Spatial Organization of Single mRNPs at Different Stages of the Gene Expression Pathway.

Contact for Reagent and Resource Sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Daniel Zenklusen ( daniel.r.zenklusen@umontreal.ca ) Method Details Reagents used, stock concentrations, working concentrations and treatment conditions Puromycin dihydrochloride (Sigma P8833) – stock at 5 mg/ml in water, Cycloheximide (Sigma C7698–1G) – stock 5 mg/ml in ethanol, Sodium Arsenite (Sigma 35000–1L-R) – stock 50 mM in water, Homoharringtonine (Sigma SML1091–10MG) – stock 10mg/ml in DMSO. The drugs were diluted in warm media to get final working concentrations and cells were treated prior to fixation as follows: Puromycin (100µg/ml for 10 min), Cycloheximide (100µg/ml for 10min), Homoharringtonine – 100µg/ml for 10 mins or 1hr and Sodium Arsenite (2mM for 1 hour). Cell culture and drug treatment HEK293 (American Type Culture Collection CRL-1573) and U2OS osteosarcoma (American Type Culture Collection HTB-96) cell lines were maintained at 37°C and 5% CO 2 in Dulbecco’s Modified Eagle Medium (DMEM) (Wisent, 319–005-CL) supplemented with 10% fetal bovine serum (FBS) (Wisent, 080–150) and passaged every 2–3 days with Trypsin (Wisent 325–043-EL). Cells were plated on poly-L-Lysine (Sigma, P8920) coated coverslips the day before treatment and fixation. On the day of the experiment, media was replaced with fresh warm media containing drug in indicated concentrations and placed back in the incubator. After treatment, the cells were briefly washed with 1xPBS, fixed with 4% paraformaldehyde in 1xPBS (pH 7.4) for 10 minutes at room temperature, washed three times with 1xPBS and stored overnight in 70% ethanol at −20°C for permeabilization. Alternatively, the cells were permeabilized using 0.1% TritonX-100 + 0.5%BSA in 1x PBS for 15mins after which they were washed 2 times with 1x PBS for 5 mins each immediately before using the samples for smFISH ( Figure S2B ). Plasmid Preparation: The phage-ubc-flag-24xSunTag-Fluc-oxBFP-AID-baUTR-24xMS2 plasmid was prepared as described in ( Bin Wu et al., 2016 ).

Generation and screening of eIF4G1 and PABPC1 mutant cell lines

Mutant cell lines were generated using CRISPR-Cas9. To produce sgRNAs targeting either eIF4G1 or PABPC1, annealed DNA oligos ( Table S1 ) were ligated into the BbsI site of plasmid pX330 (Ran 2013). Homology repair constructs containing the intended mutations and upstream and downstream homology arms (~1 kb in total) were ligated into the plasmid Lox-Stop-Lox-TOPO-∆stop ( Rakheja 2014 ), in which homology arms are cloned surrounding a puromycin resistance cassette flanked by loxP sites ( Table S1 ). HEK293 cells (5 × 105 cells in one well of a 6-well plate) were transfected with 250 ng of the pX330-sgRNA construct and 1 µg of the repair construct using Lipofectamine 2000 according to the manufacturer instructions, and then incubated in EMEM supplemented with 10% FBS in a humidified incubator at 37°C with 5% CO2. Two days following transfection, cells were trypsinized and 10% of the cells were moved into a 15-cm dish. After 24 h, puromycin was added to a final concentration of 3 µg/mL, and the media was changed daily for the next 3 days. The following day, single cells were seeded into each well of a 96-well plate on a MoFlo Astrios cell sorter (Beckman Coulter) at the Flow and Mass Cytometry Facility at SickKids Hospital, Toronto. Following expansion of single colonies, cells were harvested and screened by PCR using primers that anneal to the genome outside of the homology arm region ( Table S1 ). To excise the puromycin resistance cassette from positive clones, the cells were transfected with 1µg of pgk-Cre ( Rakheja et al., 2014 ) and incubated for 3 days before single-cell seeding, expansion, and screening for loss of the puromycin resistance gene by PCR as described above. The PCR products were analyzed by Sanger sequencing to ensure that the intended mutations were present.

Cell viability assays

Cell viability was measured using PrestoBlue Cell Viability Reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, cells were seeded in triplicate in 96-well plates at 1000 cells per well in 90µL of EMEM supplemented with 10% FBS, and then incubated at 37°C with 5% CO 2 . At 24h, 48h, and 72h after seeding, 10µL of PrestoBlue reagent was added to each well. After a further 6.5-h incubation at 37°C with 5% CO 2 , the fluorescence of each well was read on a SpectraMax M2 microplate reader (Molecular Devices).

Polysome profiling

To generate polysome profiles, cycloheximide was added to cells in a 10-cm dish to a final concentration of 100 µg/mL, and the cells were incubated for 10 min at 37°C. The cells were then placed on ice and washed twice with ice-cold PBS containing 100 µg/mL cycloheximide. Cells were lysed by shearing four times through a 26-gauge needle in 500 µL of lysis buffer (10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2 mM DTT, 500 U/ml Rnasin (Promega), EDTA-free protease inhibitor cocktail (Sigma), 100 µg/mL cycloheximide). Following centrifugation at 1300 × g for 10 min at 4°C, the supernatant was collected, flash frozen in liquid nitrogen, and stored at −80°C until further processing. Lysates were separated by loading 300 µL onto a 10–50% (w/v) sucrose gradient prepared with a Gradient Master (BioComp Instruments) and centrifuging for 2 h at 36,000 rpm in a SW41Ti rotor (Beckman Coulter) at 4°C. Gradients were fractionated on a Piston Gradient Fractionator (BioComp) coupled to an EM-1 Econo UV detector (Bio-Rad). UV profile data were recorded using Gradient Profiler software v 2.07 (BioComp). smRNA FISH Custom DNA probe sets were designed using Stellaris® Probe Designer, synthetized by Biosearch Technologies containing 3’ amine reactive group and labeled with far red dye Cy5 (GEPA25001), red dyes Cy3 (GEPA23001) from Sigma or Dylight 550 (Thermo Scientific 62263) or green dye Dy488 (Thermo Scientific 46403) as described in ( Rahman et al., 2017 ). For the mRNAs and the lncRNAs, the isoforms used to design the probes are mentioned in Figure S1 . For the mRNAs, these isoforms were verified as the predominantly expressed transcripts in HEK293 using RNA-seq datasets from human protein atlas. For the lncRNAs, the probes were designed such that they hybridize to the longer isoforms. Probe sequences are shown in Table S2 . Probe combination used are shown in Table S3 and the probe combinations used for the experiment is mentioned in the figure legends. smFISH was done as described in ( Rahman et al., 2017 ). Prior to hybridization, cells were rehydrated in 1xPBS, then washed with 10% formamide/2xSSC for 10 minutes at room temperature. The cells were hybridized with 1020 ng of each probe mix plus 40 µg of ssDNA/tRNA resuspended in the hybridization solution (10% dextran sulfate/10% formamide/2xSSC/2 mM VRC/0.1 mg/ml BSA) for 3 hours in the dark at 37°C. Post hybridization washes (2× 30 min) were carried out at 37°C with 10% formamide/2xSSC. Samples were then rinsed with 1xPBS and mounted with ProLong Gold antifade reagent with DAPI ( P36935 , Invitrogen).

Image Acquisition and pixel shift correction

Images were acquired with a 63x NA 1.46 oil objective on a Zeiss Elyra PS.1 system equipped with an Andor EMCCD iXon3 DU-885 CSO VP461 camera (1004×1002 pixels), the following filter sets: DAPI: BP420–480 + LP750 (Zeiss SR cube 07), Cy2: BP495–590+LP750 (Zeiss SR cube 13), Cy3: LP570 (Zeiss SR cube 14), Cy5: LP655 (Zeiss SR cube 10) and the following lasers: 50 mW 405 nm HR diode, 100 mW 488 nm HR diode, 100 mW 561 nm HR DPSS, 150 mW 642 nm HR diode. Each image was acquired using 3 rotations and a grid size of 42 µm for all channels. The microscope was located in a temperature-controlled room and samples were kept in the room for at least an hour before imaging to minimize thermal fluctuations. To correct for pixel shifts between channels, 0.1 µm TetraSpec beads (Invitrogen T-7279) were imaged in all channels, and the channel shift values and chromatic aberration were calculated and corrected using the built-in channel alignment tool in ZEN 2012 SP5 which uses an affine image alignment algorithm and later applied to the images. This correction was calculated for each day of imaging. Combined smFISH and Immunofluorescence for simultaneous detection of mRNA conformation and nascent translation Human U2OS osteoscarcoma cell line (American Type Culture Collection HTB-96) expressing stdMCP-Halotag, phR-scFV-GCN4-sfGFP-GB1-NLS-dWPRE, and pBabe-TIR1–9myc was prepared as described in ( Bin Wu et al., 2016 ). Single-molecule FISH immunofluorescence was performed as described in ( Bin Wu et al., 2016 ). In brief, cells were plated on 18mm diameter, #1 collagen coated coverslips (Fisher) in a 12-well dish. Cells were then transfected with 250 ng of the phage-ubc-flag-24xSunTag-Fluc-oxBFP-AID-baUTR-24xMS2 construct using X-tremeGENE 9 transfection reagent (XTG9-RO ROCHE). Six hours after transfection, IAA (Sigma-Aldrich) was added to a final concentration of 250 µM. 20 hours after transfection, fresh IAA was added to a final concentration of 250 µM. 24 hours after transfection, cells were fixed for 10 minutes in PBS + 5 mM MgCl 2 (PBSM), permeabilized for 15 minutes in PBSM + 0.1% Triton-X and 0.5 % BSA, and incubated with 100 nM MS2v5-Cy5 and 50 nM SunTagV4-Qusar 570 smFISH probe sets ( Table S2 ) and a primary antibody against GFP (GFP-1010, Aves labs, Inc.) and incubated for three hours at 37°C. After washing, cells were incubated with Alexa Fluor 488 labeled secondary antibody (ThermoFischer) and mounted in ProLong Diamond antifade reagent with DAPI (Life Technologies). Images were acquired on a custom inverted wide-field Nikon Eclipse Ti-E microscope equipped with three Andor iXon DU897 EMCCD cameras (512×512 pixels), Apochromatic TIRF 100X Oil Immersion Objective Lens/1.49 NA (Nikon MRD01991), encoded Stage with 150 micron Piezo Z (ASI), and LU-n4 four laser unit with solid state 405 nm, 488 nm, 561 nm, and 640 nm lasers (Nikon), a TRF89901 -EM ET-405/488/561/640nm Laser Quad Band Filter Set for TIRF applications (Chroma), and Nikon HTIRF system. Images were acquired using in-unit intermediate 1.5x magnification changer for a final magnification of 150x and independent, epi-illumination from the 488, 561, and 640 nm lasers. Image pixel size: XY, 106.7 nm; Z-step, 200 nm. A total of 29 cells without drug treatment (total of individual 396 mRNAs) and 40 cells (97 individual mRNAs) upon puromycin treatment were analyzed.

Immunoprecipitations and western blotting

Cells were washed with 1X PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4) and then lysed with 1 ml ice-cold lysis buffer A (100 mM KCl, 0.1 mM EDTA, 20 mM Hepes, pH 7.6, 0.4% NP-40, 10% glycerol, with freshly added 1 mM DTT and complete mini EDTA-free protease inhibitors [Roche; one tablet per 25 ml lysis buffer]) per 2.5 million cells. 50 µl was saved as the input sample. Cells were incubated with antibody (diluted according to manufacturer’s instructions) for 2 hours, rotating at 4°C. α-PABPC1 antibody was purchased from Abcam (ab21060), and α-eIF4G1 from MBL International. EZ view protein G Sepharose (Sigma) was washed twice with lysis buffer and added to lysate with 40 µl slurry used per ml of lysate. The beads and lysate were incubated with the lysate for an additional hour rotating at 4°C. The beads were washed 3X with cold lysis buffer. After the first wash, the beads were transferred to a new tube. The beads were then resuspended in protein loading dye (Life Technologies) with freshly added reducing agent, according to manufacturer’s instructions, and boiled for 3 min. 2% lysate and 10% immunoprecipitants were loaded onto an SDS-PAGE gel and probed for PABPC1 and eIF4G1. α-PABPC1 and α-eIF4G1 were used at 1:1000, and α rabbit IgG HRP (at 1:10,000) was used as the secondary antibody.

Quantification and Statistical Analysis

RNA spot detection, spot assignment and distance measurements For image analysis, 3D datasets were reduced to 2D data using maximum projections in FiJi. Spot detection was done by 2D Gaussian fitting as described in ( Thompson et al., 2002 ; Zenklusen et al., 2008 ). For 3D analysis, the spots were detected using AIRLOCALIZE as described in ( Lionnet et al., 2011 ). To separate cytoplasmic and nuclear mRNPs, masks were created in FiJi by manual segmentation using DAPI stained nuclei as reference, while ensuring that regions with overlapping spots within the same channel were not included. Assignment of the 5’, 3’ and/or the mid spots to either the cytoplasmic or the nuclear masks was done using MATLAB (MathWorks). To measure distances between different regions of mRNPs, spots from different channels were first grouped to assign neighboring spots corresponding a single RNA. This was achieved by using spots from one channel as a reference and finding spots from the other channels within a defined radius using the coordinates from 2D Gaussian fitting or 3D Gaussian fitting using a custom MATLAB script. 300nm for 2D analysis and 400nm for 3D analysis were chosen as radii to limit assigning signals from neighboring RNAs. These values were chosen as we observed very few RNAs with distances larger than these thresholds. Moreover, a threshold was required to ensure that there was no wrongful assignment of the signals. Groups with more than one spot from each channel, which could correspond to overlapping mRNPs or mRNPs close together in space, were discarded. For 2 color imaging, the 5’ signal was taken as reference and for 3 color imaging, the middle was taken as reference. Switching references yielded comparable results (not shown). 2D or 3D distances between different regions of the mRNPs were then calculated for each signal within a group.

Combined smFISH and Immunofluorescence Data Analysis

All image analysis was performed using existing or custom build packages in MatLab (MathWorks). Gaussian fitting of smFISH and immunofluorescence spot intensities was performed using FISH-quant ( Mueller et al., 2013 ). Briefly, cytoplasmic FISH spots were fit to a 3D Gaussian to determine the mRNA and translation site coordinates in each color. Both 5’-end, 3’-end, and translation site intensities were detected independently by this method. Image registration was performed by imaging 100 nm TetraSpeck Microspheres (ThermoFisher) and calibrating the field correction based on an affine transformation in a custom built MatLab package. The transformation matrix was first verified for reproducibility on other microsphere samples and then applied to mRNA samples (data not shown). Only 2D distances were considered for this analysis. To determine the end-to-end mRNA distance, we first assigned the Quasar 570 channel (SunTag Probes) to FITC channel (Alexa 488 labeled translation site) by setting a colocalization threshold of 300 nm after image correction. We then assigned the Quasar 570 to Cy5 (MS2 Probes), again with a colocalization threshold of 300 nm. We first grouped mRNA with both Cy3 and Cy5 colocalization, and then determined if there was also a colocalized translation site signal. We then binned two-color mRNA based on the presence (translating) or absence (non-translating) of translation site signal. We then determined the end-to-end distance, and, in the case of the translating mRNAs, the associated translation site intensity. Data Plotting All measurements were made for at least 2 independent biological replicates and the data plotted are representative from one of the replicates. For each measurement, at least 5 different fields were imaged with each image containing a minimum of 10 cells to make a total of at least 50 cells. For the smFISH plots, a minimum of 593 RNAs were considered for cytoplasmic plots and a minimum of 430 RNAs were considered for the nuclear plots for data from HEK293 cells and a minimum of 308 RNAs were considered for data from U2OS cells, unless mentioned otherwise. For the FISH-IF plots, a total of 323 data points for translating, 97 for puromycin and 73 for non-translating were considered. The translating mRNAs were clustered using k-means algorithm in R according to the intensity of the site of translation. After clustering, the four groups contained 64, 115, 104 and 40 RNAs from lower to higher intensity. The p-values were calculated using Kolmogorov-Smirnov test in R for the data points plotted. The center of mass plots in Figure 1G , 2D , 6D were made using R. The center of mass was calculated as the mean of the coordinates of the three regions. The different conformations were then aligned using their center of masses. For the 3-color scatter plot in Figure 2E , 6E , S5 and S7B , to get a pair of colocalization precision values, two values were chosen randomly from our data. These values were taken as the X and Y coordinates for the scatter plot. The values that served as the X and Y coordinates were used to get density plots in the same figure. The mean Radius of gyration () was calculated using: < R g > = 1 3 ∑ k = 1 3 ( r k − r m e a n ) 2 where k represents one of the three regions of the mRNP and r k the position of the corresponding position in space as determined by 2D Gaussian fitting. To calculate cell doubling times, fluorescence readings were taken at 24, 48, and 72 hrs after seeding the cells. The background fluorescence was subtracted, and the values were then normalized to the 24 hr time point. The slope of the line of best fit (after plotting in linear-log space) was determined and used to give the doubling time for each replicate. The doubling times were calculated in three independent replicates for each cell line, and then plotted as box-and-whisker plots in R.

Method Details Reagents used, stock concentrations, working concentrations and treatment conditions Puromycin dihydrochloride (Sigma P8833) – stock at 5 mg/ml in water, Cycloheximide (Sigma C7698–1G) – stock 5 mg/ml in ethanol, Sodium Arsenite (Sigma 35000–1L-R) – stock 50 mM in water, Homoharringtonine (Sigma SML1091–10MG) – stock 10mg/ml in DMSO. The drugs were diluted in warm media to get final working concentrations and cells were treated prior to fixation as follows: Puromycin (100µg/ml for 10 min), Cycloheximide (100µg/ml for 10min), Homoharringtonine – 100µg/ml for 10 mins or 1hr and Sodium Arsenite (2mM for 1 hour). Cell culture and drug treatment HEK293 (American Type Culture Collection CRL-1573) and U2OS osteosarcoma (American Type Culture Collection HTB-96) cell lines were maintained at 37°C and 5% CO 2 in Dulbecco’s Modified Eagle Medium (DMEM) (Wisent, 319–005-CL) supplemented with 10% fetal bovine serum (FBS) (Wisent, 080–150) and passaged every 2–3 days with Trypsin (Wisent 325–043-EL). Cells were plated on poly-L-Lysine (Sigma, P8920) coated coverslips the day before treatment and fixation. On the day of the experiment, media was replaced with fresh warm media containing drug in indicated concentrations and placed back in the incubator. After treatment, the cells were briefly washed with 1xPBS, fixed with 4% paraformaldehyde in 1xPBS (pH 7.4) for 10 minutes at room temperature, washed three times with 1xPBS and stored overnight in 70% ethanol at −20°C for permeabilization. Alternatively, the cells were permeabilized using 0.1% TritonX-100 + 0.5%BSA in 1x PBS for 15mins after which they were washed 2 times with 1x PBS for 5 mins each immediately before using the samples for smFISH ( Figure S2B ). Plasmid Preparation: The phage-ubc-flag-24xSunTag-Fluc-oxBFP-AID-baUTR-24xMS2 plasmid was prepared as described in ( Bin Wu et al., 2016 ).

Generation and screening of eIF4G1 and PABPC1 mutant cell lines

Mutant cell lines were generated using CRISPR-Cas9. To produce sgRNAs targeting either eIF4G1 or PABPC1, annealed DNA oligos ( Table S1 ) were ligated into the BbsI site of plasmid pX330 (Ran 2013). Homology repair constructs containing the intended mutations and upstream and downstream homology arms (~1 kb in total) were ligated into the plasmid Lox-Stop-Lox-TOPO-∆stop ( Rakheja 2014 ), in which homology arms are cloned surrounding a puromycin resistance cassette flanked by loxP sites ( Table S1 ). HEK293 cells (5 × 105 cells in one well of a 6-well plate) were transfected with 250 ng of the pX330-sgRNA construct and 1 µg of the repair construct using Lipofectamine 2000 according to the manufacturer instructions, and then incubated in EMEM supplemented with 10% FBS in a humidified incubator at 37°C with 5% CO2. Two days following transfection, cells were trypsinized and 10% of the cells were moved into a 15-cm dish. After 24 h, puromycin was added to a final concentration of 3 µg/mL, and the media was changed daily for the next 3 days. The following day, single cells were seeded into each well of a 96-well plate on a MoFlo Astrios cell sorter (Beckman Coulter) at the Flow and Mass Cytometry Facility at SickKids Hospital, Toronto. Following expansion of single colonies, cells were harvested and screened by PCR using primers that anneal to the genome outside of the homology arm region ( Table S1 ). To excise the puromycin resistance cassette from positive clones, the cells were transfected with 1µg of pgk-Cre ( Rakheja et al., 2014 ) and incubated for 3 days before single-cell seeding, expansion, and screening for loss of the puromycin resistance gene by PCR as described above. The PCR products were analyzed by Sanger sequencing to ensure that the intended mutations were present.

Cell viability assays

Cell viability was measured using PrestoBlue Cell Viability Reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, cells were seeded in triplicate in 96-well plates at 1000 cells per well in 90µL of EMEM supplemented with 10% FBS, and then incubated at 37°C with 5% CO 2 . At 24h, 48h, and 72h after seeding, 10µL of PrestoBlue reagent was added to each well. After a further 6.5-h incubation at 37°C with 5% CO 2 , the fluorescence of each well was read on a SpectraMax M2 microplate reader (Molecular Devices).

Polysome profiling

To generate polysome profiles, cycloheximide was added to cells in a 10-cm dish to a final concentration of 100 µg/mL, and the cells were incubated for 10 min at 37°C. The cells were then placed on ice and washed twice with ice-cold PBS containing 100 µg/mL cycloheximide. Cells were lysed by shearing four times through a 26-gauge needle in 500 µL of lysis buffer (10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2 mM DTT, 500 U/ml Rnasin (Promega), EDTA-free protease inhibitor cocktail (Sigma), 100 µg/mL cycloheximide). Following centrifugation at 1300 × g for 10 min at 4°C, the supernatant was collected, flash frozen in liquid nitrogen, and stored at −80°C until further processing. Lysates were separated by loading 300 µL onto a 10–50% (w/v) sucrose gradient prepared with a Gradient Master (BioComp Instruments) and centrifuging for 2 h at 36,000 rpm in a SW41Ti rotor (Beckman Coulter) at 4°C. Gradients were fractionated on a Piston Gradient Fractionator (BioComp) coupled to an EM-1 Econo UV detector (Bio-Rad). UV profile data were recorded using Gradient Profiler software v 2.07 (BioComp). smRNA FISH Custom DNA probe sets were designed using Stellaris® Probe Designer, synthetized by Biosearch Technologies containing 3’ amine reactive group and labeled with far red dye Cy5 (GEPA25001), red dyes Cy3 (GEPA23001) from Sigma or Dylight 550 (Thermo Scientific 62263) or green dye Dy488 (Thermo Scientific 46403) as described in ( Rahman et al., 2017 ). For the mRNAs and the lncRNAs, the isoforms used to design the probes are mentioned in Figure S1 . For the mRNAs, these isoforms were verified as the predominantly expressed transcripts in HEK293 using RNA-seq datasets from human protein atlas. For the lncRNAs, the probes were designed such that they hybridize to the longer isoforms. Probe sequences are shown in Table S2 . Probe combination used are shown in Table S3 and the probe combinations used for the experiment is mentioned in the figure legends. smFISH was done as described in ( Rahman et al., 2017 ). Prior to hybridization, cells were rehydrated in 1xPBS, then washed with 10% formamide/2xSSC for 10 minutes at room temperature. The cells were hybridized with 1020 ng of each probe mix plus 40 µg of ssDNA/tRNA resuspended in the hybridization solution (10% dextran sulfate/10% formamide/2xSSC/2 mM VRC/0.1 mg/ml BSA) for 3 hours in the dark at 37°C. Post hybridization washes (2× 30 min) were carried out at 37°C with 10% formamide/2xSSC. Samples were then rinsed with 1xPBS and mounted with ProLong Gold antifade reagent with DAPI ( P36935 , Invitrogen).

Image Acquisition and pixel shift correction

Images were acquired with a 63x NA 1.46 oil objective on a Zeiss Elyra PS.1 system equipped with an Andor EMCCD iXon3 DU-885 CSO VP461 camera (1004×1002 pixels), the following filter sets: DAPI: BP420–480 + LP750 (Zeiss SR cube 07), Cy2: BP495–590+LP750 (Zeiss SR cube 13), Cy3: LP570 (Zeiss SR cube 14), Cy5: LP655 (Zeiss SR cube 10) and the following lasers: 50 mW 405 nm HR diode, 100 mW 488 nm HR diode, 100 mW 561 nm HR DPSS, 150 mW 642 nm HR diode. Each image was acquired using 3 rotations and a grid size of 42 µm for all channels. The microscope was located in a temperature-controlled room and samples were kept in the room for at least an hour before imaging to minimize thermal fluctuations. To correct for pixel shifts between channels, 0.1 µm TetraSpec beads (Invitrogen T-7279) were imaged in all channels, and the channel shift values and chromatic aberration were calculated and corrected using the built-in channel alignment tool in ZEN 2012 SP5 which uses an affine image alignment algorithm and later applied to the images. This correction was calculated for each day of imaging. Combined smFISH and Immunofluorescence for simultaneous detection of mRNA conformation and nascent translation Human U2OS osteoscarcoma cell line (American Type Culture Collection HTB-96) expressing stdMCP-Halotag, phR-scFV-GCN4-sfGFP-GB1-NLS-dWPRE, and pBabe-TIR1–9myc was prepared as described in ( Bin Wu et al., 2016 ). Single-molecule FISH immunofluorescence was performed as described in ( Bin Wu et al., 2016 ). In brief, cells were plated on 18mm diameter, #1 collagen coated coverslips (Fisher) in a 12-well dish. Cells were then transfected with 250 ng of the phage-ubc-flag-24xSunTag-Fluc-oxBFP-AID-baUTR-24xMS2 construct using X-tremeGENE 9 transfection reagent (XTG9-RO ROCHE). Six hours after transfection, IAA (Sigma-Aldrich) was added to a final concentration of 250 µM. 20 hours after transfection, fresh IAA was added to a final concentration of 250 µM. 24 hours after transfection, cells were fixed for 10 minutes in PBS + 5 mM MgCl 2 (PBSM), permeabilized for 15 minutes in PBSM + 0.1% Triton-X and 0.5 % BSA, and incubated with 100 nM MS2v5-Cy5 and 50 nM SunTagV4-Qusar 570 smFISH probe sets ( Table S2 ) and a primary antibody against GFP (GFP-1010, Aves labs, Inc.) and incubated for three hours at 37°C. After washing, cells were incubated with Alexa Fluor 488 labeled secondary antibody (ThermoFischer) and mounted in ProLong Diamond antifade reagent with DAPI (Life Technologies). Images were acquired on a custom inverted wide-field Nikon Eclipse Ti-E microscope equipped with three Andor iXon DU897 EMCCD cameras (512×512 pixels), Apochromatic TIRF 100X Oil Immersion Objective Lens/1.49 NA (Nikon MRD01991), encoded Stage with 150 micron Piezo Z (ASI), and LU-n4 four laser unit with solid state 405 nm, 488 nm, 561 nm, and 640 nm lasers (Nikon), a TRF89901 -EM ET-405/488/561/640nm Laser Quad Band Filter Set for TIRF applications (Chroma), and Nikon HTIRF system. Images were acquired using in-unit intermediate 1.5x magnification changer for a final magnification of 150x and independent, epi-illumination from the 488, 561, and 640 nm lasers. Image pixel size: XY, 106.7 nm; Z-step, 200 nm. A total of 29 cells without drug treatment (total of individual 396 mRNAs) and 40 cells (97 individual mRNAs) upon puromycin treatment were analyzed.

Immunoprecipitations and western blotting

Cells were washed with 1X PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4) and then lysed with 1 ml ice-cold lysis buffer A (100 mM KCl, 0.1 mM EDTA, 20 mM Hepes, pH 7.6, 0.4% NP-40, 10% glycerol, with freshly added 1 mM DTT and complete mini EDTA-free protease inhibitors [Roche; one tablet per 25 ml lysis buffer]) per 2.5 million cells. 50 µl was saved as the input sample. Cells were incubated with antibody (diluted according to manufacturer’s instructions) for 2 hours, rotating at 4°C. α-PABPC1 antibody was purchased from Abcam (ab21060), and α-eIF4G1 from MBL International. EZ view protein G Sepharose (Sigma) was washed twice with lysis buffer and added to lysate with 40 µl slurry used per ml of lysate. The beads and lysate were incubated with the lysate for an additional hour rotating at 4°C. The beads were washed 3X with cold lysis buffer. After the first wash, the beads were transferred to a new tube. The beads were then resuspended in protein loading dye (Life Technologies) with freshly added reducing agent, according to manufacturer’s instructions, and boiled for 3 min. 2% lysate and 10% immunoprecipitants were loaded onto an SDS-PAGE gel and probed for PABPC1 and eIF4G1. α-PABPC1 and α-eIF4G1 were used at 1:1000, and α rabbit IgG HRP (at 1:10,000) was used as the secondary antibody.

Supplementary Material 1 2 Table S2. smFISH probes used in this study, Related to STAR Methods.