSpec Mini BeadBeater 16
Guanidinium Sigma G4505
Avidin Sigma A9275
Streptavidin Beads GE Healthcare  17-5113-01
Bovine serum albumin (BSA) Sigma A7906
Yeast tRNA Sigma R8508
Biotin Sigma B4501
Yeast extract Bacto 288620
peptone Bacto 211677
Glucose Sigma G8270
1 x Phosphate-Buffered saline (PBS)
0.2 M NaOH
4 x Laemmli Sample Buffer (LSB) 0.2 M Tris-Hcl pH 6.8, 8% SDS, 0.4 M DTT, 40% glycerol, 0.04% Bromophenol-Blue.
Hot phenol lysis buffer 10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS 
3 M Sodium Acetate pH 5.2
100% and 70% Ethanol (EtOH)
RNase-free water
RaPID lysis buffer 20 mM Tris pH 7.5, 150 mM NaCl, 1.8 mM MgCl2, 0.5% NP-40, 5 mg/ml Heparin, 1 mM Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl Flouride (PMSF), 10 µg/ml Leupeptin, 10 µg/ml Aprotinin, 10 µg/ml Soybean Trypsin Inhibitor, 10 µg/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease  Inhibitor.
2x Cross-linking reversal buffer 100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS.
RaPID wash buffer 20 mM Tris-HCl pH 7.5,  300 mM NaCl, 0.5% NP-40
0.5 M EDTA pH 8
Silver Stain Plus Kit Bio-Rad  161-0449 For detecting proteins in polyacrylamide gels
SD selective medium  1.7 g/l Yeast nitrogen base with out amino acids and ammonium sulfate, 5 g/l Ammonium sulfate, 2% glucose, 350 mg/l Threonine, 40 mg/l Methionine, 40 mg/l Adenine, 50 mg/l Lysine, 50 mg/l Tryptophan, 20 mg/l Histidine, 80 mg/l Leucine, 30 mg/l Tyrosine, 40 mg/l Arginine
Anti-eEF3 (EF3A,YEF3) Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School) 1:5,000
Anti GFP antibody Santa Cruz sc-8334 1:3,000
Anti rabbit IgG-HRP conjugated SIGMA A9169 1:10,000

  1. Hogan, D. J., Riordan, D. P., Gerber, A. P., Herschlag, D., Brown, P. O. Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system. PLoS Biol. 6, e255 (2008).
  2. Tsvetanova, N. G., Klass, D. M., Salzman, J., Brown, P. O. Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae. PloS One. 5, (2010).
  3. Castello, A., et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell. 149, 1393-1406 (2012).
  4. Gerstberger, S., Hafner, M., Tuschl, T. A census of human RNA-binding proteins. Nat Rev Genet. 15, 829-845 (2014).
  5. Baltz, A. G., et al. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol Cell. 46, 674-690 (2012).
  6. Mitchell, S. F., Parker, R. Principles and properties of eukaryotic mRNPs. Mol Cell. 54, 547-558 (2014).
  7. Licatalosi, D. D., Darnell, R. B. RNA processing and its regulation: global insights into biological networks. Nat Rev Genet. 11, 75-87 (2010).
  8. Eliyahu, E., Lesnik, C., Arava, Y. The protein chaperone Ssa1 affects mRNA localization to the mitochondria. FEBS Lett. 586, 64-69 (2012).
  9. Ascano, M., Gerstberger, S., Tuschl, T. Multi-disciplinary methods to define RNA-protein interactions and regulatory networks. Curr Opin Genet Dev. 23, 20-28 (2013).
  10. Denman, R. B. mRNPs take shape by CLIPPING and PAIRING. BioEssays. 28, 1132-1143 (2006).
  11. McHugh, C. A., Russell, P., Guttman, M. Methods for comprehensive experimental identification of RNA-protein interactions. Genome Biol. 15, 203 (2014).
  12. Bernstein, D. S., Buter, N., Stumpf, C., Wickens, M. Analyzing mRNA-protein complexes using a yeast three-hybrid system. Methods. 26, 123-141 (2002).
  13. SenGupta, D. J., et al. A three-hybrid system to detect RNA-protein interactions in vivo. Proc Natl Acad Sci USA. 93, 8496-8501 (1996).
  14. Yosefzon, Y., et al. Divergent RNA binding specificity of yeast Puf2p. RNA. 17, 1479-1488 (2011).
  15. Mitchell, S. F., Jain, S., She, M., Parker, R. Global analysis of yeast mRNPs. Nat Struct Mol Biol. 20, 127-133 (2013).
  16. Zielinski, J., et al. In vivo identification of ribonucleoprotein-RNA interactions. Proc Natl Acad Sci USA. 103, 1557-1562 (2006).
  17. Bell, T. J., Eberwine, J. Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation. Methods Mol Biol. 1324, 457-468 (2015).
  18. Leppek, K., Stoecklin, G. An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins. Nucleic Acids Res. 42, e13 (2014).
  19. Slobodin, B., Gerst, J. E. A novel mRNA affinity purification technique for the identification of interacting proteins and transcripts in ribonucleoprotein complexes. RNA. 16, 2277-2290 (2010).
  20. Samra, N., Atir-Lande, A., Pnueli, L., Arava, Y. The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner. BMC Mol Biol. 16, 17 (2015).
  21. Loya, A., et al. The 3'-UTR mediates the cellular localization of an mRNA encoding a short plasma membrane protein. RNA. 14, 1352-1365 (2008).
  22. Haim-Vilmovsky, L., Gadir, N., Herbst, R. H., Gerst, J. E. A genomic integration method for the simultaneous visualization of endogenous mRNAs and their translation products in living yeast. RNA. 17, 2249-2255 (2011).
  23. Eldad, N., Yosefzon, Y., Arava, Y. Identification and characterization of extensive intra-molecular associations between 3'-UTRs and their ORFs. Nucleic Acids Res. 36, 6728-6738 (2008).
  24. Slobodin, B., Gerst, J. E. RaPID: an aptamer-based mRNA affinity purification technique for the identification of RNA and protein factors present in ribonucleoprotein complexes. Methods Mol Biol. 714, 387-406 (2011).
  25. Schmitt, M. E., Brown, T. A., Trumpower, B. L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 18, 3091-3092 (1990).
  26. Eldad, N., Arava, Y. A ribosomal density-mapping procedure to explore ribosome positions along translating mRNAs. Methods Mol Biol. 419, 231-242 (2008).
  27. Arava, Y., Seger, R., Fbeta Walker, M. D. GRFbeta, a novel regulator of calcium signaling, is expressed in pancreatic beta cells and brain. J Biol Chem. 274, 24449-24452 (1999).
  28. Arava, Y., Adamsky, K., Ezerzer, C., Ablamunits, V., Walker, M. D. Specific gene expression in pancreatic beta-cells: cloning and characterization of differentially expressed genes. Diabetes. 48, 552-556 (1999).
  29. Bavli-Kertselli, I., Melamed, D., Bar-Ziv, L., Volf, H., Arava, Y. Overexpression of eukaryotic initiation factor 5 rescues the translational defect of tpk1w in a manner that necessitates a novel phosphorylation site. FEBS J. 282, 504-520 (2015).
  30. Eliyahu, E., Melamed, D., Arava, Y. Genome-wide analysis of RNA extracted from isolated mitochondria. Methods Mol Biol. 714, 287-299 (2011).
  31. Brunelle, J. L., Green, R. One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE). Methods Enzymol. 541, 151-159 (2014).
  32. Gundry, R. L., et al. Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Current protocols in molecular biology. Chapter 10, Unit 10.25 (2009).
  33. Medzihradszky, K. F. In-solution digestion of proteins for mass spectrometry. Methods Enzymol. 405, 50-65 (2005).
  34. Bachler, M., Schroeder, R., von Ahsen, U. StreptoTag: a novel method for the isolation of RNA-binding proteins. RNA. 5, 1509-1516 (1999).
  35. Oeffinger, M. Two steps forward--one step back: advances in affinity purification mass spectrometry of macromolecular complexes. Proteomics. 12, 1591-1608 (2012).
  36. Ross, A. F., Oleynikov, Y., Kislauskis, E. H., Taneja, K. L., Singer, R. H. Characterization of a beta-actin mRNA zipcode-binding protein. Mol Cell Biol. 17, 2158-2165 (1997).
  37. Deshler, J. O., Highett, M. I., Schnapp, B. J. Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum. Science. 276, 1128-1131 (1997).
  38. Bertrand, E., et al. Localization of ASH1 mRNA particles in living yeast. Mol Cell. 2, 437-445 (1998).
  39. Aizer, A., et al. Quantifying mRNA targeting to P-bodies in living human cells reveals their dual role in mRNA decay and storage. J Cell Sci. 127, 4443-4456 (2014).
  40. Gadir, N., Haim-Vilmovsky, L., Kraut-Cohen, J., Gerst, J. E. Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae. RNA. 17, 1551-1565 (2011).
  41. Lopez de Heredia, ., M, R. P., Jansen, RNA integrity as a quality indicator during the first steps of RNP purifications : a comparison of yeast lysis methods. BMC Biochem. 5, 14 (2004).
  42. Lee, J. S., Kallehauge, T. B., Pedersen, L. E., Kildegaard, H. F. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Sci Rep. 5, 8572 (2015).
  43. Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P., Del Bene, F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res. 24, 142-153 (2014).
  44. Oda, Y., Huang, K., Cross, F. R., Cowburn, D., Chait, B. T. Accurate quantitation of protein expression and site-specific phosphorylation. Proc Natl Acad Sci USA. 96, 6591-6596 (1999).
  45. de Godoy, L. M. SILAC yeast: from labeling to comprehensive proteome quantification. Methods Mol Biol. 1156, 81-109 (2014).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved