Cell Culture. HEK293T cells from the ATCC (passages <25) were cultured in a 1:1 DMEM/MEM mixture (Cellgro) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37℃ under 5% CO2. For fluorescence microscopy imaging experiments, cells were grown on 7 × 7-mm glass coverslips in 48-well plates. For APEX-PS experiments, cells were grown on 15-cm glass-bottomed Petri dishes (Corning). To improve the adherence of HEK293T cells, glass slides and plates were pre-treated with 50 mg/mL fibronectin (Millipore) for 20 min at 37℃ before cell plating and washing three times with Dulbecco’s PBS (DPBS) (pH 7.4). HEK293T cells stably expressing APEX2-NLS, APEX2-NIK3x, APEX2-OMM, APEX2-NES, ERM-APEX2 and TurboID-NES were generated in our previous studies 17, 24.
APEX labeling and crosslinking in living cells. For both western blot analysis and proteomic analysis, HEK293T cells stably expressing the APEX2 fusion construct of interest were cultured in 15-cm dish for 18-24 h to about 90% confluency. APEX labeling was initiated by changing to fresh medium containing 500 μM biotin-phenol (Iris Biotech GMBH) and incubating at 37℃ under 5% CO2 for 30 min. H2O2 (Sigma Aldrich) was then added to a final concentration of 1 mM and the plate was gently agitated for 1 min. For OMM-localized RBP profiling under PUR treatment, HEK293T cells stably expressing APEX2-OMM were treated with 200 μM puromycin and 500 μM biotin-phenol at 37℃ under 5% CO2 for 30 min and then APEX labeling was initiated by H2O2 treatment for 1 minute.
For FA crosslinking, media was aspirated and the APEX labeling reaction was quenched by addition of 2 mL azide-free quenching solution (10 mM ascorbate and 5 mM Trolox in DPBS). Cells were incubated at room temperature for 1 min, then media was removed by aspiration and 10 mL of crosslink-quench solution (0.1% (v/v) formaldehyde, 10 mM sodium ascorbate and 5 mM Trolox in DPBS) was added. After 1 min, media were aspirated and cells were again incubated in 10 mL fresh crosslink-quench solution for 9 min at room temperature with gentle agitation. The crosslinking reaction was terminated in 125 mM of glycine for 5 min at room temperature. Cells were washed twice with 10 mL room-temperature DPBS, harvested by scraping, pelleted by centrifugation, and either processed immediately or flash frozen in liquid nitrogen and stored at -80°C for further analysis.
For UV crosslinking, the reaction was quenched by replacing the medium with an equal volume of quenching solution (10 mM ascorbate, 5 mM Trolox and 10mM sodium azide in DPBS). Cells were washed with quenching solution for three times and media were aspirated UV cross-linking was performed on PBS-washed cells by UV irradiation at 254 nm with 400 mJ/cm2 (CL-1000 Ultraviolet Crosslinker, UVP). Cells were washed twice with 10 mL ice-cold DPBS, harvested by scraping, pelleted by centrifugation, and either processed immediately or flash frozen in liquid nitrogen and stored at -80°C before further analysis.
For the validation of TurboID-PS shown in Figure S1J, HEK293T cells stably expressing TurboID-NES were cultured for 18-24 h. The biotin labeling was initiated by adding a final concentration of 50 μM biotin for 10 min. The labeling was stopped by transferring the cells to ice and washing five times with ice-cold DPBS. The cells were crosslinked by addition of 10 mL crosslinking solution (0.1% (v/v) formaldehyde in DPBS) for 10 min at room temperature with gentle agitation. The crosslinking reaction was terminated in 125 mM of glycine for 5 min at room temperature. Cells were washed and collected as described above.
Phase separation. Cell pellets were resuspended in 1 mL Trizol and the homogenized lysate was transferred to a new tube. After incubating at room temperature for 5 min to dissociate, 200 μL of chloroform (Fisher Scientific) were added and vortexed, and the sample was centrifuged for 15 min at 12,000 g at 4 °C. The upper, aqueous phase (containing non-crosslinked RNAs) and the lower, organic phase (containing non-cross-linked proteins) was removed. Interface (containing the protein–RNA complexes) was resuspended in 1 mL Trizol and subjected to two more cycles of phase separation. Finally, the interface was precipitated by 1 mL methanol and pelleted by centrifugation at 14,000g, room temperature for 10 min. The precipitated proteins was resuspended in 100 μL of 100 mM triethylammonium bicarbonate (TEAB), 1 mM MgCl2, 1% SDS and incubated at 95°C for 20 min. The samples were cooled at room temperature for 5 min and digested with 2 μg RNase A, T1 mix (2 mg/mL of RNase A and 5,000 U/mL of RNase T1, Thermo Fisher Scientific) for 2 h at 37°C. Another 2 μg of RNase mix was added and incubated overnight at 37°C. The resulting solution was subjected to the final round of phase separation and the RBPs in organic phase was recovered by precipitation in 4.5 mL methanol with centrifugation at 14,000g, room temperature for 20 min. The protein pellets were resuspended in 1 mL methanol, transferred to a new 1.5 mL Eppendorf tube and pelleted by centrifugation at 14,000g, room temperature for 10 min. The protein pellets were resuspended in 1 mL RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1× protease inhibitor cocktail (Sigma-Aldrich), and 1 mM PMSF) with sonication using a Misonix sonicator (0.5 s on, 0.5 s off, for a total of 10s on). The total RBP solution was then subjected for Western blotting or streptavidin enrichment.
Streptavidin bead-based enrichment. To enrich biotinylated material from the total RBP solution, 300 μL streptavidin-coated magnetic beads (Pierce) were washed twice with RIPA buffer, then incubated with the 1 mL total RBP solution with rotation for 2 h at room temperature. The beads were subsequently washed twice with 1 mL of RIPA lysis buffer, once with 1 mL of 1 M KCl, once with 1 mL of 0.1 M Na2CO3, once with 1 mL of 2 M urea in 10 mM Tris-HCl (pH 8.0), and twice with 1 mL RIPA lysis buffer. For Western blotting analysis, the enriched proteins were eluted by boiling the beads in 75 μL of 3× protein loading buffer supplemented with 20 mM DTT and 2 mM biotin. For proteomic analysis, the beads were then resuspended in 1 mL fresh RIPA lysis buffer and transferred to a new Eppendorf tube. The beads were then washed with 1 mL wash buffer (75 mM NaCl in 50 mM Tris HCl pH 8.0) twice. The beads were resuspended in 50 μL of wash buffer and shipped to Steve Carr’s laboratory (Broad Institute) on dry ice for further processing and preparation for LC-MS/MS analysis.
Gels and Western blots. For blotting shown in Figure 1C-D, Figure 6A and Figure S1, 5% of cells were subjected to total cell lysates analysis. the pellets were lysed by resuspending in 200 μL RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1× protease inhibitor cocktail (Sigma-Aldrich), and 1 mM PMSF) by gentle pipetting and incubating for 5 min at 4°C. Lysates were clarified by centrifugation at 10,000 r.p.m. for 10 min at 4°C. Protein concentration in clarified lysate was estimated with Pierce BCA Protein Assay Kit (ThermoFisher Scientific). 5% of total RBP solution was collected as the samples after phase separation. The total cell lysates, samples after phase separation and streptavidin enrichment were resolved on a 9% SDS-PAGE gel. The silver-stained gel shown in Figure S1A and H were generated using Pierce Silver Stain Kit (ThermoFisher Scientific).
For all Western blots, after SDS-PAGE, the gels were transferred to nitrocellulose membrane, and then stained by Ponceau S (5 min in 0.1% (w/v) Ponceau S in 5% acetic acid/water). The blots were then blocked in 5% (w/v) milk (LabScientific) in TBS-T (Tris-buffered saline, 0.1% Tween 20) for at least 30 min at room temperature. For streptavidin blotting, the blots were stained with 0.3 μg/mL streptavidin-HRP in 3% BSA (w/v) in TBS-T for 1 h at 4°C. The blots were washed three times with TBS-T for 5 min each time before to development. For validation of the specificity of APEX-PS in Figure 1D and S1I, the blots were stained with primary antibodies in 3% BSA (w/v) in TBS-T for 2 h in room temperature or overnight at 4°C. The primary antibodies include anti-SRSF1 (1:2500 dilution, ab129108, Abcam), anti-hnRNPC (1:2500 dilution, ab133607, abcam), anti-GRSF1 (1:2500 dilution, ab205531, Abcam) and anti-ETS-2 (1:1000 dilution, sc-365666, Santa Cruz Biotechnology). For validation of SYNJ2BP and EXD2 in Figure 6A and 6B, the blots were stained with anti-SYNJ2BP (1:2000 dilution, HPA000866-100UL, Sigma-Aldrich) and anti-EXD2 (1:2000 dilution, HPA005848-100UL, Sigma-Aldrich) in 3% BSA (w/v) in TBS-T for 2 h in room temperature or overnight at 4°C. For the evaluation of protein synthesis in Figure 6F, 6G, 7E and 7F, the blots were stained with anti-UQCR11 (1:1000 dilution, MBS715423, MyBioSource), anti-MTFP1 (1:2500 dilution, ab198217, Abcam), anti-PET117 (1:2500 dilution, PA5-61574, ThermoFisher Scientific), anti-RAB5IF (1:1000 dilution, PA543332, ThermoFisher Scientific), anti-MRPS17 (1:2500 dilution, ab175207, Abcam) and anti-HSP60 (1:2500 dilution, ab190828, Abcam) in 3% BSA (w/v) in TBS-T for 2 h in room temperature or overnight at 4°C. After washing three times with TBS-T for 5 min each, the blots were stained with secondary antibodies in 3% BSA (w/v) in TBS-T for 2 h in room temperature. The blots were washed three times with TBS-T for 5 min each time before to development with Clarity Western ECL Blotting Substrates (Bio-Rad) and imaging on the ChemiDoc XRS+ System (Bio-Rad).
On-bead trypsin digestion of biotinylated proteins. To prepare samples for mass spectrometry analysis, proteins bound to streptavidin beads were washed twice with 200 μL of 50 mM Tris HCl buffer (pH 7.5) followed by two washes with 2 M urea/50 mM Tris (pH 7.5) buffer. The final volume of 2 M urea/50 mM Tris buffer (pH 7.5) was removed and beads were incubated with 50 μL of /50 mM Tris and 0.5 μg trypsin for 30 mins at 37°C with shaking. After 30 mins, the supernatant was removed and transferred to a fresh tube containing LysC digest. The streptavidin beads were washed once with 50 μL of 50 mM Tris buffer (pH 7.5) and the wash was combined with the on-bead digest supernatant and digested on shaker for at least 3 hours at 37 degrees. The eluate was reduced with 4 mM DTT for 30 min at 25°C with shaking. The samples were alkylated with 10 mM iodoacetamide for 45 min in the dark at 25°C with shaking. Then 0.5 μg of trypsin was added to the sample and the digestion continued overnight at 25°C with shaking. After digestion, samples were acidified (to pH < 3.0) by adding formic acid such that the sample contained ~1% formic acid. Samples were desalted on C18 StageTips and evaporated to near dryness in a vacuum concentrator.
TMT labeling and fractionation of peptides. Desalted peptides were labeled with TMT (11-plex) reagents 63. Peptides were reconstituted in 100 μL of 50 mM HEPES. Each 0.8 mg vial of TMT reagent was reconstituted in 41 μL of anhydrous acetonitrile and added to the corresponding peptide sample for 1 h at room temperature. Labeling of samples with TMT reagents was completed with the design shown in Figure 2A and Figure 5A. TMT labeling reactions were quenched with 8 μL of 5% hydroxylamine at room temperature for 15 min with shaking, evaporated to dryness in a vacuum concentrator, and desalted on C18 StageTips. For each TMT 11-plex cassette, 50% of the sample was fractionated by basic pH reversed phase using StageTips while the other 50% of each sample was reserved for LC-MS analysis by a single-shot, long gradient. One StageTip was prepared per sample using 2 plugs of Styrene Divinylbenzene (SDB) (3M) material. The StageTips were conditioned two times with 50 μL of 100% methanol, followed by 50 μL of 50% MeCN/0.1% formic acid, and two times with 75 μL of 0.1% formic acid. Sample was resuspended in 100 μL of 0.1% formic acid and loaded onto the stageTips and washed with 100 μL of 0.1% formic acid. Following this, sample was washed with 60 μL of 20 mM NH4HCO3/2% MeCN, this wash was saved and added to fraction 1. Next, sample was eluted from StageTip using the following concentrations of MeCN in 20 mM NH4HCO3: 10%, 15%, 20%, 25%, 30%, 40%, and 50%. For a total of 6 fractions, 10 and 40% (fractions 2 and 7) elutions were combined, as well as 15 and 50% elutions (fractions 3 and 8). The six fractions were dried by vacuum centrifugation.
Liquid chromatography and mass spectrometry. Fractionated peptides were resuspended in 8 μL of 0.1% formic acid and were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) using an Q-Exactive Plus Orbitrap MS (ThermoFisher Scientific) coupled online to an Easy-nLC 1200 (ThermoFisher Scientific). Four microliters of each sample was loaded onto a microcapillary column (360 μm outer diameter × 75 μm inner diameter) containing an integrated electrospray emitter tip (10 μm), packed to approximately 20 cm with ReproSil-Pur C18-AQ 1.9 μm beads (Dr. Maisch GmbH) and heated to 50 °C. The HPLC solvent A was 3% MeCN, 0.1% formic acid, and the solvent B was 90% MeCN, 0.1% formic acid. The SDB fractions were measured using a 110 min MS method, which used the following gradient profile at 200 nL/min: (min:%B) 0:2; 1:6; 85:30; 94:60; 95:90; 100:90; 101:50; 110:50 (the last two steps at 500 nL/min flow rate). The Q-Exactive Plus Orbitrap MS was operated in the data-dependent acquisition mode acquiring HCD MS/MS scans (resolution = 35,000, quadrupole isolation width of 0.7 Da) after each MS1 scan (resolution =70,000, 300-1800 m/z scan range) on the 12 most abundant ions using an MS1 target of 3×106 and an MS2 target of 5×104. The maximum ion time utilized for MS/MS scans was 120 ms and the HCD normalized collision energy was set to 30. The dynamic exclusion time was set to 20 s, and the peptide match and isotope exclusion functions were enabled. Charge exclusion was enabled for charge states that were unassigned, 1 and >6.
Mass spectrometry data processing. Collected data were analyzed using Spectrum Mill software package v6.1pre-release (Agilent Technologies). Nearby MS scans with the similar precursor m/z were merged if they were within ± 60 s retention time and ±1.4 m/z tolerance. MS/MS spectra were excluded from searching if they failed the quality filter by not having a precursor MH+ in the range of 750 - 4000. All extracted spectra were searched against a UniProt database (12/28/2017 containing human reference proteome sequences, common laboratory contaminants, and mycoplasma ribosomes. Search parameters included: parent and fragment mass tolerance of 20 p.p.m., 50% minimum matched peak intensity, and‘calculate reversed database scores enabled. The digestion enzyme search parameter used was Trypsin Allow P, which allows K-P and R-P cleavages. The missed cleavage allowance was set to 3. TMT labeling was required at lysine, but peptide N termini were allowed to be either labeled or unlabeled. Allowed variable modifications were protein N-terminal acetylation, pyro-glutamic acid, deamidated N, and oxidized methionine. Individual spectra were automatically assigned a confidence score using the Spectrum Mill autovalidation module. Score at the peptide mode was based on target-decoy false discovery rate (FDR) of 1%. Protein polishing autovalidation was then applied using an auto thresholding strategy. Relative abundances of proteins were determined using TMT reporter ion intensity ratios from each MS/MS spectrum and the mean ratio is calculated from all MS/MS spectra contributing to a protein subgroup. Proteins identified by 2 or more distinct peptides and a protein score of at least 20 were considered for the dataset.
Analysis of proteomic data for nucleus and nucleolus. To determine the cutoff in each biological replicate, we adopted the ratiometric analysis as previously described 37. The original identified proteins are shown in Table S1. For the assignment of nuclear RBPs, a list of known RBPs were firstly collected from RNA binding Gene Ontology Term (GO:0003723) and several previous datasets 6, 7, 10, 12, 13, 32, 34, 35. The true-positives (TPs) were the known RBPs with nuclear annotation in the following GO terms: GO:0016604, GO:0031965, GO:0016607, GO:0005730, GO:0001650, GO:0005654, GO:0005634. For the false-positives (FPs), a list of 6815 proteins with non-nuclear annotation in the following GO terms: GO:0015629, GO:0016235, GO:0030054, GO:0005813, GO:0045171, GO:0000932, GO:0005829, GO:0005783, GO:0005768, GO:0005929, GO:0005794, GO:0045111, GO:0005811, GO:0005764, GO:0005815, GO:0015630, GO:0030496, GO:0070938, GO:0005739, GO:0072686, GO:0005777, GO:0005886, GO:0043231; and are not annotated with the following GO terms: GO:0016604, GO:0031965, GO:0016607, GO:0005730, GO:0001650, GO:0005654, GO:0005634. The non-nuclear proteins that were not annotated as known RBP were assigned as FPs. For each replicate, the proteins were first ranked in a descending order according to the TMT ratio (128N/126C, 128C/127N, 129N/127N). For each protein on the ranked list, the accumulated true-positive count and false-positive count above its TMT ratio were calculated. A receiver operating characteristic (ROC) curve was plotted accordingly for each replicate (Figure S2B). The cutoff was set where true-positive rate - false-positive rate (TPR-FPR) maximized. Post-cutoff proteomic lists of the three biological replicates were intersected and proteins enriched in at least two biological replicates were collected. The potential glycosylated proteins were removed according to the annotation of glycoproteins or locations exclusively in the secretory pathway (e.g. ER/Golgi lumen, plasma membrane, extracellular regions) to obtain the nuclear RBPome list (Table S2).
For the assignment of nucleolar RBPs, TPs were known RBPs with nucleolar annotation (GO:0005730). The FPs were non-nuclear proteins without RBP annotation. For each replicate, the APEX-PS-NIK3x sample was not only compared to the negative controls (e.g. omitting H2O2 or enzyme), but also compared with the APEX-PS-NLS sample. The proteins were first ranked in a descending order according to the TMT ratio and cutoff was assigned by ROC analysis as described above (Figure S3A). The two types of comparison were intersected for each replicate (130C/126C and 130C/128N for replicate 1; 131N/129C and 131N/128C for replicate 2; 131C/129C and 131C/129N for replicate 3). The resulting lists of the three biological replicates were intersected and the potential glycosylated proteins were removed to obtain the final nucleolar RBP list (Table S3).
For the analysis of nuclear specificity of the nuclear and nucleolar RBPs (Figure 2H and 3F), we collected a list of 6889 human protein with nuclear annotations in the following GO terms: GO:0016604, GO:0031965, GO:0016607, GO:0005730, GO:0001650, GO:0005654, GO:0005634. The number of nuclear proteins presented in each dataset was determined. For the analysis of RNA binding specificity of nuclear RBPs (Figure 2G and 3G), the number of known RBPs presented in each dataset was determined. For the sensitivity analysis of nuclear RBPome (Figure 2I), a gold standard list of nuclear RBPs was manually curated (Table S2) according to previous literature3 and the coverage of APEX-PS, serIC and RBR-ID was determined. For comparing APEX-PS profiling with global phase separation profiling (Figure 2J and 3H), the protein abundance36 of overlapped RBPs and novel RBPs identified by APEX-PS was compared according to previous datasets. The analysis of the RNA types associated with nuclear and nucleolar RBPs (Figure 2F and S3C) was performed as previous studies10. Briefly, the RBPs identified by oligodT pulldown methods 6, 7, 12, 32, 34, 35 were assigned as poly(A) RNA binding proteins. The RNA binding types of the remaining RBPs were manually evaluated based on previous literature (Table S2 and S3). For the analysis of RBDs (Figure 4A, B and S4A, B), the domains of nuclear and nucleolar RBPs were obtained from Pfam (Table S2 and S3). The classification of classical and non-classical RBDs was based on previous studies 7, 39. The numbers of RBPs containing at least one classic RBD, only containing non-classical RBDs or without any RBDs were determined for both annotated RBPs and RBP orphans (Figure 4A and S4A). The number of nuclear and nucleolar RBPs containing each RBD was shown in Figure 4B and S4B, respectively. To identify RBPs with higher RNA binding activities, the nuclear RBPs were ranked in a descending order according to the +FA/-FA ratio (Mean value of 128N/127C, 128C/127C and 129N/127C for nuclear RBPs shown in Figure 4D). The ROC analysis was performed using RBPs with RRM as TPs and RBPs with non-classical RBDs as FPs. The nuclear RBPs with +FA/-FA ratio below the cutoff were assigned with high RNA binding affinity (Table S2).
Analysis of proteomic data for OMM. The original identified proteins are shown in Table S4. To assign OMM RBPs under –PUR and +PUR conditions, a curated list of known OMM proteins45 was used as TPs and mitochondrial matrix proteins identified by APEX-mito profiling16 were assigned as FPs. For each replicate, the APEX-PS-OMM sample was not only compared to the negative control omitting H2O2, but also compared with the APEX-PS-NES sample. The proteins were first ranked in a descending order according to the TMT ratio and cutoff was assigned by ROC analysis as described above (Figure S5B). For assignment of OMM RBPs under basal condition, proteins above the cutoff of 127C/126C and 127C/131N were intersected for replicate 1 and proteins above the cutoff of 128N/126C and 128N/131N were intersected for replicate 2. For assignment of OMM RBPs under PUR treatment, proteins above the cutoff of 129C/128C and 129C/131C were intersected for replicate 1 and proteins above the cutoff of 130N/128C and 130N/131C were intersected for replicate 2. The resulting lists of the two biological replicates were intersected and the potential glycosylated proteins were removed to obtain the final OMM RBP list under basal and PUR condition, respectively (Table S5).
For the analysis of mitochondria specificity of the OMM RBPs (Figure 5E), a list of mitochondrial proteins were collected from MitoCarta database, GOCC terms containing mitochondrial annotations, mitochondrial matrix proteome and IMS proteome identified by APEX profiling. The number and percentage of mitochondrial proteins in human proteome, OMM proteins identified by APEX2-OMM profiling and the OMM RBPs under basal and PUR conditions was determined. For the analysis of OMM RBPs involved in mitochondrial-ER contact (Figure S5C), the number of OMM RBPs overlapped with proteins in mitochondrial-ER contact identified by split-TurboID46 was determined. For the analysis of RNA binding specificity of OMM RBPs (Figure 5F), the number of known RBPs described above in OMM RBPs was compared with that of OMM proteins identified by APEX profiling45.
Immunofluorescence staining and fluorescence microscopy. For fluorescence imaging experiments in Figure 2B, HEK293T cells expressing APEX2-NLS and APEX2-NIK3x were plated and labeled as described above. Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. Cells were then washed with PBS for three times and permeabilized with cold methanol at -20°C for 5-10 min. Cells were washed again three times with PBS and blocked for 1 h with 3% BSA in DPBS (‘‘blocking buffer’’) at room temperature. For APEX2-NLS imaging, cells were then incubated with anti-V5 antibody (1:1000 dilution, ThermoFisher Scientific) in blocking buffer for 1 h at room temperature. After washing three times with DPBS, cells were incubated with DAPI/secondary antibody (Alexa Fluor488), and neutravidin-Alexa Fluor647 in blocking buffer for 30 min. For APEX2-NIK3x imaging, cells were incubated with DAPI, and neutravidin-Alexa Fluor647 in blocking buffer for 30 min. Cells were then washed three times with DPBS and imaged. For fluorescence imaging in Figure S6B, HEK cells were treated with 200 μM puromycin for 30 min. Cells were fixed, washed and blocked as described above. Cells were incubated with anti-TOM20 (1:500 dilution, Santa Cruz Biotechnology) and anti-SYNJ2BP (1:500 dilution, Sigma-Aldrich) in blocking buffer for 1 h at room temperature. . After washing three times with DPBS, cells were incubated with DAPI/secondary antibody in blocking buffer for 30 min. Cells were then washed three times with DPBS and imaged. Fluorescence confocal microscopy was performed with a Zeiss AxioObserver microscope with 60× oil immersion objectives, outfitted with a Yokogawa spinning disk confocal head, Cascade II:512 camera, a Quad-band notch dichroic mirror (405/488/568/647), and 405 (diode), 491 (DPSS), 561 (DPSS) and 640 nm (diode) lasers (all 50 mW). DAPI (405 laser excitation, 445/40 emission), Alexa Fluor488 (491 laser excitation, 528/38 emission) and AlexaFluor647 (640 laser excitation, 700/75 emission) and differential interference contrast (DIC) images were acquired through a 60x oil-immersion lens. Acquisition times ranged from 100 to 2,000 ms. All images were collected and processed using SlideBook 6.0 software (Intelligent Imaging Innovations).
Metabolic labeling of RNA-protein complexes. For the validation of SYNJ2BP and EXD2 as RBPs (Figure 6B), HEK293T cells were grown to ~80% confluence in 15-cm dish and treated with 1 mM 5-EU for 16 h. The cells were washed with PBS for three times, followed by irradiation with 254-nm UV light at 150 mJ/cm2 (CL-1000 Ultraviolet Crosslinker, UVP). The cells were then lysed in 1 mL of 50 mM Tris-HCl (pH 7.5) buffer with sonication and subjected to centrifugation with 20000 g for 10 min to remove the debris. The lysates were reacted with 100 μM azide-PEG3-biotin (Click Chemistry Tools), 500 μM CuSO4, 2 mM THPTA (Sigma-Aldrich) and 5 mM sodium ascorbate (freshly prepared) for 2 h at r.t. with vortex, followed by adding 5 mM EDTA to stop the reaction. The lysates were precipitated with 8 vol of methanol at -80 °C for 1 h and washed twice with precooled methanol. The pellets were then resuspended in 1 mL RIPA lysis buffer with sonication and enriched by streptavidin beads overnight as we described above. After washing with RIPA buffer for three times, the beads were boiled in protein loading buffer with 2 mM biotin for 10 min. The samples were then analyzed by western blot with anti-SYNJ2BP and anti-EXD2 antibodies.
RNA-immunoprecipitation sequencing (RIP-seq). The RIP experiments were performed as described and high throughput sequencing services were provided by Cloud-Seq Biotech (Shanghai, China). The SYNJ2BP RIP was performed with SYNJ2BP antibody (Sigma Aldrich) and RNA-seq libraries were generated using the TruSeq Stranded Total RNA Library Prep Kit (Illumina) according to the manufacturer’s instructions and the library quality was evaluated with BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an illumina Hiseq instrument with 150 bp paired-end reads. The relative enrichment of each mRNA was obtained from the fold change of gene-level FPKM (fragments per kilobase of transcript per million mapped reads) values.
Cross-linking immunoprecipitation (CLIP). To validation of SYNJ2BP mRNA targets under –PUR and +PUR conditions, HEK293T cells were treated with 0 or 200 μM puromycin for 30 min. The cells were washed with 5 mL PBS for three times, crosslinked by 254-nm UV light at 150 mJ/cm2. Then the cells were lysed in 500 μL CLIP lysis buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and EDTA-free protease inhibitor mixture). The cell lysates were incubated on ice for 10 min and cleared by centrifugation at 13000 g for 15 min at 4 °C. 125 μL Dynabeads protein G (ThermoFisher Scientific) were washed with 500 μL lysis buffer for twice and incubated with 10 μg anti-SYNJ2BP antibody at r.t. for 45 min. The lysates were then incubated with the antibody-conjugated beads overnight at 4 °C. The beads were washed twice with 900 μL high salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate) and twice with 500 μL wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20). The beads were resuspended in 54 μL water, and then mixed with 33 μL 3X proteinase digestion buffer, 10 μL proteinase K (20 mg/mL, ThermoFisher Scientifi) and 3 μL Ribolock RNase inhibitor. The 3X proteinase digestion buffer were freshly prepared as follow: 330 μL 10X PBS, pH = 7.4 (Ambion); 330 μL 20% N-laurylsarcosine sodium solution (Sigma-Aldrich); 66 μL of 0.5 M ETDA; 16.5 μL of 1 M DTT; 357.5 μL water. Proteinase digestion was performed at 42 °C for 1 h and 55 °C for 1 h with vigorous mixing and the supernatant was collected. The recovered RNAs were purified using RNA clean and concentrator -5 kit (Zymo Research) and subjected to further analysis.
Generation of SYNJ2BP KO cells stably expressing APEX2 constructs. The non-targeted guide and SYNJ2BP KO HEK293T cells were generated previously 45. For preparation of lentiviruses, HEK293T cells in 6-well plates were transfected at 60%–70% confluency with the lentiviral vector pLX304 containing APEX2-OMM or APEX-NES (1,000 ng), the lentiviral packaging plasmids dR8.91 (900 ng) and pVSV-G (100 ng), and 8 mL of Lipofectamine 2000 for 4 h. About 48 h after transfection the cell medium containing lentivirus was harvested and filtered through a 0.45-mm filter. The non-targeted guide and SYNJ2BP KO HEK293T cells were then infected at ~50% confluency, followed by selection with 8 mg/mL blasticidin in growth medium for 7 days before further analysis.
APEX RNA labeling at OMM. APEX labeling was performed as described above in non-targeted guide and SYNJ2BP KO HEK293T cells stably expressing APEX2-OMM or APEX2-NES. The RNA was extracted from cells using the RNeasy plus mini kit (QIAGEN) following the manufacture protocol, including adding β-mercaptoethanol to the lysis buffer. The cells were sent through the genomic DNA (gDNA) eliminator column supplied with the kit. A modification to the protocol was replacing the RW1 buffer with RWT buffer (QIAGEN) for washing. The extracted RNA was eluted into RNase-free water and RNA concentrations were determined using the Nanodrop (ThermoFischer Scientific).
To enrich biotinylated RNAs, we used 10 μL Pierce streptavidin magnetic beads (ThermoFischer Scientific) per 25 mg of RNA. The beads were washed 3 times in B&W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.1% TWEEN 20), followed by 2 times in Solution A (0.1 M NaOH and 0.05 M NaCl), and 1 time in Solution B (0.1 M NaCl). The beads were then suspended in 100-150 mL 0.1 M NaCl and incubated with 100-125 mL RNA on a rotator for 2 h at 4°C. The beads were then placed on a magnet and the supernatant discarded. Beads were washed 3 times in B&W buffer and resuspended in 54 mL water. A 3X proteinase digestion buffer was made (1.1 mL buffer contained 330 mL 10X PBS pH = 7.4 (Ambion), 330 μL 20% N-Lauryl sarcosine sodium solution (Sigma Aldrich), 66 mL 0.5M EDTA, 16.5 mL 1Mdithiothreitol (DTT, ThermoFischer Scientific) and 357.5 mL water). 33 μL of this 3X proteinase buffer was added to the beads along with 10 mL Proteinase K (20 mg/mL, Ambion) and 3 mL Ribolock RNase inhibitor. The beads were then incubated at 42°C for 1 h, followed by 55°C for 1 h on a shaker. The RNA was then purified using the RNA clean and concentrator -5 kit (Zymo Research) and subjected to further analysis.
RT-qPCR. For the RT-qPCR analysis of CLIP and APEX RNA labeling experiments, the enriched RNA was first reverse transcribed following the Superscript III reverse transcriptase (ThermoFischer Scientific) protocol using random hexamers as primers. The resulting cDNA was then tested using qPCR using the primers above in 2X SYBR Green PCR Master Mix (ThermoFischer Scientific), with data generated on Lightcycler 480 (Roche).
Azidohomoalanine labeling. To evaluate the impact of SYNJ2BP on protein synthesis of its clients (Figure 6G, 7E and 7F), cells were cultured in methionine-free medium supplemented with 1 mM azidohomoalanine (AHA). Cells were lysed in RIPA buffer and protein concentration was normalized to 2 mg/mL. 1 mL lysates were reacted with 100 μM biotin-PEG4-alkyne, premixed 2-(4-((bis((1-tertbutyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol1-yl)-acetic
acid (BTTAA)-CuSO4 complex (500 μM CuSO4, BTTAA:CuSO4 with a 2:1 molar ratio) and 2.5 mM freshly prepared sodium ascorbate for 2 h at room temperature. The resulting lysates were precipitated by 8 mL methanol at -80°C overnight and the precipitated proteins were centrifuged at 8000 g for 5 min at 4 °C. The proteins were washed twice with 1 mL cold methanol and resuspended in 1 mL RIPA buffer with sonication. The biotinylated proteins were further captured by 200 μL streptavidin magnetic beads for 2 h. The beads were washed as described above and proteins were eluted by boiling the beads in 75 μL of 3× protein loading buffer supplemented with 20 mM DTT and 2 mM biotin. The resulting samples were analyzed by western bloting with antibodies indicated.
Complex III and IV activity assay. Complex III activity was assayed using a mitochondrial complex III activity assay kit (Sigma Aldrich) and complex IV activity was determined using a complex IV human enzyme activity microplate assay kit (Abcam). HEK293T expressing non-targeted Cas9 and SYNJ2BP knockout cells were obtained from our previous study 45 were plated in 15 cm dish. Cell pellets were lysed and mitochondrion was purified according to the manufacturer’s protocol in a mitochondrial isolation kit for cultured cells (Abcam). The activity was determined by following the manufacturer’s protocol with a standard curve.
Cell proliferation assays. In order to determine the effect of SYNJ2BP on cell proliferation (Figure S7A), HEK293T expressing non-targeted Cas9 and SYNJ2BP knockout cells were obtained from our previous study 45. The MTS assay was performed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega), following the manufacturer’s instructions. 1 × 104 cells per well were plated in 96-well plates with 100 μL fresh medium per well. The cells were cultured for 1-3 days, and the medium was freshly changed every 24 h. 20 μL of CellTiter 96 AQueous One Solution Reagent was added into each well and incubated for 4 h.
To evaluate the impact of SYNJ2BP knockout on cell viability under CHX and PUR treatment (Figure 7A and S7B), 1 × 104 cells per well were plated in 96-well plates with 100 μL fresh medium per well. After 24 h, the cells were treated with desired concentration (0, 25, 50, 100, 200 and 400 μM) of drugs for 12 h and then changed into normal medium for another 12 h. For glucose/galactose cell viability assay (Figure 7B), 2000 cells per well were plated in 96-well plates with 100 μL fresh medium per well. After 24 h, cells were washed with DPBS and the growth medium was replaced with medium containing 10% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin and DMEM without glucose supplemented with 10 mM galactose or 10 mM glucose, as well as 200 μM drugs. After 24 h, the cells were changed into the mediums without drugs for 48 h and 20 μL of CellTiter 96 AQueous One Solution Reagent was added into each well and incubated for 4 h.
To evaluate the cellular recovery from heat stress (Figure 7H), 1 × 104 cells per well were plated in 96-well plates with 100 μL fresh medium per well. After 24 h, the cells were incubated at 42°C for 1 h and then incubated at 37°C for 1-3 days. For the sodium arsenite stress, cells were treated with 400 μM sodium arsenite for 1 h and then cultured in the normal medium for 1-3 days. 20 μL of CellTiter 96 AQueous One Solution Reagent was added into each well and incubated for 4 h. The absorbance at 490 nm was recorded using a 96-well plate reader. Each biological experiment has five technical replicates and three biological replicates were performed.
Statistical analysis. For comparison between two groups, P values were determined using two-tailed Student’s t tests, *P < 0.05; **P < 0.01; ***P < 0.001; N.S. not significant. For all box plots (Figure 3c, Supplementary Figure 4c, 5c, 5d and 6d), P values were calculated with Wilcoxon rank sum by R (*P < 0.05; **P < 0.01; ***P < 0.001). Error bars represent means ± SD.
Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Additional data beyond that provided in the Figures and Supplementary Information are available from the corresponding author upon request.