Mapping of Nab3 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2
Using CRAC, we compared the transcriptomic occupancy of Nab3 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 had indeed abrogated binding of Nab3 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.). Overall design: Two biological replicates of CRAC experiments performed in a parental Saccharomyces cerevisiae BY4741 strain (PIC2-GFP) and two derived mutants: mut-Nab3-BS and mut-NNS-BS. While mut-Nab3-BS lacks Nab3 RNA-binding sites in PIC2, the other strain has no Nab3 or Nrd1 RNA-binding sites in PIC2 (mut-NNS-BS). All strains encoded NAB3-HTF (HIS6-TEV-3xFLAG). When SC-Ura cultures containing 1% (w/v) raffinose reached an OD600 of 1, cells were UV cross-linked (254 nm; 500 mJ/cm2) in the Vari-X-linker (UVO3) and rapidly filtered on 0.8 µm membranes (Merck) using a filtration device connected to a vacuum pump. Membranes were collected in 50 mL tubes, flash frozen by submersion into liquid nitrogen and stored at -80ºC until the day of the experiment.   On the day of the experiment, pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1 EDTA-free protease inhibitor cocktail (Roche) per 50 mL and 5 mM ß-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM ß-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM ß-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM ß-mercaptoethanol) and incubated at 4 °C overnight while rotating at 12 rpm.   Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM ß-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 1 hour. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3' adapter (5'App-NAGATCGGAAGAGCACACGTCTG-ddC 3'), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for six hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5'end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-?ATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5' linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5' adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5'-ACACGACGCTCTTCCGATCTNNNCGCTTAGCN-3' and 5'-ACACGACGCTCTTCCGATCTNNNGACTTAGCN-3' or 5'-ACACGACGCTCTTCCGATCTNNNTAAGCN-3' for PIC2-GFP repeats 1 and 2 respectively; 5'-ACACGACGCTCTTCCGATCTNNNCACTAGCN-3' and 5'-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3' for mut-Nab3-BS replicates 1 and 2 respectively;  and 5'-ACACGACGCTCTTCCGATCTNNNGTGACAN-3' for both mut-NNS-BS libraries. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).