Quantitative analysis of protein-RNA interactions in fission yeast

Protocol Quantitative analysis of protein-RNA interactions in fission yeast Characterizing the interactions between RNAs and proteins in vivo is key to better understand how organisms regulate gene expression. Here we describe a robust and quantitative protocol to measure specific RNA-protein interactions in a native context using RNA immunoprecipitation (RIP). We provide a comprehensive experimental framework to detect cotranslational interactions and detail the quantitative analysis of purified RNAs by PCR and high-throughput sequencing. Although we developed the protocol in fission yeast, it can be readily implemented in other yeast species. Alberto Elı ´as- Villalobos, Caia Duncan, Juan Mata, Dominique Helmlinger [email protected] (A.E.-V.) [email protected] (D.H.) Highlights Simple and robust detection of RNA- protein interactions in anativecontext Ribonucleoprotein complex isolated by immunoprecipitation Quantitative analysis of purified RNA by PCR or highthroughput sequencing Detailed experimental pipeline to demonstrate cotranslational interactions Elı ´as-Villalobos et al., STAR Protocols 3, 101373 June 17, 2022 ª2022 The Author(s). https://doi.org/10.1016/ j.xpro.2022.101373 ll OPEN ACCESS Protocol Quantitative analysis of protein-RNA interactions in fission yeast Alberto Elı ´as-Villalobos, 1,2,3,5, *Caia Duncan, 4,5 Juan Mata, 4 and Dominique Helmlinger 1,6, * 1 CRBM, University of Montpellier, CNRS, Montpellier, France 2 Instituto de Biomedicina de Sevilla (IBiS), Universidad de Sevilla-Hospital Universitario Virgen del Rocı ´o-CSIC, Seville, Spain 3 Departamento de Gene ´tica, Universidad de Sevilla, Seville, Spain 4 Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK 5 Technical contact 6 Lead contact *Correspondence: [email protected] (A.E.-V.), [email protected]r (D.H.) https://doi.org/10.1016/j.xpro.2022.101373 SUMMARY Characterizing the interactions between RNAs and proteins in vivo is key to better understand how organisms regulate gene expression. Here, we describe a robust and quantitative protocol to measure specific RNA-protein interactions in a native context using RNA immunoprecipitation (RIP). We provide a comprehensive experimental framework to detect cotranslational interactions and detail the quantitative analysis of purified RNAs by PCR and high-throughput sequencing. Although we developed the protocol in fission yeast, it can be readily implemented in other yeast species. For complete details on the use and execution of this protocol, please refer to Toullec et al. (2021). BEFORE YOU BEGIN Timing: 30 min to 2 h 1. Make sure you have all reagents for buffer preparation, additives, and commercial kits, including: a. Per sample, 100 mL of Pan mouse magnetic beads and 3 mL(3mg) of your selected mouse monoclonal antibody. Note: This protocol has been set up using Pan mouse magnetic beads and anti-influenza virus hemagglutinin (anti-HA), anti-human c-Myc protein (anti-MYC) or anti-FLAG peptide (anti-FLAG) mouse monoclonal antibodies. Other antibodies should work but may require optimization by adjusting antibody and extract quantities. b. 50 mg/mL heparin stock solution. c. 40 units of RNase inhibitor per sample. d. 4.4 units of Turbo DNase per sample. e. Acid-washed glass beads to be added up to the meniscus of the lysed extract (typically, 500 mLto1mLpersample). f. 1.5 mL tubes. g. 2 mL screw-cap tubes. h. 5 mL tubes. i. 50 mL tubes. j. Needle (25G). STAR Protocols 3, 101373, June 17, 2022 ª2022 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 ll OPEN ACCESS k. Bunsen burner. l. PureLink TM RNA Mini Kit (or equivalent). m. RNase free water. n. Ethanol. o. Coomassie reagent for protein quantification. p. Spectrophotometer. q. Heat block at 70C. r. EDTA-free protease inhibitors cocktail, Pepstatin A, Bestatin, PMSF, (optional) phosphatase inhibitors. s. Centrifuge at 4C. t. If assessing co-translational interactions, prepare stock solutions of puromycin (50 mg/mL) and GTP (200 mM). You can also use EDTA or ATG-mutated strains. Refer to Figures 1 and 2for details about the effect of each perturbation on translation. Note: The PureLink TM MicroKitcanalsobeusedfortheextractionoftheIPRNA(seespecific instructions below). Note: For further information about the mechanism of action and the use of puromycin see Aviner (2020). 2. Prepare all buffers and reagents, such as stock solutions of the protease inhibitors Bestatin (1 mg/mL; 1,0003), Pepstatin A (1 mg mL; 1,0003) and PMSF (100 mM; 1003). Keep all buffers at 4C, except when containing detergents, such as Nonidet P-40 (NP40), in which case keep at ambient temperature. 3. 3–4 days before starting, streak your strains on appropriate media and let them grow at 32C. Verify your markers. We recommend the use of epitope tagged strains. Note: This protocol uses Schizosaccharomyces pombe as an experimental system. Adjust this step according to the growth conditions of your favorite organism. Note: To increase the reproducibility of the procedure, we recommend to always keep similar amounts of inoculated biomass from plates and harvest the same number of yeast cells between samples and experiments. Figure 1. Types of protein-RNA interactions detected by this RIP protocol (A) Direct interaction between the protein bait tagged with an epitope (HA in this example) and an RNA. (B) Indirect interaction between an epitope-tagged bait and its target RNAs through an RNA-binding protein (RBP). (C) Cotranslational interaction between an epitope-tagged protein bait and a nascent polypeptide detected through indirect binding to the translated mRNA. ll OPEN ACCESS 2STAR Protocols 3, 101373, June 17, 2022 Protocol Figure 2. Experimental strategies to test whether protein-mRNA interactions detected by RIP are dependent on translation (A) Cotranslational interaction between a protein bait and an mRNA in a wild-type strain in the absence of any treatment (control condition). (B) Impact of mutating the ATG of the targeted mRNA on the co-translational binding of the bait. (C) Effect of puromycin addition on translation and on the detection of cotranslational interactions. Puromycin, which partly resembles the 30end of an amino-acylated tRNA, enters the A site of the ribosome and transfer to the growing polypeptide, leading to the formation of a puromycylated chain and causing premature chain release. (D) Effect of EDTA on ribosome integrity and on the detection of cotranslational interactions. EDTA, which is added in vitro to the extraction buffers used in the protocol, causes dissociation of the small and large ribosomal subunits. ll OPEN ACCESS STAR Protocols 3, 101373, June 17, 2022 3 Protocol KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Myc antibody, clone 9E11 Abcam Cat#ab56; RRID: AB_304976 Mouse monoclonal anti-FLAG antibody, clone M2 Sigma Cat#F1804; RRID: AB_262044 Mouse monoclonal anti-HA antibody, clone 16B12 BioLegend Cat#901514; RRID: AB_2565336 Chemicals, peptides, and recombinant proteins Bestatin Sigma Cat#B8385 Pepstatin A Sigma Cat#P5318 Phenylmethanesulfonyl fluoride (PMSF) Sigma Cat#P7626 b-Glycerol phosphate disodium salt hydrate Sigma Cat#G9422 Sodium orthovanadate (Na 3 VO 4 ) Sigma Cat#450243 Sodium fluoride (NaF) Sigma Cat#201154 Dynabeads Pan-Mouse IgG Thermo Fisher Scientific Cat#11041 Promega Recombinant RNasinRibonuclease Inhibitor Thermo Fisher Scientific Cat#PR-N2511 Igepal CA-630 Sigma Cat#56741 Heparin sodium salt from porcine intestinal mucosa Sigma Cat#H3393 cOmplete EDTA-free cocktails tablets Roche Cat#04693132001 Puromycin hydrochloride (optional) Santa Cruz Biotechnology Cat#sc-108071B Adenine sulphate Formedium Cat#DOC0229, Uracil Formedium Cat#DOC0213, L-Lysine Formedium Cat#DOC0160 L-Histidine monohydrochloride monohydrate Sigma Cat#H8125 L-Leucine Formedium Cat#DOC0156 Bacto yeast extract Thermo Fisher Scientific-Gibco Cat# 212750 D-(+)-Glucose Sigma Cat# G7021 Tris base EUROMEDEX Cat#26-128-3094-B Hydrochloric acid (HCl) Sigma Cat#320331 Potassium chloride (KCl) Sigma Cat#P9541 Magnesium chloride (MgCl2) Sigma Cat# M8266 Glycerol Sigma Cat#G6279 Igepal CA630 Sigma Cat#I8896 Bromophenol blue sodium salt Sigma Cat#B5525 Polyoxyethylene ether W-1 (for qPCR master mix) Sigma Cat#P7516 Bovine Serum Albumin (BSA) (for qPCR master mix) Sigma Cat#A4378 dATP (for qPCR master mix) Thermo Fisher Scientific-Invitrogen Cat#10216018 dCTP (for qPCR master mix) Thermo Fisher Scientific-Invitrogen Cat#10217016 dGTP (for qPCR master mix) Thermo Fisher Scientific-Invitrogen Cat#10218014 dTTP (for qPCR master mix) Thermo Fisher Scientific-Invitrogen Cat#10219012 Potassium chloride (KCl) (for qPCR master mix) Merck-Sigma-Supelco Cat#1.04936.1000 Magnesium chloride (MgCl 2 ) (for qPCR master mix) Merck-Sigma-Supelco Cat#1058331000 SYBR Green (for qPCR master mix) FMC BioProducts / Cambrex Cat#50513 Glycerol (for qPCR master mix) Thermo Fisher Scientific-MP Biomedicals Cat#ICN800687 2-Amino-2-methyl-1,3-propanediol for qPCR master mix Sigma Cat#A9074 Hydrochloric acid fuming 37% for qPCR master mix Sigma-Merck Cat#1003171000 Platinum Taq DNA polymerase for qPCR master mix Thermo Fisher Scientific-Invitrogen Cat#10966-034 Critical commercial assays SuperScript III First-Strand System Thermo Fisher Scientific Cat#18080051 Purelink RNA Mini kit Thermo Fisher Scientific Cat#12183018A Purelink RNA Micro Scale kit Thermo Fisher Scientific Cat#12183016 TURBO DNA-free kit Invitrogen Cat#AM1907 Experimental models: Organisms/strains Schizosaccharomyces pombe strains See Table S4 at Toullec et al., 2021. Derivatives from h- 972 strain (ATCC 24843) (Continued on next page) ll OPEN ACCESS 4STAR Protocols 3, 101373, June 17, 2022 Protocol Note: Any other chemical or media additive were bought from Sigma-Merk or Formedium, respectively. MATERIALS AND EQUIPMENT After preparing the media, autoclave it for 10 min at 113C and let it cool down to room temperature before using it. To prepare solid media, add 2% w/v of bacto agar before autoclaving. CRITICAL: NP40 (or its alternative Igepal CA-630) is highly toxic through oral, skin, or eye contact. Manipulate with extreme care, using gloves and protection glasses and following the instructions of the manufacturer. Alternatives: NP40 can be replaced by Igepal CA-630 (same concentration of stock and working solutions). Continued REAGENT or RESOURCE SOURCE IDENTIFIER Oligonucleotides Oligonucleotides See Table S5 at Toullec et al., 2021. N/A Software and algorithms GraphPad Prism version 9.2.0 https://www.graphpad.com/ Liquid Yeast Extract with Supplements (YES) media Reagent Final concentration Amount Bacto yeast extract 0.5% w/v 5 g Glucose 3% w/v 30 g Supplements*0.225 g/L each 1.125 g Adenine 0.225 g/L 0.225 g Uracil 0.225 g/L 0.225 g Lysine 0.225 g/L 0.225 g Histidine 0.225 g/L 0.225 g Leucine 0.225 g/L 0.225 g ddH 2 O n/a Up to 1 L Total n/a 1 L *It is possible to use either a powder mix containing all supplements (adenine, uracil, lysine, histidine and leucine) or add each of them individually. Immunoprecipitation buffer (IP buffer) Reagent Final concentration Amount Tris-HCl pH 8.0 (1 M) 20 mM 1 mL KCl (1 M) 140 mM 7 mL MgCl 2 (1 M) 1.8 mM 0.1 mL NP40 (10%) 0.1% 0.5 mL Heparin (50 mg/mL) 0.2 mg/mL 0.2 mL ddH 2 O n/a 41.2 mL Total n/a 50 mL Keep heparin stock solution aliquoted at 20C; avoid repeated cycles of thawing and freezing. All other stock solutions can be kept at room temperature. Nonetheless, to be able to prepare a cold IP buffer at will, we recommend keeping stock solutions at 4C, with the exception of NP40. Once prepared, keep IP buffer at 4C until use. ll OPEN ACCESS STAR Protocols 3, 101373, June 17, 2022 5 Protocol Keep all stock solutions at 20CandIPbufferat4 C. Once prepared, keep complete IP buffer at 4C until use. CRITICAL: PMSF is a highly toxic neurotoxin. Prepare the stock solution (100 mM in DMSO) in a fume hood. To add it to your buffer, take the stock out of the 20Cand warm the tube in your hands, wearing gloves, until no PMSF crystals are visible. Alternatively, warm the tube in a table-top incubator preset to RT. Solubilized PMSF should be added last, as it rapidly degrades in aqueous solution. Add PMSF and mix gently immediately to prevent precipitation. CRITICAL: NaVO 4 and NaF are highly toxic via oral and can cause skin and eye irritation. Prepare 1 mg/mL (1,0003) stock solutions in a fume hood, wearing gloves and glasses. Keep stock solutions at 20C. To add it to your buffer, take the stock solutions out of the 20C and warm the tube in your hands, wearing gloves. Keep solutions on ice until added to the buffer. Immunoprecipitation wash buffer with heparin and PMSF (W1) Add 3 mL of heparin (50 mg/mL) and 30 mLofPMSF(1003)to3mLofWbuffer. Immunoprecipitation wash buffer with heparin (W2) Add 1.5 mL heparin (50 mg/mL) to 1.5 mL of W buffer. Complete immunoprecipitation buffer (complete IP buffer) Reagent Volume per sample (without phosphatase inhibitors) Volume per sample (with phosphatase inhibitors)* IP buffer 1039.5 mL 962.5 mL Heparin (50 mg/mL) 1.1 mL 1.1 mL Turbo DNase 2.2 mL 2.2 mL EDTA-free protease inhibitors cocktail (253stock solution; 1 pill into 2 mL sterile distilled water) 44 mL44mL Bestatin 1,00031.1 mL 1.1 mL Pepstatin 1,00031.1 mL 1.1 mL PMSF 100311 mL11mL Beta-glycerol phosphate 203–55mL Na 3 VO 4 1,0003–11mL NaF 1,0003–11mL Total 1,100 mL 1,100 mL *If needed. Immunoprecipitation wash buffer (W buffer) Reagent Final concentration Amount Tris-HCl pH 8.0 (1 M) 20 mM 1 mL KCl (1 M) 140 mM 7 mL MgCl 2 (1 M) 1.8 mM 0.1 mL Glycerol (50%) 10% 10 mL NP40 (10%) 0.01% 0.1 mL ddH 2 O n/a 31.8 mL Total n/a 50 mL All stock solutions can be kept at room temperature. Nonetheless, to be able to prepare a cold W buffer at will, we recommend keeping stock solutions at 4C, with the exception of NP40 and optionally glycerol 50%. ll OPEN ACCESS 6STAR Protocols 3, 101373, June 17, 2022 Protocol Note: Polyoxyethylene ether W-1 can be replaced by a combination of two detergents: Brij 56 (0.09%, Sigma P5759) and Brij 58P (0.15%, Sigma P5884). STEP-BY-STEP METHOD DETAILS Cell growth and beads preparation—Day 1 and day 2 Timing:5–10min(Day1)and1h(Day2) Note: This protocol is an adaptation of previous protocols (Gerber et al., 2004;Duncan and Mata, 2011). Note: This protocol will detect specific protein-RNA interactions occurring directly, indirectly or co-translationally (Figure 1). Note: Upon harvesting yeast cells, keep your samples and buffers cold (0C–4C), unless otherwise indicated. 1. Inoculate one single colony into 5 mL of YES rich media (or your favorite/required media). Let it grow at 32C for 24 h to reach saturation. Note: Timing should be adjusted depending on media, strain and growth rate, in order to obtain a saturated culture. Ideally, a concentration of 1 310 8 cells/mL should be reached. Note: Use colonies of similar size for each strain and between biological replicates. Maintaining the same timing from streaking out your strains from the 80C to inoculation helps. If mutants form bigger or smaller colonies as compared to control/wild-type strain, adjust the amount of biomass for each inoculate. CRITICAL: Always include at least one negative control. Either a non-tagged strain or a strain containing another, unrelated protein tagged with the same epitope can be used. We recommend using both when performing RIP for the first time or when testing a bait for the first time. The former gives an idea of the non-specific binding of RNAs to antibody-coated beads. The latter serves as a control for non-specific binding due to stickiness of nucleic acids to proteins and epitopes. If using antibodies against an endogenous protein, a deletion mutant can be used as a negative control. 2. Measure OD at 595 nm and dilute the culture in 60 mL of fresh media to reach a cell concentration of 1.5 310 7 cells/mL at the time of harvesting the culture (step 10), aiming for at least 4–5 doubling. Incubate at 32C. 103qPCR master mix Reagent Reference Final concentration Polyoxyethylene ether W-1 Sigma P7516 0.24% Bovine Serum Albumin (BSA) Sigma A4378 500 mg/mL dNTPs Invitrogen 10216, 10217, 10218, 10219 300 mM KCl Merck proanalysis 1.04936.1000 50 mM MgCl 2 Merck proanalysis 1058331000 30 mM SYBR Green FMC BioProducts 50513 1/3000 Glycerol ICN 800687 16.24% 2-Amino-2-methyl-1,3-propanediol buffer at pH 8.3 using HCl (Merck proanalysis 1.00317.1000) Sigma A9074 400 mM Platinum Taq DNA polymerase Invitrogen 10966-034 0.4 U/mL ll OPEN ACCESS STAR Protocols 3, 101373, June 17, 2022 7 Protocol Note: Beware that if additional conditions are required, such as testing cotranslational interactions by using EDTA and/or puromycin (Figure 2),thevolumeshouldbeadjusted accordingly. For example, to check the cotranslational dependency of the interaction using puromycin, inoculate 120 mL of media per strain. Note: To allow proper oxygenation of the culture, its volume should be less than about 20% of the flask. CRITICAL: If using slow- or fast-growing mutants, adjust the cell number to guarantee that all cultures reach the concentration of 1.5 310 7 cells/mL at the same time. Performing prior proliferation assays is recommended to optimize this step. 3. Prepare IP buffer. Keep on ice. 4. For each sample: Wash 100 mL of Pan mouse IgG magnetic beads 3 times with 500 mL IP buffer each time: a. Take 100 mL slurry of beads into a 1.5 mL tube. b. Remove the supernatant using the magnetic stand. c. Add 500 mL ice-cold IP buffer. d. Mixbyconstantlyinvertingthetubefor1min. e. Remove IP buffer using the magnetic stand. f. Repeat steps c-e twice. 5. Resuspend the beads in 500 mLice-coldIPbuffer. 6. Add 3 mL(3mg) of your favorite mouse monoclonal primary antibody. 7. Incubate overnight at 4Crotating. Cell harvesting, lysis, and immunoprecipitation (part 1)—Day 3 Timing: 3–4 h Goal: To obtain sufficient material for subsequent RNA isolation and immunoprecipitation. 8. Prepare complete IP buffer. Keep on ice. Note: If assessing cotranslational interactions using EDTA (Figure 2), also prepare complete IP buffer containing 25 mM EDTA (from a 0.5 M pH 8.0 stock in distilled water; filter sterilized). Always use this buffer (complete IP buffer+EDTA) instead of complete IP buffer for EDTA- treated samples during the rest of the protocol. For a more rigorous assessment of the cotranslational nature of protein-RNA interaction, we recommend using either puromycin or an ATG mutant version of a known target gene, if possible. 9. Count and annotate the concentration of the cultures. It should be about 1.5 310 7 cells/mL. 10. Transfer 50 mL of culture to a 50 mL tube. Note: Adjust small differences in the required volume for each strain. Use the less concentrated culture at first and adjust the other cultures accordingly. To minimize variability and/or allow comparisons, it is recommended to use a similar number of cells for each sample and subsequent replications of the experiment. Optional: When using puromycin (Figure 2), transfer 2 350 mL of culture to two different flasks. Then add 1 mL of puromycin hydrochloride stock solution (50 mg/mL in sterile distilled water) to one of them and 1 mL of sterile distilled water to the other one. Incubate for 15 minutes at 32C before harvesting them as indicated in step 10. ll OPEN ACCESS 8STAR Protocols 3, 101373, June 17, 2022 Protocol iv. Put the tubes in the thermocycler, start the program and let it run for the 65C/5 min step andjust30sfromthe4 C/90 s step. Pause the thermocycler and proceed with the following steps. v. Take out your PCR tubes and keep them on ice while adding and mixing the components indicated in the next step. vi. Per sample, add a mix containing: 10 mLof53superscript buffer, 1 mLofDTT(0.1M), 1mLofRNasinand1mL of Superscript III. vii. Mixbypipetting. viii. Put back the tubes in the thermocycler and re-start the program from the 4C/90 s step (ofwhichthereshouldbe60sleft). Pause point: The resulting cDNA samples can be now stored at 20C until proceeding with qPCR, microarray or high-throughput sequencing. Quantification of RNA binding to the bait by qPCR—Day 4 or day 5 Binding levels of the protein bait to the RNAs of interest are quantified using qPCR. The relative binding frequency is estimated by calculating the fraction of input cDNA recovered in the IP. Note: This protocol was designed to test for interaction between a specific bait and candidate RNAs. For an unbiased screen, use a RIP-chip or RIP-seq protocol, in which cDNAs are analyzed either through microarray hybridization or high throughput sequencing, respectively (see Costello et al., 2015).Here,wealsoprovideaquickguideonhowtoproceedwitha global approach in the ‘‘limitations’’ section below. Regardless, whenever possible, we recommend performing an initial check by qPCR using a positive control RNA. Note: For qPCR, we typically use a 50-fold dilution of total cDNA and a 3-fold dilution of IP cDNA samples, but higher dilutions of IP cDNA up to 10-fold work equally well. We used 103home-made SYBR Green Master Mix (see materials and equipment), 96 well plates and Stratagene MX3000P and MX3005P qPCR machines. Our amplicons are typically below 200 bp in size. Optimization might be required when using other dilutions, qPCR reagents, machine or amplicon length. 87. For each primer pair, prepare dilutions of one of the input cDNAs in sterile distilled water to make a standard curve and calculate primer pair efficiency. Note: To prepare the standards, we use input cDNA from a non-tagged strain which has grown and has been processed in standard conditions. For an initial standard of a first-time used pair of priming oligonucleotides, we recommend making 4, 20, 100, 500, 2,500 and 12,500-fold dilutions of one of the input cDNAs. Once the primers are validated, we reduce the number of dilutions for the standard down at least three. Alternatives: We strongly recommend using the same type of material for the standard as for the samples that are analyzed. However, if this is not possible, dilutions of genomic DNA can be used to prepare the standard for all primer pairs. 5, 0.5, 0.05, 0.005 and 0.0005 ng/mL concentrated genomic DNA samples could be used in a first try to prepare the standards. Always use clean, high-quality genomic DNA preparations. Note: We include a standard curve for each primer pair in the same plate than the samples. 88. Prepare a master mix for each primer pair that you want to test in your qPCR experiment, by mixing the reagents indicated in the table below. ll OPEN ACCESS STAR Protocols 3, 101373, June 17, 2022 15 Protocol Note: Primers specific for a negative control should be included. For example, a gene which has no functional relationship with the bait and does not interact with it. If there is no previous information, include several potential negative controls for your first assay. Note: As a positive control, primers targeting the mRNA of the bait itself can be used. We have successfully used this type of control for proteins with N-terminal epitope tag used for immunoprecipitation, as well as with long C-terminal tags, such as HA3-TAP2 using an HA antibody for IP. Beware that if using a short C-terminal tag in the bait, this positive control might not work because the native polypeptide quickly detaches from polysomes, hence its own mRNA, upon synthesis of the C-terminal epitope tag. If using an antibody against an endogenous protein, the success of this strategy depends on where the epitope is in the sequence. Note: To calculate the volume needed for each primer pair master mix consider the number of samples you have, 3 technical replicates and the standard (also performed in triplicates). 89. Prepare a 50-fold dilution of the input cDNAs and a 3-fold dilution of your IP cDNA into sterile distilled water. 90. Prepare an equivalent dilution of the control DNase-treated total RNA samples in which reverse transcriptase was not added during the reverse transcription reaction (see Note in step 85.b) to be used as control for genomic DNA contamination. We will name these samples No-RT samples. 91. Distribute 20 mL of the master mix prepared in step 88 per well. Mix well before dispensing. 92. Distribute 5 mL of the diluted cDNA or No-RT samples in each of the corresponding wells. Mix well before dispensing. 93. Cover the PCR plate with compatible tube caps or a transparent film as required by the qPCR machine instrument and mix by hand. 94. BrieflyspindowntheqPCRplateinacentrifugeat200g. 95. Run the plate into the qPCR machine using the appropriate program according to the primer pairs used and machine instructions. We used the program below for our assays described in Toullec et al. (2021): Note: It is possible to keep the plate at 4Cinthedark.Wehavehadsuccessfulrunsafterupto 2hours. PCR reaction master mix Reagent Amount per well Amount per sample (3 technical replicates) 103SYBR Green PCR Master Mix 2.5 mL 7.5 mL Primer 1 (10 mM) 0.5 mL 1.5 mL Primer 2 (10 mM) 0.5 mL 1.5 mL ddH 2 O 16.5 mL 49.5 mL PCR cycling conditions Steps Temperature Time Cycles Initial Denaturation 95C 10 min 1 Denaturation 95C 30 s 40 cycles Annealing 60C 1 min Extension 72C 1 min Melting curve 70Cto95 C, increment 0.5C, measure fluorescence. Hold 12C forever ll OPEN ACCESS 16 STAR Protocols 3, 101373, June 17, 2022 Protocol Note: A melting curve at the end of the program is important to obtain the dissociation curve of the PCR product. Only a single specific product should form. 96. Calculate the efficiency of the primers. Most qPCR machine software do this automatically. The efficiency should be between 90%–110% (3.6 Rslope R3.1) to proceed with quantification. Note: To manually calculate the efficiency of your primers, first calculate the equation of their standard curve to obtain the slope. The slope is derived from a graph that plots the cycle threshold (Ct) values against the Log10 of a given amount of DNA template. Efficiency (%) = [10^(-1/slope) – 1] 3100. A slope of -3.32 indicates an amplification efficiency of 100%. 97. After exporting the data in the selected format, calculate the starting quantities by relying on the standard curve of each primer pair. Most qPCR machine software do this automatically and allow to directly export these data. 98. Calculate the mean of the starting quantities of each sample by averaging the three (or more) technical replicates. 99. Correct each input and IP value for the corresponding dilutions and calculate a ‘‘percent IP’’ value, which is a proxy of the RNA IP efficiency relative to the amount of RNA in the input and calculated by dividing each IP value by its corresponding input value (see tables below). EXPECTED OUTCOMES A successful RIP-qPCR should give a robust and reproducible enrichment of the targeted RNA over background. ‘‘Percent IP’’ values can be, however, variable and close to background. Therefore, Input and IP quantities or volumes at each step Step Input IP IN/IP Starting material for RNA extraction 400 mg protein extract 4,000 mg protein extract 0.1 RNA extraction final volume 33 mL40mL 1.212 DNase 10 mg (max. 86.67% of total volume of RNA sample) 87.5% of total volume of RNA samples TBD (max. 0.991) RT 1 mg=3mL of DNase-treated RNA) 33.5 mL (95,71% of DNase) 0.105 qPCR 50-fold dilution of cDNA 3-fold dilution of cDNA 0.06 plate 5 mL/25 mL total, per well 5 mL/25 mL total, per well 1 Total – – 7.6 310 -4 Input and IP dilution factors Input dilution factor relative to IP Input and IP individual dilution factors at each step Step Input IP Input IP RNA extraction starting material 30.1 1 30.1 31 RNA extraction final volume 31.212 1 31.212 31 DNase TBD, maximum is 30.991 (max = 26 mL of RNA samples = 86.67% of total volume) 1 (87.5% of total volume of RNA sample) Max 30.867 30.875 RT 30.105 (10% of DNase; 3 mL) 1 (95.71% of DNase) 30.1 30.957 qPCR 30.06 (50-fold) 1 (3-fold) 30.02 30.33 plate 1 (5 mL/25 mL total, per well) 1 (5 mL/25 mL total, per well) 3131 ‘‘Total dilution factor’’ 30.00076 (7.6 310 -4 )1 30.00021 30.276 IN/IP relative dilution factor*30.00076 (7.6 310 -4 )30.00076052219 (7.6 310 -4 ) *For each IP, we use 0.00076-times less input (0.076%). Thus, the IP/IN ratio must be multiplied by 0.00076 ([IP value] 31)/([input value] 37.6 310 -4 )] ll OPEN ACCESS STAR Protocols 3, 101373, June 17, 2022 17 Protocol including negative and positive controls and a sufficient number of biological replicates is essential to carefully interpret these values. The level of enrichment will depend on several parameters: The frequency of bait binding to its target RNAs (dynamics). In other words, the percentage of cells in the population where the protein bait is bound to a specific RNA at a given time. Each specific bait will have a different binding frequency to its target RNAs. Baits that share RNA targets might, therefore, give rise to different enrichment results by RIP-qPCR. The epitope used. IP efficiency depends, among other parameters, on the strength of the epitopeantibody interaction. Thus, a RIP-qPCR assay usingthesamebaitbuttaggedwithdifferentepitopes could result in significant differences in the final enrichment results. The nature of the bait. Depending on whether using an RNA binding protein or a protein that indirectly binds RNA through intermediate factors, the dynamics and efficiency of the RIP-qPCR could change. Comparing ‘‘percent IP’’ values between test and negative control samples, we consider that enrichment below 5-fold correspond to background signal, based on the background levels that we have typically observed in RIP-qPCR using an unrelated tagged protein as bait (Toullec et al., 2021). The identification of co-translational RNA-protein interactions can be used to predict protein-pro- tein interactions and to identify new components of protein complexes (Duncan and Mata, 2014). Nonetheless, several aspects limit the use of this protocol with this aim (see below). QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analysis of RIP-qPCR data is performed using either a t-test when comparing two means or analysis of variance (ANOVA) when comparing more than two means, across one (one-way ANOVA) or two and more variables (two-way ANOVA and up). Post-hoc tests are used for pairwise comparison. In this protocol, we typically used a significance level of 0.01 and a minimum of four biological replicates, which correspond to four independent experiments. We recommend representing quantitative percent IP values as individual data points overlaid with the mean and the standard deviation. LIMITATIONS RIP followed by qPCR, microarray hybridization or high-throughput sequencing has become a standard method for studying protein-RNA interactions in different organisms (Jayaseelan et al., 2014; Gagliardi and Matarazzo, 2016;Kachaev et al., 2017;Bierhoff, 2018;Seo and Chua, 2019). Nonetheless, RIP has the following limitations. Because the RIP-qPCR protocol presented here does not rely on crosslinking to stabilize protein- RNA interactions, its success will depend on the strength and dynamics of the interaction. We implemented this protocol with the goal of detecting cotranslational interactions between interacting proteins (Toullec et al., 2021), which are transient and engage only a small fraction of the bait present in the cell. RIP-qPCR is therefore a sensitive method but we do not know what range of affinities is detectable with this methodology. Indeed, we do not have affinity measurements for such cotranslational interactions, which are challenging to reconstitute in vitro. Other direct or indirect interactions, weak and/or transient, may be missed by this method and might therefore require some form of stabilization to be captured, such as crosslinking. The use of puromycin or EDTA makes this protocol suitable for studying co-translational protein- RNA interactions (Figure 2). However, it does not allow to distinguish between direct binding of the protein bait to RNA and indirect binding, for example, through an RNA-binding protein (Figure 1). ll OPEN ACCESS 18 STAR Protocols 3, 101373, June 17, 2022 Protocol Although the identification of co-translational protein-RNA binding can predict protein-protein interactions, biochemical validation is needed using, for instance, co-immunoprecipitation or colocalization of the proteins of interest (Duncan and Mata, 2014). Also, because the interactions revealed by RIP might not be direct (for example, a preformed protein complex might interact with a nascent peptide), complementing RIP with mass spectrometry or imaging-based techniques is crucial when studying new partners of a protein bait, new components of a protein complex, or protein complex assembly mechanisms, as shown in (Duncan and Mata, 2014;Kassem et al., 2017;Kamenova et al., 2019;Elı ´as-Villalobos et al., 2019;Toullec et al., 2021;Lautieretal.,2021). This protocol has been used with epitope-tagged proteins only, for which highly efficient antibodies are commercially available. Using specific antibodies directed against the protein of interest or home-made antibodies might impact the quality of the results and require optimization. Strong RNA-binding proteins frequently co-elute with non-specific RNAs during purifications. Individual qPCRs can quickly become cumbersome to verify the specificity of protein-RNA interactions, unless using several control mRNAs clearly indicate that only a specific type of mRNA binds to the bait. Alternatively, a genome-wide analysis will be required to estimate how many RNAs bind to the protein of interest and address this issue. The several controls proposed along this protocol, including a non-tagged strain, a strain tagged in an unrelated bait and including in the qPCR analysis mRNAs to which the bait should not bind, would also help determining the specificity of the detected interactions and the requirement or not of alternative approaches. Considerations for genome-wide analyses of RIP: In its present form, this protocol is not directly suitable for high-throughput analyses since it is conceived to check for the binding capacity of a bait to specific RNAs of interest. To identify unknown RNA binding targets of an RBP there are currently two genome-wide derivatives of this protocol, RIP-chip and RIP-seq. RIP-chip requires specialized equipment and carefully designed microarrays. Due to these limitations, RIP-seq has become the dominant technique. While the objective of this protocol is not to describe either technique in detail, we describe in a supplemental file the main modifications and considerations for combining this protocol with established high-throughput sequencing pipelines (Methods S1). TROUBLESHOOTING Problem 1 Low protein yield (step 26). Potential solution Low protein yield is typically caused by inefficient cell lysis (see problem 2). Alternatively, it can be caused by protein degradation during sample processing. Make sure that the samples remain at low temperature, below 4C, throughout the procedure, by prechilling all tubes and buffers before use and by adding protease inhibitors at the indicated steps. Note that when using media lacking nutrients, for example for quiescent cells, protein and RNA yields will drop (see problem 5). Problem 2 Inefficient cell lysis (step 17). Potential solution In S. pombe and other yeasts, cell lysis can be monitored by direct observation under a light microscope. It is important to verify the integrity of yeast cells before and after bead-beating by monitoring cells under a light microscope, using 5 mL of the extract spread on a glass slide. Efficient cell lysis corresponds to about 90% of lysed cells, which appear as black and refringent. Alternatively, if there are only cellular debris, estimate the number of intact cells before and after lysis. Lysis efficiency should reach close to 90%. Otherwise, repeat the bead-beating procedure until reaching this threshold. In this case, to avoid heating of the samples and protein degradation, place samples ll OPEN ACCESS STAR Protocols 3, 101373, June 17, 2022 19 Protocol on ice between each lysis cycle. Alternative lysis methods, such as cryogrinding can be used, but larger culture volumes are typically required to end up with sufficient material for subsequent steps and to avoid variability between samples. Problem 3 Low RNA yield (steps 57 and 84). Potential solution RNA degradation is frequently caused by inappropriate manipulation of RNA-containing samples or absence of RNA inhibitors. This can be minimized by wearing gloves when manipulating your samples, using nuclease-free filter-tips and making sure that a sufficient amount of RNase inhibitors is added at the indicated steps. Using media lacking nutrients can impact protein and RNA yields - see problem 5. Problem 4 High variability in RIP signal (step 99). Potential solution This RIP protocol involves many steps and differences in any of them can lead to significant changes in RIP signal. It is thus crucial to be precise and rigorous to avoid variations across samples and experiments. Protein and RNA degradation is another frequent source of variability (see problems 1,3,and5). Therefore, it is important to make sure that the samples remain at low temperature throughout the procedure, to prechill all tubes and buffers before use and to add protease and RNase inhibitors at the indicated steps. Problem 5 Low protein or RNA yields when using media lacking nutrients (steps 1, 26, 57, and 84). Potential solution The physiological state of the yeast culture can significantly impact the synthesis and stability of RNAs and proteins. For example, nutrient starvation is typically accompanied by an increased expression and activation of proteases and nucleases as well as global changes in gene expression (Nakashima et al., 2002,2006;Kohda et al., 2007;Margueratetal.,2012). In this case, Western blotting and RT-qPCR should be used to control for variations in the levels of the protein bait and the RNA targets, respectively. In our experience, with S. pombe, protein degradation becomes detectable following a 60 min shift to starvation medium. In this case, adding higher amounts of protease and RNase inhibitors can help. In addition, scaling up the amount of starting material by increasing the number of cells grown can also help. Finally, using the RNA micro kit instead of the RNA mini kit allows increasing the concentration of the RNA samples (see first ‘‘Note’’ under the heading ‘‘isolation of immunoprecipitated RNA – day 3’’). RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dominique Helmlinger ([email protected]). Materials availability This study did not generate new unique reagents. Data and code availability This study did not generate/analyze datasets. ll OPEN ACCESS 20 STAR Protocols 3, 101373, June 17, 2022 Protocol SUPPLEMENTAL INFORMATION Supplemental information can be found online at https://doi.org/10.1016/j.xpro.2022.101373. ACKNOWLEDGMENTS We thank all members of the laboratory for helpful discussions. A.E.-V. was a recipient of a postdoctoral fellowship from the Fondation pour la Recherche Me´ dicale (SPF20130526854) at the CRBM and of a Maria Zambrano grant, funded by the European Union – NextGenerationEU, at the Universidad de Sevilla. J.M. and C.D. were supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC BB/G011869/1 and BB/S015833/1). This work was supported by funds from the Agence Nationale de la Recherche (ANR-15-CE12-0009-01 and ANR-15-CE11-0022-03) and the Fondation ARC (PJA 20181208277) to D.H. AUTHOR CONTRIBUTIONS Conceptualization: A.E.-V and C.D.; investigation: A.E.-V. and C.D.; writing – original draft, A.E.-V.; writing–reviewandediting,A.E.-V., C.D., J.M., and D.H.; funding acquisition, J.M. and D.H.; supervision, J.M. and D.H. DECLARATION OF INTERESTS The authors declare no competing interests. REFERENCES Aviner, R. (2020). 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