Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Summary

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.

Abstract

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.

Introduction

RNA-binding proteins (RBPs) represent about 10% of S. cerevisiae proteins^1,2 and about 15% of mammalian proteins^3-5. 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 degradation^6-8. Thus, the distinct set of RBPs bound....

Protocol

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-PCR^20,23. 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 inducible promoter (methionine depletion) should also be introduced into the cells²⁴. An identical strain, excluding the introduced MS12 lo....

Results

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.......

Discussion

Various methods use the isolation of specific mRNAs to identify their associated proteins^{11,34 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 complexes^36,37. An effective approach of this type was presented recently, which enabled the identification of novel proteins that bind a regulatory RNA motif¹⁸. A dr.......

Materials

List of materials used in this article

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
1x Phosphate-Buffered saline (PBS)
0.2 M NaOH
4x 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