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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....
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 .......
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.......
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.......
The authors declare no competing financial interests.
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.
....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 |
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