Studying RNA-Binding Protein Interactions with Target mRNAs in Eukaryotic Cells: Native Ribonucleoprotein Immunoprecipitation (RIP) Assays

Abstract

Post-transcriptional regulation of mRNA can potently dictate protein expression patterns in eukaryotic cells. This mode of regulation occurs through cis-acting regulatory regions in the mRNA transcript that mediate direct interactions with trans-acting RNA-binding proteins (RBPs). This mRNA/protein interaction can be studied in numerous ways that range from in vitro to in vivo through messenger ribonucleoprotein immunoprecipitation (mRNP-IP or RIP) assays. This modified immunoprecipitation approach is an important and sensitive method to determine the regulation of gene expression by specific RBPs under different cellular stressors.

1. Introduction

Immunoprecipitation (IP) assays have been a cornerstone of molecular biology research. Identifying the interaction of proteins with other proteins (co-IP assay) and with chromatin (chromatin IP or ChIP) has been critical for the understanding of interactions that regulate gene expression. Herein, we describe a specific and sensitive assay to capture and profile specific mRNA targets that are directly regulated by RNA-binding proteins (RBPs). The utility of this technique has been widely demonstrated in the literature and has been recently extended to the study of mRNA/protein interactions in tumor xenografts in vivo [1].

This chapter module focuses specifically on ribonucleoprotein immunoprecipitation (RNP-IP or RIP) assays done to determine mRNA/protein interactions (Fig. 1). However, it is noteworthy to mention that other methodologies have been developed to map the precise binding sites of a specific RBP in the transcriptome (for more information and an assay protocol, please refer to ref. 1), including a methodology known as Photoactivatable Ribonucleoside-Enhanced Cross-linking and IP (PAR-CLIP) assay, which complements RIP analysis in elucidating RNP regulatory interactions [2, 3].

Fig. 1.

(a) Stepwise schematic of the RIP assay. RBP-bound transcripts can be identified by RNA-seq or analyzed by RT-qPCR using specific probes. (b) Validation of cell lysates and post-IP samples from HuR RIP by Western blot analysis to detect HuR, α-tubulin, and lamin A/C. (c) RT-qPCR analysis of RBP-bound mRNAs. An established mRNA target is typically used as a positive control, while non-target mRNAs serve as negative controls

RIP analysis allows for a sensitive, fast, and robust profiling of mRNA/protein interactions [4]. In addition, this technique can be effectively employed to compare and profile the regulons of a specific RBP upon different cellular stressors (e.g., hypoxia, DNA damage, nutrient deprivation). These assays have been widely used by cancer researchers to determine acute and potent changes in gene expression mediated by RBPs [5, 6].

In brief, this technique along with other molecular biology protocols can determine endogenous targets of RNA-binding proteins (e.g., HuR, TTP, AUF1) and may also be used to determine the significance of these targets when a cancer cell is under a specific stress. First, using RIP analysis, one can evaluate the impact of a stress (e.g., a chemotherapeutic agent or hypoxia) upon the ability of an RBP (e.g., HuR) to associate with a target mRNA (Fig. 1). Second, the RIP assay can be used to study novel and established targets, by studying the RNA using sequencing approaches (RNA-seq), microarray analysis, and conventional quantitative (real-time) RT-PCR. The influence of the RBP upon the target mRNA can then be evaluated by silencing or ectopically overexpressing the RBP, and the consequences of RBP manipulation upon the target can be studied by assessing target mRNA stability and translation efficacy. Ultimately, the results must be validated by scoring of cancer-specific clinical specimens via immunohistochemistry to identify associations between a specific RBP and the protein of interest encoded by the target mRNA.

2. Materials

1. Protein A or G Sepharose beads (Table 1) (see Note 1).

2. Antibody of choice. Commercial vendors (e.g., MLB International) have RIP-grade, validated antibodies.

3. Normal IgG isotype control.

4. 1× phosphate-buffered saline (PBS).

5. Non-enzymatic cell dissociation agent (e.g., Cellstripper by Corning).

6. Dithiothreitol (DTT, 0.1 M).

7. Ethylenediaminetetraacetic acid (EDTA, 0.5 M, pH 8.0).

8. DNaseI (RNase-free, 2 units/μl).

9. RNase inhibitor (20 units/μl).

10. Proteinase K (20 mg/ml).

11. Acid phenol:chloroform.

12. Sodium acetate (NaOAc, 3 M, pH 5.5).

13. 100 % ethanol, molecular biology grade.

14. 70 % ethanol in sterile, nuclease-free water.

15. Sodium dodecyl sulfate (SDS, 20 %) in sterile, nuclease-free water.

16. Glycogen coprecipitant (alternatively, yeast tRNA may be used).

17. Sterile, nuclease-free water.

18. Digitonin detergent (4 mg/ml prepared in 100 % ethanol).

19. Tabletop microcentrifuge.

20. Microcentrifuge tubes (1.5-ml capacity).

21. Rotary shaker.

Table 1.

Relative affinity of immobilized proteins A and G for various antibody species and subclasses of polyclonal and monoclonal IgGs

Species	Protein A	Protein G
Monoclonal
Human
IgG1	++++	++++
IgG2	++++	++++
Mouse
IgG1	+	++++
IgG2a	++++	++++
IgG2b	+++	+++
Rat
IgG1	−−−−	+
IgG2a	−−−−	++++
IgG2b	−−−−	++
IgG2c	+	++
Polyclonal
Rabbit	++++	+++
Goat	-	++
Rat	+/−	++
Mouse	++	++
Human IgG	++++	++++

−−−− (weak or no binding), ++++ (strong binding)

Adapted from: http://www.kpl.com/docs/techdocs/purifigg.pdf

2.1. Solutions

1. All solutions should be prepared in sterile, nuclease-free water.

2. NT2 buffer: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 0.05 % Nonidet P-40 (or IGEPAL-CA630).

3. 100× protease inhibitor cocktail: 5 mg phenylmethanesulfonyl fluoride (PMSF), 100 μg aprotinin, 100 μg leupeptin, 100 μg pepstatin. Bring up to 1 ml in 100 % ethanol, aliquot, and store at −20 °C.

4. Homemade cell lysis buffer: 10 mM Tris–HCl pH 7.5, 100 mM NaCl, 2.5 mM MgCl₂, 40 μg/ml digitonin, 20 units/ml RNase inhibitor, 1× protease inhibitor cocktail (see Note 2).

5. Immunoprecipitation buffer: Prepare the following per sample. 700 μl of NT2 buffer, 10 μl of 0.1 M DTT, 2.5 μl of RNase inhibitor (20 units/μl), and 33 μl of 0.5 M EDTA (pH 8.0).

6. Proteinase K master mix: Prepare the following per sample. 100 μl of NT2 buffer, 5 μl of proteinase K (20 mg/ml), 0.5 μl of 20 % SDS, 1 μl of RNase inhibitor (20 units/μl).

7. Glycogen master mix: Prepare the following per sample. 25 μl sodium acetate (NaOAc, 3 M, pH 5.5), 625 μl 70 % ethanol, 3 μl Glycogen.

3. Methods

All steps should be performed on ice. Cells should be 50–70 % confluent at the time of harvest. NT2 buffer should be freshly made each day.

3.1. Preparation of Antibody-Bound Beads

1. Thoroughly mix the vial of beads to ensure proper mixing.

2. Transfer 100 μl of bead solution to a sterile, nuclease-free 1.5-ml centrifuge tube.

3. Centrifuge the bead solution at 1,200 × g, at 4 °C, for 1 min. Discard supernatant.

4. Add 200 μl of NT2 buffer to the centrifuge tube.

5. Centrifuge the bead solution at 1,200 × g, at 4 °C, for 1 min. Discard supernatant.

6. Repeat steps 4 and 5 two additional times for a total of three washes with NT2 buffer.

7. Add 320 μl of NT2 buffer to the centrifuge tube.

8. Add 30 μg of antibody of choice, or isotype control IgG, to the centrifuge tube.

9. Rotate the centrifuge tube in a rotary shaker, end over end overnight, at 4 °C.

10. Centrifuge the bead/antibody mixture at 5,000 × g, at 4 °C, for 5 min. Discard supernatant.

11. Add 1 ml of NT2 buffer to the centrifuge tube.

12. Centrifuge the bead/antibody mixture at 5,000 × g, at 4 °C, for 5 min. Discard supernatant.

13. Repeat steps 11 and 12 one additional time.

14. Leave the bead/antibody mixture on ice until step 1 of Subheading 3.5.

3.2. Harvesting of Cells

1. Aspirate media from cell culture (see Note 3).

2. Wash cells with PBS.

3. Collect cells using Cellstripper or equivalent non-enzymatic cell dissociation reagent.

4. Centrifuge the cells at 400 × g, at 4 °C, for 5 min. Discard supernatant.

5. Resuspend cell pellet in 500 μl of PBS.

6. Centrifuge the cells at 400 × g, at 4 °C, for 5 min. Discard supernatant.

7. Repeat steps 5 and 6 two additional times for a total of three washes in PBS.

3.3. Preparation of Cytoplasmic Protein Lysate and DNase I Treatment

1. Prepare cytoplasmic protein lysate. If using a commercial kit, follow manufacturer’s protocols with the following modification: Add RNase inhibitor to lysis buffer at final concentration of 20 units/ml. If using homemade cell lysis buffer, resuspend cell pellet in 200 μl of lysis buffer and leave sample on ice for 2 min. Centrifuge the samples at 2,000 × g, at 4 °C, for 8 min, and collect supernatant.

2. To each sample, add 30 units of DNase I and 30 units of RNase inhibitor.

3. Incubate the samples at 37 °C for 10 min, with periodic mixing (via gentle tapping of the tube).

4. Take a 10–20 μl aliquot of the cytoplasmic protein lysate. Save this sample for Western blot analysis.

3.4. Clearing of Cytoplasmic Protein Lysate

1. Repeat steps 1–6 of Subheading 3.1 as written, with the exception of using 50 μl of bead solution instead of 100 μl.

2. Add the cytoplasmic protein lysate to the washed beads.

3. Rotate the centrifuge tube in a rotary shaker, end over end for 30 min, at 4 °C.

4. Centrifuge the sample at 16,000 × g, at 4 °C, for 5 min. Collect supernatant. This is the precleared lysate.

3.5. Immunoprecipitation

1. Add 750 μl of immunoprecipitation buffer to the bead/antibody mixture (from Subheading 3.1). Add the precleared lysate.

2. Rotate the centrifuge tube in a rotary shaker, end over end for 2 h, at 4 °C (see Note 4).

3. Centrifuge the sample at 5,000 × g, at 4 °C, for 2 min. Discard supernatant.

4. Add 1 ml of NT2 buffer to the centrifuge tube.

5. Centrifuge the sample at 5,000 × g, at 4 °C, for 2 min. Discard supernatant.

6. Repeat steps 4 and 5 four additional times, for a total of five washes with NT2 buffer.

7. Take a 10–20 μl aliquot of the beads. Save this sample for Western blot analysis. This is the IP sample (see Note 5).

3.6. Proteinase K Treatment

1. Add 100 μl of proteinase K master mix to each IP sample.

2. Incubate the sample at 55 °C for 1 h, with periodic mixing (via gentle tapping of the tube).

3. Centrifuge the sample at 5,000 × g, at 4 °C, for 5 min. Collect supernatant (~100 μl) into a new 1.5-ml centrifuge tube.

4. Resuspend the remaining bead mixture (in the old centrifuge tube) with 200 μl of NT2 buffer.

5. Centrifuge the old centrifuge tube at 5,000 × g, at 4 °C, for 2 min. Collect supernatant (~200 μl), and transfer to the new centrifuge tube from step 3.

3.7. Phenol Extraction

1. Add 300 μl of acid phenol:chloroform to the centrifuge tube. Vortex vigorously for 1 min. The sample should have a uniform, opaque, white color (see Note 6).

2. Centrifuge the sample at 16,000 × g, at room temperature, for 1 min. Collect ~250 μl (125 μl × 2) of the top aqueous phase into a new 1.5-ml centrifuge tube. To the above collected aqueous phase, add Glycogen master mix.

3. Mix the sample by inversion.

4. Incubate the sample at −20 °C overnight.

5. Centrifuge the sample at 16,000 × g, at 4 °C, for 30 min. RNA should precipitate as a blue pellet. Carefully discard supernatant.

6. Resuspend the RNA pellet in 1 ml of 70 % ethanol.

7. Centrifuge the sample at 16,000 × g, at 4 °C, for 30 min. RNA should precipitate as a blue pellet. Carefully discard supernatant.

8. Invert the centrifuge tube over a paper towel for 5 min to air-dry the RNA pellet.

9. Resuspend the RNA in 20 μl of nuclease-free water. Smaller volumes can be used if expecting low yield.

10. Measure the concentration and purity of the RNA sample (see Note 7).

11. Store RNA at −80 °C.

4. Notes

1. Magnetic beads can be used if desired. The appropriate bead selection for each antibody type is shown in Table 1.

2. The cell lysis buffer recipe is optimized for cytoplasmic lysate extraction. This mode of extraction does not compromise the cellular nuclei, which can be lysed following centrifugation (see detailed protocol Subheading 3.3). Commercially available kits for nuclear–cytoplasmic fractionation are usually comprised of mild hypotonic buffers similar to the one in this protocol. We have found that commercial kits are more effective for cytoplasmic extraction by minimizing nuclear contamination into the cytoplasmic extracts. For example, RIP-grade kits for preparation of cytoplasmic proteins include NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) and CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich).

3. Regarding the starting cell number and confluence before the RIP, we have found that a 150-mm plate of cells at 50–70 % confluence is generally optimal because it ensures that there are enough protein and RNA cargo for the IP step.

4. This step can go on longer, if desired up to 12–16 hr.

5. Validation of the IP efficiencies by Western blotting is critical. Mix the cytoplasmic protein lysate (obtained in Subheading 3.3) and the IP sample (obtained in Subheading 3.5) with the appropriate amount of loading buffer, boil the samples for 5 min, and perform Western blotting to confirm that the IP reaction indeed enriched the RBP of interest in the beads. Additionally, in a successful validation of a cytoplasmic protein extraction, the cytoplasmic lysate should stain positive for the RBP of choice and α-tubulin but negative for lamin A/C. For Western blot validation of RBP of interest, it is important to use antibodies of a different species than that used in the IP reaction in order to avoid excessive background signal from the heavy and light immunoglobulin chains (e.g., if rabbit antibody was used for IP, use mouse antibody for Western blotting).

6. The bottle of acid phenol:chloroform has two layers of liquid. Make sure to take 300 μl from the bottom layer.

7. For best results, it is recommended that the extracted RNA be analyzed in a Bio Analyzer to check for quality and purity before cDNA synthesis, RNA-seq, or microarray hybridization.