Computational protocol: Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers

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[…] Coupling of the compound derivatives to the NHS-activated beads (GE Healthcare) was performed by incubation of 1 µmol of compound with 1 ml of bead solution. After binding overnight at room temperature in DMSO and 1.5% triethylamine, any remaining reactive groups were blocked with 33% (final volume) ethanolamine for 24 h at room temperature. Subsequent washing steps were performed with DMSO. Binding efficiency was estimated by comparing the optical densities of the compound solutions before and after coupling at the maximal absorbance wavelength 292 nm.Heavy and light SILAC labeled SMA type 1 fibroblasts were lysed in 50 mM HEPES pH 7.5 buffer containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.5% NP-40, protease inhibitors (Roche), sonicated using a Branson Digital Sonifier on ice. Samples were then cleared by centrifugation at 12,000 × g for 10 min at 4 °C. Heavy or light supernatants were loaded in quintuples onto Sepharose® beads immobilized with the active or inactive derivative for 1 h at 4 °C under rotation. Beads were washed three times with lysis buffer, treated with SDS-PAGE sample buffer for 5 min at 85 °C, and identical volume of both elutions (active and inactive beads) were combined. For the experiment using RNAse, an additional step was added before elution, using RNAse A (Roche) at 1.5 U μl−1 for 20 min at room temperature. Eluates were separated on 4–20% Tris-Glycine SDS-PAGE. After protein fixation in 40% ethanol and 10% acetic acid, gels were stained overnight with Coomassie Brilliant blue. Each gel lane was cut into seven slices between the 20 and 150 kDa region, in-gel digested as described above, and stored at −20 °C prior to analysis. Samples were re-constituted in 2% acetonitrile/5% formic acid and analyzed by liquid chromatography–mass spectrometry (LC–MS) using an Easy-nLC coupled to an LTQ Orbitrap Velos Pro (Thermo Fisher Scientific) equipped with a Digital PicoView 550 source (New Objective, Woburn, MA) and an active background ion reduction device (ABIRD, ESI Source Solutions, Woburn, MA). Peptides were loaded onto an Aqua C18 (Phenomenex, Torrance, CA) trapping column (packed in-house, 10 mm length × 100 μm inner diameter, 5 μm particle size) and separated on a Reprosil-Pur C18-AQ (Dr. Maisch GmbH, Ammerbuch, Germany) analytical column (200 mm length × 75 μm inner diameter, 3 μm particle size, 120 Å) using the following gradient at 250 nl per min: 0–2% B in 2 min, 2–35% B in 46 min, 35–80% in 2 min, and 80% B for 10 min (buffer A: 0.6% acetic acid; buffer B: 0.6% acetic acid/acetonitrile). The eluent was electrosprayed via uncoated SilicaTip emitters by applying 1.7-2-0 kV spray voltage via a liquid junction. The mass spectrometer was operated in the data dependent mode to automatically switch between MS and MS/MS. Survey full scan MS spectra were acquired from m/z 350 to 1700 in the Orbitrap with a resolution of R = 60,000 (at m/z 400) after accumulation to a target value of 1 × 106 in the linear ion trap for a maximum time of 500 ms. The detected ions were recalibrated on-the-fly using the ambient air polysiloxane at m/z 445.120024 as lock-mass (51). The 10 most intense ions with a charge state > 1 + and a threshold above 1000 were selected for collision induced dissociation (CID) in the linear ion trap. Ions were accumulated to a target value of 10,000 using the predictive ion trap fill time mode, isolated with a width of 2 amu, CID performed at a normalized collision energy of 35% with an activation Q of 0.25 for 10 ms and wideband activation mode enabled. Fragmented ions with a m/z width of ±10 ppm width were placed on an exclusion list for 30 s and a maximal size of 500. Raw files were processed using Mascot Distiller 2.5.1.0 (Matrix Science) and peak lists were searched against the human UniProt/SwissProt database (December 2013 release, 20,274 sequences) using Mascot server. Trypsin/P was used as an enzyme, allowing for a maximum of one miscleavage, 20 ppm and 0.5 Da as precursor and fragment mass tolerances, respectively. Carbamidomethylated cysteine was selected as fixed, while oxidized methionine, was set as variable modifications. SILAC variable modifications Lys8 and Arg10 (heavy) were set in the SILAC quantification method. Individual peptide to spectrum matches were filtered using maximum expect p-value corresponding to a false discovery rate of ≤1% as determined by enabling the decoy database search. SILAC peptide quantitation and ratio generation was performed with Mascot Distiller using the following settings: precursor ion protocol, Simpson’s rule as integration method, minimal Rho correlation of 0.7, and an XIC threshold of 0.1. Ratios median was calculated for protein with a minimum of two quantified peptides. Peptide level protein quantitation reports were generated in html format for further processing. HTML reports were loaded into R 3.1.0 for statistical analysis, and only protein hits with at least two peptides, a Mascot Score > 31, found in at least 7 out of 10 samples and were used in subsequent analysis. The SDS band where a given protein was identified with highest abundance was kept, and protein entries in the other bands were discarded. Ratios of reversed peptides ratios were reversed, and all ratios were log2-transformed. One-sided t tests were performed, with the null hypothesis that the mean of the log-transformed ratios of a given protein equals 0. The corresponding alternative hypothesis states that the original, not-transformed ratios H:L (after H:L reversion, where appropriate) are significantly greater than 1:1. The p-values of the one-sided t-test were adjusted for multiple testing following Benjamini and Hochberg. Proteins were considered as significantly and relevantly enriched if both the adjusted p-value ≤ 5 % and the ratio between groups > 1:1.5 were found.From pull-down experiments with active and inactive compound, proteins were selected that showed at least 1.5 fold enrichment for the active compound, with an adjusted p-value of 0.05 or less. This produced a list of 430 proteins. Using the GO_SLIM collection of gene ontology annotations (http://ceur-ws.org/Vol-1546/paper_44.pdf), these proteins were found to be enriched for 8 categories based on Fisher’s exact test and using a p-value cutoff of 0.001. Each of them is characterized by the negative logarithm of the enrichment p-value on the x axis of Supplementary Fig. ; the full is given in Supplementary Table . The category “translation” scored best, but is not related to the compound’s mode of action. Reasoning that proteins involved in the translational machinery would be associated through their interactions with RNA, we produced a second list of 339 proteins by manually removing all ribosomal proteins from the original list. Repeating the enrichment analysis, we obtained drastically smaller or at best very similar enrichment p-values (y axis in Supplementary Fig. ), with the exception of the “RNA splicing” category, which scored substantially better. We conclude that this category is the strongest hit in our search that is independent of the (inevitable) presence of ribosomal proteins in our data set. [...] 100 pmol of 5 biotinylated ESE2 sequence (5′-AAAAAGAAGGAAGG-3′) was bound to 50 μl of streptavidin magnetic beads according to the manufacturer’s instructions (PierceTM Magnetic RNA–protein pull-down kit; Thermo Scientific; Rockford, USA). SMA type 1 human fibroblasts pellets were resuspended in 10 mM HEPES pH7.8, 1 mM MgCl2, 10 mM KCl, protease inhibitors (cOmplete™ Protease Inhibitor Cocktail EDTA free Roche) and homogenized with a potter. Lysates were centrifuged at 500 × g for 10 min at 4 °C. Pelleted nuclei were resuspended in 50 mM HEPES pH 7.3 buffer containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.5% NP-40, protease inhibitors, RNase inhibitors (Protector, Roche), and sonicated using a Q125 sonicator (Qsonica). Samples were then cleared by centrifugation at 300 × g for 10 min at 4 °C and the supernatant was incubated for 1 h 30 min at 4°C with bead-immobilized ESE2 sequence as bait. For competition experiments, lysates were pre-incubated for 1 h 30 min with increasing amounts of SMN-C6 or SMN-C7 (0; 0.5; 1; 5 and 10 μM final concentration) in triplicates. After washing 3 times in lysis buffer, proteins were eluted in reducing SDS-PAGE sample buffer for 5 min at 85°C. Eluates were separated on 4–20% Tris-Glycine SDS-PAGE and stained with Coomassie Brilliant blue. Gel lanes were cut in five bands spanning from 20 to 120 kDa. For protein in-gel digestion, an adapted protocol from Shevchenko et al. was used. Proteins were reduced with 50 mM dithiothreitol for 45 min at 56 °C, alkylated with 55 mM iodoacetamide for 1 h in the dark, and digested with trypsin (Promega) overnight at room temperature. Peptides were extracted twice with 1:2 (v/v) acetonitrile/25 mM ammonium bicarbonate, and 1:2 (v/v) acetonitrile/5% formic acid, respectively, for 15 min at 37 °C. Dried samples were re-constituted in 2% acetonitrile/5% formic acid and run in quintuples by LC–MS, using an EASY-nLC 1000 ultrahigh pressure liquid chromatography) coupled to an Orbitrap Fusion Mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were concentrated on an Acclaim PepMap C18 trapping column (100 μm × 20 mm, 5 μm particle size). Peptides were separated on an Acclaim PepMap C18 EASY-spray column (75 μm × 500 mm, 2 μm particle size) using the following gradient at 300 nl per min: 7–50% B in 45 min, 50–80% B in 2 min, 80% B for 13 min, corresponding to a total time of 60 min (buffer A: 0.1% formic acid; buffer B: 0.1% formic acid/acetonitrile). Data were on-the-fly recalibrated using ambient air hexacyclodimethylsiloxane at m/z 445.12002. The ten most intense precursor ions, with charge states between 2 and 6, a minimum intensity of 5E3, were mono-isotopically selected for higher-energy collisional dissociation, using a quadrupole isolation of m/z 0.7, automatic gain control target of 1E4, maximum IT of 35 ms, collision energy of 30%, and ion trap readout with rapid scan rate. Only a single charge state per precursor was selected for MS2. Interrogated precursor ions were dynamically excluded for 75 s using a ±10 ppm mass tolerance. Raw files were processed using Progenesis QI 2.1 (Nonlinear Dynamics; Newcastle, UK) and Mascot Server 2.5.1 (Matrix Science, London, UK) together with the UniProt/SwissProt human protein database (08.2015 release, 20,204 entries without splice variants) using trypsin/P as an enzyme, a maximum of two missed cleavage sites, 10 ppm, and 0.5 Da as the precursor, and fragment ion tolerances, respectively. Carbamidomethylated cysteines (+57.02146 Da) were set as static while oxidized methionines (+15.99492 Da) were set as dynamic modifications. For statistical analysis of the raw data and in order to detect proteins displaced with increasing free SMN-C6 and SMN-C7 concentration, the concepts of contrast tests, combined with a moderated linear model, were applied. Monotonic contrasts were used to compare the protein abundance values above and below each concentration point, and the maximum of the resulting series of t-statistic values was determined for each protein quantitation group. To obtain the p-values, the concentration labels were permuted 1000 times based on the step-down minP algorithm, modified for one-sided tests, and adjusted for multiple testing. Proteins with adjusted p-values below 5% were considered as specific binders. Computations were performed in R (version 3.3.2; http://www.r-project.org/). […]

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