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 to a particular transcript at any time point determines its processing and ultimately its fate.

The identification of RBPs associated with an mRNA could significantly improve our understanding of processes underlying their post-transcriptional regulation. Diverse genetic, microscopic, biochemical and bioinformatics methods have been used to identify proteins involved in mRNA regulation (reviewed in^9-11). However, only a few of these methods enable the identification of proteins associated with a particular target mRNA. Of note is the Yeast Three Hybrid system (Y3H), which utilizes the mRNA of interest as bait to screen an expression library in yeast cells. Positive clones are usually observed through a growth selection or reporter expression^12-14. The key advantage of this method is the large number of proteins that can be scanned in a cellular environment and the ability to measure the strength of the RNA-protein interaction. Drawbacks include the relatively large number of false positive results due to non-specific binding, and the high potential for false negative results due, in part, to misfolding of the fusion protein prey or the bait RNA.

An alternative to the genetic approach is affinity purification of RNA with its associated proteins. Poly A-containing mRNAs can be isolated through the use of oligo dT columns, and their associated proteins are detected by mass spectrometry. The RNA-protein interaction is conserved in its cellular context by crosslinking, which makes short-range covalent bonds. The use of the oligo dT column yields a global view of the entire proteome that is associated with any poly A-containing mRNA^3,5,15. However, this does not provide a list of proteins that are associated with a particular mRNA. Very few methods are available to accomplish such an identification. The PAIR method entails the transfection of nucleic acid with complementarity to the target mRNA^16,17. The nucleic acid is also attached to a peptide, which allows crosslinking to RBPs in close vicinity to the interaction site. After crosslinking, the RBP-peptide-nucleic acid can be isolated and subjected to proteomics analysis. Recently, an aptamer-based methodology was successfully applied to extracts from mammalian cell lines¹⁸. An RNA aptamer with improved affinity to streptavidin was developed and fused to a sequence of interest (AU-rich element (ARE) in this case). The aptamer-ARE RNA was attached to streptavidin beads and mixed with cell lysate. Proteins that associated with the ARE sequence were purified and identified by mass spectrometry (MS). Although this method detected associations that occur outside the cellular settings (i.e., in vitro), it is likely to be modified in the future so as to introduce the aptamers into the genome and thus enable the isolation of proteins associated with the mRNA while in the cellular milieu (i.e., in vivo). In yeast, where genetic manipulations are well established, the RaPID method (developed in Prof. Jeff Gerst's lab) provides a view of in vivo associations¹⁹. RaPID combines the specific and strong binding of the MS2 coat protein (MS2-CP) to the MS2 RNA sequence, and of the streptavidin-binding domain (SBP) to streptavidin conjugated beads. This enables efficient purification of MS2-tagged mRNAs through streptavidin beads. Moreover, expression of 12 copies of MS2 loops allows up to six MS2-CPs to bind simultaneously to the RNA and increase the efficiency of its isolation. This protocol was therefore suggested to enable the identification of novel mRNA-associated proteins once the eluted samples are subjected to proteomics analysis by mass spectrometry.

We recently utilized RaPID to identify novel proteins associated with the yeast PMP1 mRNA²⁰. PMP1 mRNA was previously shown to be associated with the ER membrane and its 3' untranslated region (UTR) was found to be a major determinant in this association²¹. Thus, RBPs that bind PMP1 3' UTR are likely to play an important role in its localization. RaPID followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) resulted in the identification of many new proteins that interact with PMP1²⁰. Herein, we provide a detailed protocol of the RaPID methodology, important controls that need to be done, and technical tips that may improve yield and specificity.

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 loops, should be used as a control for the detection of non-specific signals.

1. RaPID Purification

1. Grow 500 ml of yeast cells (use 250-1,000 ml of yeast cells depending on the expression level of the gene of interest) expressing MS2-tagged mRNA²⁰ and MS2-CP-GFP-SBP fusion protein²⁴ to OD₆₀₀ 0.8 - 1.0 at 30 ^oC in the appropriate growth medium (e.g., Synthetic Dextrose (SD) selective medium).
2. Centrifuge cells at 3,000 x g for 4 min at RT and discard the supernatant.
3. Wash cells in 1x phosphate buffered saline (PBS), centrifuge as before (step 1.2), resuspend in an equal volume (step 1.1) of SD without methionine, and incubate for 45 - 60 min at 30 ^oC to induce expression of the MS2-CP-GFP-SBP fusion protein.
   Note: Longer induction time may cause aggregation of GFP.
4. Set aside 10 ml of cells and extract RNA from this sample using the simple "hot phenol" method²⁵. Caution: Phenol is highly toxic and should be handled according to its safety instructions.
   Note: This RNA is a good reference for the quality of isolated RNA (Figure 1A).
5. For 500 ml cells, add 1.35 ml 37% formaldehyde to a final concentration of 0.1% to crosslink the RNA-protein complexes. Continue incubation at 30 ^oC for 10 min. Caution: Formaldehyde is highly toxic and should be handled according to its safety instructions.
6. Add 26.5 ml of 2.5 M glycine to a final concentration of 0.125 M and incubate for 3 min to stop crosslinking. From this step on, put everything on ice to minimize RNA degradation.
7. Centrifuge cells at 3,000 x g for 4 min at 4^oC and wash with 45 ml cold 1x PBS.
8. Resuspend cells in 1 ml RaPID Lysis buffer per 100 ml of initial cell culture.
9. Split into aliquots of 500 µl in screw-capped microfuge tubes and add ~ 400 mg of chilled glass beads to each aliquot (roughly a full 0.2 ml PCR tube).
10. Lyse cells in a bead beater for 3 min. Immediately put the lysate on ice.
    Note: Cell lysis releases cellular RNases, which are the major cause of RNA degradation. RaPID Lysis buffer contains a high concentration of RNase inhibitors, such as the nonspecific RNase inhibitor heparin and specific inhibitors. Working quickly and keeping everything cold is also recommended.
11. Transfer the lysate to new tubes. Pierce a small hole in the bottom of each microfuge tube with a hot needle (0.8 mm x 40 mm) and place the microfuge tubes on top of the 15 ml tubes. Use the head of a 5 ml syringe as an adaptor between the microfuge tubes and the 15 ml tubes. Centrifuge the tubes assembly at 3,000 x g for 1 min at 4 ^oC.
12. Transfer the flow-through from the 15 ml tube to a new 1.5 ml tube and centrifuge at 10,000 x g for 10 min at 4 ^oC to clear cell debris.
13. Pool supernatants from all 1.5 ml tubes into a single 15 ml tube, and set aside 1/50 and 1/100 volume of the lysate for RNA and protein isolation, respectively.
    Note: These will serve as the "Input" samples for subsequent analysis (Figure 1).
14. Add 300 µg of avidin per 500 ml of initial yeast culture and incubate for 30 min (this can be reduced to 10 min) at 4 ^oC under constant rolling (10 rpm).
    Note: Avidin blocks biotin and biotinylated proteins from binding to the streptavidin beads.
15. Pre-wash the streptavidin beads before incubation with the cell lysate.
    1. Transfer about 300 µl of the streptavidin beads slurry to a 1.5 ml microcentrifuge tube and remove the upper supernatant. This will result in 250 µl of beads. Wash the beads twice with 1 ml of RaPID lysis buffer. Centrifuge the streptavidin beads at 660 x g for 2 min at 4^oC between each step.
    2. Block the beads by incubating for 1 hr with 0.5 ml of RaPID lysis buffer, 0.5 ml of 10 mg/ml BSA and 10 µl of 10 mg/ml yeast tRNA. Beads can be left O/N in blocking solution at 4^oC with rotation if necessary.
    3. Wash twice with 1 ml of RaPID Lysis buffer.
       Note: Magnetic beads can also be used to reduce time and background. These, however, have lower capacity than sepharose beads.
16. Add 250 µl of the pre-washed streptavidin beads to the avidin-containing lysate (from step 1.14) and incubate 1 hr at 4 °C with constant rotation (10 rpm).
    Note: This incubation time is a compromise between providing sufficient binding time and minimizing chances for RNA degradation.
17. Centrifuge at 660 x g for 2 min at 4 ^oC to clear the streptavidin beads and transfer the supernatant to a new 15 ml tube. Set aside 1/50 and 1/100 volume of the lysate for RNA and protein isolation, respectively.
    Note: These will be the "Unbound" samples.
18. Wash the beads with 1 ml of RaPID Lysis buffer, shake gently, transfer to a new 1.5-ml microcentrifuge tube, and centrifuge as in step 1.17. Repeat this step three times.
19. Wash the beads with 1 ml of RaPID Wash buffer, and this time rotate (10 rpm) for 5 min at 4 °C. Repeat this step twice.
20. Wash the beads with 1 ml of 1x PBS and centrifuge as above.
21. Wash the beads again with 120 µl of 1x PBS. Centrifuge as above, and take the entire supernatant for RNA and protein extraction as the "Wash" sample.
    Note: This last wash sample will indicate the stringency of the washes and should be of the same volume as the Elution sample. This will simplify processing and loading in subsequent assays.
22. To elute RNA and RNA-associated proteins from the column, add 120 µl of 6 mM biotin in 1x PBS and incubate for 45 min at 4 °C with constant rotation (10 rpm).
23. Centrifuge the beads as above and transfer the eluted material to a new 1.5 ml tube. Spin the eluted sample again and transfer the upper phase to a new 1.5 ml tube to ensure no carryover of beads.
24. Take samples for RNA or protein extraction.
    Note: The volume taken should depend on the following assay and expression level of the RNA or protein of interest. As a guideline, for northern analysis²⁶, take 80% of the sample, for Western blot^23,27 or RT-PCR²⁸, take 20%, and for mass spectrometry²⁹ to discover new proteins, take the entire sample.

2. RNA Extraction

1. Add an equal volume of 2x Crosslinking Reversal Buffer and incubate for 45 min at 65 °C. Continue directly for RNA extraction.
   Note: The Crosslinking Reversal Buffer contains ethylenediaminetetraacetic acid (EDTA), which significantly improves RNA extraction²⁰.
2. Use the standard phenol:chloroform method²⁶ to extract RNA from the "input" and "unbound" samples (steps 1.13 and 1.17).
   Note: These samples contain a large amount of the RNase inhibitor heparin, which can inhibit subsequent Reverse Transcriptase-(RT)utilizing assays (e.g., RT-PCR), therefore, LiCl precipitation³⁰ is advised to remove it. The "wash" and "elution" samples (steps 1.21 and 1.24) contain low amounts of RNA and are precipitated through the following steps.
3. Add an equal volume of 8M Guanidinium-HCl (GuHCl) and two volumes of 100% ethanol to the "wash" and "elution" samples. Incubate at -80 °C for at least 2 hr.
   Note: Guanidinium will efficiently denature proteins and thereby inhibit RNases and proteases in the sample.
4. Centrifuge at 20,000 x g, 4 °C for 30 min and discard the supernatant. Wash the pellet with cold 80% ethanol and centrifuge again at 20,000 x g, 4 °C for 15 min.
5. Dissolve the RNA pellet in 400 µl of RNase free water and precipitate again to remove any leftover GuHCl or other contaminants. Add 40 µl of 3 M sodium acetate, pH 5.2 and 800 µl of 100% ethanol, incubate at -20 °C for at least 1 hr.
6. Centrifuge and wash as in step 2.4 and remove all ethanol with a 10 µl tip. Resuspend the RNA pellet in 10 µl RNase free water. Take extra care as the RNA pellet is usually not visible.
   Note: Samples can be used for northern analysis as described in²³ (Figure 1).

3. Protein Preparation for Western Blot or Mass Spectrometry Analysis

1. Add 1/3 volume of 4x Laemmli Sample Buffer (LSB) to protein samples and incubate 45 min at 65 °C to reverse the crosslinking of RNA and proteins.
   Note: Samples can be stored at -20 °C.
2. Run proteins on a SDS polyacrylamide gel (SDS-PAGE)³¹. Adjust the percentage of the gel according to the size of proteins to be analyzed.
   Note: 10% acrylamide gel gives a wide range of separation and is good as a first approximation.
3. Transfer proteins to the membrane as in the standard immunoblot analysis²⁷ for control of isolation efficiency (Figure 1C). Stain with either Coomassie Blue R250 or silver stain before mass spectrometry analysis.
   Note: Silver stain is the preferred choice because it is more sensitive and allows better detection (Figure 1B). Staining can be done with either manually prepared solutions or commercial kits. The incubation time of the gel in the final dye solution should be determined experimentally. Long incubation may reveal low abundant proteins, but can cause highly expressed proteins such as MS2-CP-SBP-GFP to be over stained and may mask surrounding proteins in their vicinity. Additionally, a high background might occur. Follow the reaction carefully, and add Stop Solution at the appropriate time.
4. Cut out bands from the gel and transfer them to 1.5 ml tubes. Store samples at -20 °C, or send for protein extraction and trypsin digestion followed by LC-MS/MS analysis³².
5. As an alternative proteomic approach to exclude the gel separation step, digest eluted material from step 1.24 in solution using trypsin and subject to LC-MS/MS analysis³³.
   Note: Omitting the SDS-PAGE separation simplifies the protocol and increases yield. However, samples might contain traces of the detergent NP-40, which inhibits mass spectrometry analysis. To overcome this problem, perform the following steps:
   1. Run protein on SDS-PAGE³¹ for 10 - 15 min.
   2. Cut out the entire portion of the lane (i.e., where the proteins are located).
      Note: The protein sample might include a significant amount of the MS2-CP-SBP protein that may obscure signals of low abundance proteins.

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