Characterization of ribosome stalling and no-go mRNA decay stimulated by the fragile X protein, FMRP

Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal noncanonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNA•ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not poly(A)-binding protein, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of four putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes but only stimulates NGD of a small select set of transcripts, revealing a minor role of FMRP that would be misregulated in fragile X syndrome.

using MagicMedia E. coli Expression Medium (Thermo Fisher # K6803) supplemented with 50 µg/mL kanamycin and 35 µg/mL chloramphenicol for auto-induction.A 5 mL starter culture in LB media supplemented with 50 µg/mL kanamycin, 35 µg/mL chloramphenicol, and 1% glucose (w/v) was inoculated with a single colony and grown overnight at 37°C, 250 rpm. 1 mL of a warm and fresh overnight starter culture was then used to inoculate 50 mL of room temperature MagicMedia and incubated for 48-72 hrs at 18°C, 160 rpm in a 250 mL baffled flask.After autoinduction, cultures were pelleted and stored at -20°C for purification later.Recombinant proteins were purified using a dual affinity approach, first using the C-terminal His6-tag, then the Nterminal MBP-tag.Cell pellets were resuspended and lysed with BugBuster Master Mix (Sigma # 71456) using the recommended 5 mL per 1 g wet cell pellet ratio for 10 min at room temperature with gentle end-over-end rotation (10-15 rpm).Lysates were placed on ice and kept cold moving forward.Lysates were cleared by centrifugation for 20 min at 18,000 rcf in a chilled centrifuge (4°C).Lysates were then incubated with HisPur Cobalt Resin (Thermo Fisher # 89965) in a Peirce centrifugation column (Thermo # 89897) for 30 min at 4°C with gentle endover-end rotation.Columns were centrifuged in a pre-chilled (4°C) Eppendorf 5810R for 2 min at 700 rcf to eliminate the flow through and then were washed 5X with two resin-bed volumes of ice-cold Cobalt IMAC Wash Buffer (50 mM Na 3 PO 4 , 300 mM NaCl, 10 mM imidazole; pH 7.4) in a pre-chilled (4°C) Eppendorf 5810R for 2 min at 700 rcf.His-tagged proteins were then eluted in a single elution step with two resin-bed volumes of ice-cold Cobalt IMAC Elution Buffer (50 mM Na 3 PO 4 , 300 mM NaCl, 150 mM imidazole; pH 7.4) by gravity flow.Eluates were then incubated with Amylose resin (NEB # E8021) in a centrifugation column for 2 hrs at 4°C with gentle end-over-end rotation (10-15 rpm).Columns were washed 5X with at least two bedvolumes of ice-cold MBP Wash Buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA; pH 7.4) by gravity flow.MBP-tagged proteins were then eluted by a single elution step with two resin-bed volumes of ice-cold MBP Elution Buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 10 mM maltose; pH 7.4) by gravity flow.Recombinant proteins were then desalted and buffer exchanged into Protein Storage Buffer (25 mM Tris-HCl, 125 mM KCl, 10% glycerol; pH 7.4) using a 7K MWCO Zeba Spin Desalting Column (Thermo Fisher # 89892) and, if needed, concentrated using 10K MWCO Amicon Ultra-4 (EMD Millipore # UFC803024).Recombinant protein concentration was determined by Pierce Detergent Compatible Bradford Assay Kit (Thermo Fisher # 23246) with BSA standards diluted in Protein Storage Buffer as well as SDS-PAGE and Coomassie staining before aliquoting in single use volumes, snap freezing in liquid nitrogen, and storage at -80°C.

mRNP formation and in vitro translation
In vitro translation was performed in the dynamic linear range as previously described but adapted to translate mRNPs (9, 23, and Kearse et al., 2016).30 nM in vitro transcribed nLuc reporter mRNA was diluted in RNA Dilution Buffer (10 mM Tris-HCl, 5 mM Mg(OAc) 2 , 100 mM KCl; pH 7.4).In a total of 4 µL, 30 fmol of nLuc reporter mRNA was mixed with 0-10 picomol of recombinant protein and 100 picomol of UltraPure BSA (Thermo Fisher # AM2618) on ice for 1 hr.UltraPure BSA stock was diluted in protein storage buffer and its addition was necessary to prevent non-specific binding of the reporter mRNA to the tube.For in vitro translation reactions, 6 µL of a Rabbit Reticulocyte Lysate (RRL) master mix was added to each 4µL mRNP complex.10 μL in vitro translation reactions were performed in the linear range using 3 nM mRNA in the Flexi RRL System (Promega # L4540) with final concentrations of reagents at 30% RRL, 10 μM amino acid mix minus leucine, 10 μM amino acid mix minus Methionine, 0.5 mM Mg(OAc) 2 , 100 mM KCl, 8 U murine RNase inhibitor (NEB # M0314), 0-1 µM recombinant protein, and 10 µM UltraPure BSA.Reactions were incubated for 30 min at 30°C, terminated by incubation on ice and diluted 1:5 in Glo Lysis Buffer (Promega # E2661).25 μL of prepared Nano-Glo reagent (Promega # N1120) was mixed with 25 μL of diluted reaction and incubated at room temperature for 5 min in the dark (with gentle shaking during the first minute), and then read on a Promega GloMax Discover Multimode Microplate Reader.FFLuc mRNA was treated and translated exactly the same; FFLuc luminescence was measured exactly the same but used ONE-Glo (Promega # E6110) instead of Nano-Glo.

mRNA•ribosome dissociation assays with puromycin
We have previously described in detail and in a complete methods manuscript the use of the ability of puromycin to dissociate ribosomes from reporter mRNA with a low-speed sucrose cushion (9,23).nLuc reporter mRNA translation was performed as described above except that translation was limited to 15 min at 30°C.Samples were then placed on ice for 3 min before the addition of 0.1 mM puromycin (final) and further incubation at 30°C for 30 min.Control samples lacking puromycin (water added instead) were kept on ice.Cycloheximide (1.43 mg/mL final) was then added to all samples to preserve ribosome complexes on mRNAs and halt puromycin incorporation.In a separate tube, FFLuc reporter mRNA was translated as described above (3 nM mRNA conditions) for 15 min at 30°C and was terminated by the addition of 1.43 mg/mL cycloheximide (final) and incubation on ice.
The treated nLuc and FFLuc translation reactions (from above) were combined on ice and then mixed with an equal volume (28 µL) of ice-cold 2X Ribosome Dilution Buffer (40 mM Tris-HCl, 280 mM KCl, 20 mM MgCl 2 , 200 μg/ml cycloheximide, 2 mM DTT; pH 7.4).The entire 56 μl volume was then layered on top of 130 μl of ice-cold 35% (w/v) buffered sucrose (20 mM Tris-HCl, 140 mM KCl, 10 mM MgCl 2 , 100 μg/mL cycloheximide, 1 mM DTT; pH 7.4) in a prechilled 7 mm x 20 mm thick-walled polycarbonate ultracentrifuge tubes (Thermo Scientific # 45233) and centrifuged in a S100AT3 rotor at 4°C for 60 min at 50,000 x g (43,000 rpm) in a Sorvall Discovery M120 SE Micro-Ultracentrifuge.The supernatant was then discarded and each pellet was resuspended in 0.5 mL of TRIzol (Thermo Fisher # 15596018).Total RNA was extracted from each pellet following the manufacturer's protocol with glycogen (Thermo Fisher # R0561) added at the isopropanol precipitation step.The resulting RNA pellet was resuspended in 30 μL nuclease-free water.16 μL of extracted RNA was converted to cDNA using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad # 1708841).cDNA reactions were then diluted 10-fold with nuclease-free water and stored at -20°C or used immediately.RT-qPCR was performed in 15 μL reactions using iTaq Universal SYBR Green Supermix (Bio-Rad # 1725124) in a Bio-Rad CFX Connect Real-Time PCR Detection System with 1.5 μL diluted cDNA and 250 nM (final concentration) primers.nLuc reporter mRNA abundance was normalized to the spikedin control FFLuc mRNA using the Bio-Rad CFX Maestro software (ΔΔCt method).Abundance of total signal was calculated using Q n = 2 ΔΔCt and P = 100 × Q n /Q total as previously described (Pringle et al., 2019).Primers for RT-qPCR can be found in Table S6.

Cell culture and siRNA knockdowns
N2A cells were obtained from ATCC (# CCL-131) and maintained in high glucose DMEM (Thermo # 11995065) supplemented with 10% heat-inactivated FBS and 1% penicillinstreptomycin in standard tissue culture-treated plastics at 37°C with 5% CO 2 .N2A cells were seeded in 12-well plates 24 hrs prior to knockdown, and then transfected with Silencer Select siRNAs (Thermo) using Lipofectamine RNAiMAX (Thermo # 13778150) following the manufacturer's recommendation.At the time of transfection, N2A cells were at ~20% confluency.24 hrs post siRNA transfection, the media was changed.After 72 hr transfection, total RNA or protein was harvested by TRIzol or RIPA buffer, respectively.Silencer Select siRNAs used in this study are listed in Table S5.

Sucrose gradient ultracentrifugation
In vitro translation reactions were scaled up 10-fold to 100 µL.After 30 min at 30°C, reactions were transferred to ice, 1 µL of 100 mg/mL cycloheximide was added (~1 mg/mL final), and samples were snap frozen in liquid nitrogen and stored at -80°C.When ready to perform sucrose gradients, samples were then thawed on ice, 5 µL of 10 mM CaCl 2 (~0.5 mM final) and 5.3 µL of 3,000 U/mL S7 micrococcal nuclease (Thermo # EN0181; stock at 30,000 U/mL in PBS; ~150 U/mL final) was added.After nuclease digestion at 25°C for 10 min, samples were quenched by adding 2.2 µL of 50 mM EGTA (~1 mM final).Samples were then diluted with an equal volume of ice-cold 2X Polysome Dilution Buffer (40 mM Tris-HCl, 280 mM KCl, 20 mM MgCl 2 , 2 mM DTT, 200 µg/mL cycloheximide; pH 7.4), gently mixed, and layered on top of a linear 10-50% (w/v) buffered sucrose gradient (20 mM Tris-HCl, 140 mM KCl, 10 mM MgCl 2 , 1 mM DTT, 100 μg/mL cycloheximide; pH 7.4) in a 14 mm × 89 mm thin-wall Ultra-Clear tube (Beckman # 344059) that was formed using a Biocomp Gradient Master.Gradients were centrifuged at 35K rpm for 120 min at 4°C in a SW-41Ti rotor (Beckman) with maximum acceleration and no brake using a Beckman Optima L-90 Ultracentrifuge.Gradients were subsequently fractionated into 0.5 mL volumes using a Biocomp piston fractionator with a TRIAX flow cell (Biocomp) recording a continuous A 260 nm trace.
When assaying N2A cell lysates after 72 hr knockdown, whole cell lysates were prepared using RIPA buffer.Briefly, cells were placed on a bed of ice and media was aspirated before a gentle 1 mL ice-cold PBS rinse.Cells were then lysed in 300 μL of ice-cold RIPA buffer for 10 min at 4°C with gentle rocking.The entire lysate (including any apparent cell debris) was mixed with 100 μL of 4X reducing SDS sample buffer and heated to 70°C for 15 min.Samples were then homogenized by syringing 6X through a 28G needle.30 µL was then separated by Tris-Glycine SDS-PAGE, transferred on 0.2 µm PVDF, and probed as described above.Rabbit anti-ZNF598 (Thermo # PA5-59777) was used at 1:1,000.Rabbit anti-FMRP (Abcam # ab17722) was used at 1:1,000.Rabbit anti-GAPDH (Cell Signaling # 5174) was used at 1:1,000.HRP-conjugated goat anti-rabbit IgG (H+L) (Thermo # 31460) was used at 1:10,000 for ZNF598 and FMRP, and at 1:30,000 for GAPDH.Chemiluminescence was performed with SuperSignal West Pico PLUS and imaged using an Azure Sapphire Biomolecular Imager.

RNA-seq and mRNA decay analyses via Roadblock-qPCR
After 72 hr knockdown, total RNA was extracted with TRIzol reagent following the manufacturer's protocol.RNA concentration and purity was determined by UV spectroscopy (i.e., Nanodrop).RNA library preparation (with RNA quality validation and rRNA depletion included) and sequencing was conducted by Novogene.Adapters and poorly sequenced reads were removed from raw sequencing data using TrimGalore (Kechin et al., 2017).The quality of sequencing reads following trimming and filtration was assessed using FastQC (Andrews, 2015).Processed sequencing reads were aligned to the mouse reference genome (GRCm39) using STAR (Dobin et al., 2013).Sequence coverage BigWig files were generated using STAR alignment BAM output using the deepTools tool bamCoverage, using counts per million (CPM) normalization (Ramirez et al., 2014).Genomic features were quantified by counting the number of reads aligning to exons of genes using FeatureCounts (Liao et al., 2013).Normalization and differential gene expression analysis was then preformed using the R package DESeq2 (Love et al., 2014).The pipeline used for the pre-processing, alignment, and post-alignment analysis of RNA-seq data can be found at https://doi.org/10.5281/zenodo.8302724.Any other custom scripts used in this analysis are available upon request.All raw RNA-seq data has been deposited in the NCBI Gene Expression Omnibus (GEO).
Roadblock-qPCR was used to determine mRNA half-lives and was performed as described by the Thoreen lab (40).72 hr post knockdown, media was replaced with pre-warmed completed supplemented with 400 μΜ 4SU (stock at 80 mM in DMSO; Sigma #T4509-100MG).Timepoints were taken immediately (0 hr), 2, 4, 6, and 8 hrs later by aspirating media and adding 1 mL of TRIzol.After extracting total RNA by following the manufacture's recommendations, 3 μg of RNA was modified in NEM Reaction Buffer (50 mM Tris-HCl, 1 mM EDTA, and 50 mM NEM; pH8) in a 50 μL reaction at 42°C for 90 min. 1 M NEM (Sigma # 3876) was made with 100% EtOH, aliquoted, and stored at -20°C.The reaction was quenched by addition of 20 mM DTT (final) and RNA was purified using an RNA Clean & Concentrator-5 Kit (Zymo # R1013) with a final elution volume of 15 μL. 1 μg of recovered RNA was used to generate cDNA for RT-qPCR using the iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad # 1708841).cDNA reactions were then diluted 10-fold with nuclease-free water and stored at −20°C or used immediately.RT-qPCR was performed in 15 μl reactions using iTaq Universal SYBR Green Supermix (Bio-Rad# 1725124) in a Bio-Rad CFX Connect Real-Time PCR Detection System with 1.5 μl diluted cDNA and 250 nM (final concentration) primers.Target mRNA levels were normalized to 18S rRNA, and half-lives were calculated using one phase decay trend lines calculated by nonlinear regression in GraphPad Prism 10.0.3.All data are reported and the 95% confidence interval was included as a watermark when appropriate.
Steady state mRNA levels in Figure 4 and Figure S9 were normalized to 18S rRNA and GAPDH, respectively.
All RT-qPCR primer sequences are available in Table S6.were treated identically (i.e., gels were run in parallel with primary antibodies dilutions, secondary antibody dilutions, & ECL chemiluminescence regent were made in batch and split equally, and blots were imaged for the same amount of time in parallel).E-F) Fold change of RPS6 (E) and RPL7 (F) levels in each fraction with and without S7 nuclease treatment from panels C & D. Signal from each fraction was normalized to their respective MBP-mEGFP spikein control and then compared between the two conditions.G) Anti-FMRP, anti-PABP, anti-RPS6, and anti-RPL7 Western blot analysis of fraction #11 that contains disomes.Recombinant MBP-mEGFP was spiked-in and used as a loading control.H-I) Quantification of the indicated proteins in panel G. Bands were first normalized to MBP-mEGFP and then set relative to untreated.Data are shown as mean ± SD. n = 3 biological replicates.Comparisons were made using a two-tailed unpaired t test with Welch's correction.individually or in combination as indicated.Steady state mRNA levels were measured by RT-qPCR with GAPDH as the reference gene.Only Id3, Dbh, Map3k8, and Tbr1 were reproducible by RT-qPCR with both independent siRNAs.Double knockdown did not result in increased levels over single knockdowns, suggesting that FMRP and ZFP598 act in the same decay pathway for Id3, Dbh, Map3k8, and Tbr1.Data are shown as mean ± SD. n = 3 biological replicates.Comparisons were made using an ordinary one-way ANOVA with Tukey's multiple comparisons.
Figure S1.nLuc mRNA pelleting through the sucrose cushion is translation dependent.A) Relative quantification of nLuc reporter mRNA pelleted through a 35% (w/v) sucrose cushion after a low-speed centrifugation.Lane 1 is a negative control lacking nLuc mRNA.Lane 2 is a negative control containing mRNA in RRL but not incubated at 30°C to start translation.Lane 3 is nLuc mRNA in RRL translated for 15 min at 30°C.Lane 4 is nLuc mRNA in RRL translated for 15 min at 30°C and then incubated with 0.1 mM puromycin (final) for 30 min at 30°C.Data are shown as mean ± SD. n=3 biological replicates.B) Relative quantification of nLuc reporter mRNA pelleted through a 35% (w/v) sucrose cushion after a low-speed centrifugation with WT NT-hFMRP (1 µM final) with and without translation (15 min at 30°C).Data are shown as mean ± SD. n=3 biological replicates.Comparisons were made using a two-tailed unpaired t test with Welch's correction.

Figure S2 .
Figure S2.The collision reporter mRNA, but not the control reporter mRNA, generates nuclease-resistant collided ribosomes.A-D) Replicates for polysome analysis of translated control and collision reporter mRNAs with nuclease treatment related to Figure 2. The collision reporter mRNA generates nuclease-resistant collided disomes and trisomes, with a concurrent decrease in monosomes as compared to the control reporter.E) Anti-RPS6 and anti-RPL7 Western blots of fraction #11 that contains disomes.Recombinant MBP-mEGFP was spiked in and used as a loading control.

Figure S3 .
Figure S3.WT NT-hFMRP does not cause detectable nuclease-resistant ribosome collisions on nLuc mRNA in RRL.A-C) Polysome analysis of in vitro translation reactions with nuclease treatment and the indicated recombinant proteins at 0.3 μM final (A-C) or 1 µM final (D-F).G) Anti-RPS6 and anti-RPL7 Western blots of fraction #11 that contains disomes.Recombinant MBP-mEGFP was spiked in and used as a loading control.

Figure S4 .
Figure S4.WT NT-hFMRP does not cause detectable nuclease-resistant ribosome collisions in RRL at 0.3 μM on nLuc mRNA.A-I) Replicates of polysome analysis of in vitro translation reactions with nuclease treatment and the indicated recombinant proteins at 0.3 μM final related to Figure S3.J) Anti-RPS6 and anti-RPL7 Western blots of fraction #11 that contains disomes.Recombinant MBP-mEGFP was spiked in and used as a loading control.

Figure S5 .
Figure S5.WT NT-hFMRP does not cause detectable nuclease-resistant ribosome collisions in RRL at 1 μM on nLuc mRNA.A-I) Replicates of polysome analysis of in vitro translation reactions with nuclease treatment and the indicated recombinant proteins at 1 μM final related to Figure S3.J) Anti-RPS6 and anti-RPL7 Western blots of fraction #11 that contains disomes.Recombinant MBP-mEGFP was spiked in and used as a loading control.

Figure S6 .
Figure S6.WT NT-hFMRP does not cause detectable nuclease-resistant ribosome collisions in RRL at 1 μM on FFLuc mRNA.A) In vitro translation of FFLuc reporter mRNAs pre-incubated with protein storage buffer (Buffer) or the indicated recombinant protein (1 µM final).Data are shown as mean ± SD. n = 3 biological replicates.Comparisons were made using a two-tailed unpaired t test with Welch's correction.B-F) Polysome analysis of in vitro translation reactions with nuclease treatment and the indicated recombinant proteins at 1 μM final.Duplicate samples are shown.

Figure S7 .
Figure S7.FMRP co-sediments with nuclease-resistant disomes from cells.A-B) Polysome analysis of N2A cell lysates without (A) and with (B) S7 nuclease treatment.C-D) Anti-RPS6 and anti-RPL7 Western blots of fractions 1-16 of untreated (C) or nuclease-treated cell lysates (D).The first lane contains fractions 1 & 2 combined and the second lane contains fractions 3 & 4 combined.The third lane and on contain single fractions.Recombinant MBP-mEGFP was spiked-in and used as a loading control.Western blots between the two conditions

Figure S8 .
Figure S8.N2A cells naturally have nuclease-resistant disomes.A-D) Replicates of polysome analysis of N2A cell lysates without (A & C) and with (B & D) S7 nuclease treatment related to Figure S7.

Figure S10 .
Figure S10.Dbh, Rrh, and Tbr1 mRNAs are stabilized upon Fmr1 and Zfp598 KD compared to scramble control conditions in N2A cells.A-F) Roadblock-qPCR was used to measure mRNA half-lives (t 1/2 ) of Dbh mRNA (A, B), Rrh mRNA (C, D), and Tbr1 mRNA (E, F) in N2A cells.The Scramble negative control is shown in black (and is the same in A & B, in C & D, and in E & F), Fmr1 KD in red, and Zfp598 KD in blue.n=6 biological replicates.One phase decay trend lines calculated by nonlinear regression are shown.However, due to the mRNA levels not decreasing below 50% during the 8 hr time course, the t 1/2 for these mRNAs could not be determined.Nevertheless, in all three examples, the one phase decay trend lines for both Fmr1 KD and Zfp598 KD are markedly above the Scramble negative control.