Characterization of Sucrose Gradient Sedimentation.

A) A summary of protocol for isolating the RNA Granules (RG) from C57 and FMR1-KO mouse whole brain homogenate using sucrose gradient fractionation. B) SDS-page stained with Coomassie brilliant blue showing the distribution of proteins from each fraction of the sucrose gradient from WT mouse and rat brains. Equal volumes of resuspended ethanol precipitates (fractions 1-10) and resuspended pellet were used. C) Representative immunoblots for WT and FMR1-KO mouse starting materials (brain homogenate), ribosomal cluster (RC; fraction 5/6) and RNA Granules (RG; pellet). D-F) The Quantification of S6 (N = 6) was normalized to the level of S6 in the starter fraction. The quantification of UPF1 (N = 6), Stau2 59kD (N = 5) and Stau2 52kD (N = 5) from RC and RG was normalized to the level of S6 in that fraction, and then normalized again to the ratio of that protein and S6 in the starter fraction. H-K) The Quantification of enrichment of RG from RC (RG/RC) between FMR1-KO and WT samples for s6 (N = 6; p = 0.008), UPF1 (N = 6; p>0.05), Stau2 59kD (N = 5; p>0.05), and Stau2 52kD (N = 5;p>0.05). Error bars are Standard Error of the Mean; Student’s T-test.

Loss of FMR1 does not affect anisomycin-purmycin competition.

A) A summary of protocol for anisomycin and puromycin competition on the RNA granule fraction. B) Top: Representative immunoblot stained with antibodies to puromycin (anti-puro) to showcase the inhibition of puromycylation (100 uM) by anisomycin (100 uM) in liver polyribosomes. Bottom: Corresponding membrane stained with ponceau before immunoblotting. The experiment was replicated twice with similar results. C) Top: Representative immunoblot that stained with anti-puromycin (anti-puro) to showcase the inhibition of puromycylation (100 uM) by anisomycin (100 uM) in Rat RG, WT mouse RG and FMR1-KO mouse RG. Bottom: Corresponding membrane stained with ponceau before immunoblotting. D) Quantification of percentage puromycylation resistant to anisomycin inhibition in Rat RG (N = 3), WT mouse RG (N = 4), FMR1-KO RG (N = 4). All groups are insignificant from each other (one way ANOVA, p > 0.05).

Higher Nuclease reduces size of RPFs in WT RNA granules.

A) A summary of protocol for ribosomal footprinting procedure. B) Representative image for size distribution of normalized footprint reads from high magnesium and high nuclease treatment group (M) and normal magnesium and normal nuclease treatment group (WT) for RPFs derived from the RNA granule fraction. C) Representative image for read coverage for M WT RNA Granule and WT RNA Granule UTR, untranslated region; CDS, coding sequence. D) Representative image for the number of read extremities (shading) for each read length (Y-axis) based on the distance from start(left) to stop(right) with the 5’ end (top) and 3’ end (bottom) for M WT RNA Granule and WT RNA Granule. E) Representative image for the periodicity statistics for each read coverage RPFs. Though the representative images above only included one replicate for the result, the data are shown in all replicates for both WT and FMR1-KO groups.

High Magnesium buffer does not affect ribosome structure, but leads to less sedimentation of secretory mRNAs.

(A) Composite cryo-EM maps of class 1 (A) and class 2 (B) 80S ribosomes found in the GF after purification in high magnesium buffer and RNase I treatment. The top panels show a side view of the two classes of ribosome particles contained in the sample. The bottom panels show top views of the same cryo-EM maps. The 40S and 60S subunits are shown as transparent densities for easier viewing of the position of the tRNA molecules in each class. B) Gene Ontology (GO) terms of the significant genes from the differential expression gene (DEG) analysis of WT RG and WT M RG for 1) top: Biological Function 2) middle: Cellular Components and 3) bottom: Molecular

Assessment of RPF abundance, occupancy and enrichment in RGs of WT and FMRI-KO mice.

A) A summary of protocol to generate RPF abundance, occupancy and enrichment. B) GO terms of the WT M RG (left) and FMR1-KO (right)for abundance (top), occupancy (Middle) and enrichment (bottom). For each graph, GO terms from the top 500 genes: Biological Function (top), Cellular Components (middle), and Molecular Function (bottom).

Comparison of putative stalled mRNAs vs Total mRNAs in RPFs from the RG of WT and FMR1-KO mice.

Comparison of mRNAs associated to ribosome resistant of initiation inhibitor run-off (Shah et al., 2020) and FMRP-CLIPped mRNAs (Maurin et al., 2018; Darnell et al., 2011) to all other mRNAs. A) WT Granule abundance, B) FMR1-KO Granule M abundance, C) WT Granule Occupancy, D) FMR1-KO Granule Occupancy, E) WT Granule Enrichment, and F) FMR1-KO Granule Enrichment. P-values from each set were calculated by performing student t-test between the mRNAs that matched to the published dataset and the ones that did not.

Comparison of putative stalled mRNAs between WT and FMR1-KO mice.

Fold change between A) WT and FMR1 fold change in RG abundance (Shah average = 0.047; Maurin average = 0.086; Darnell average = 0.079), B) WT and FMR1 fold change in RG occupancy, C) WT and FMR1 fold change in RG enrichment, and D) WT and FMR1 fold change in RC abundance in comparison to mRNAs associated to ribosome resistant of initiation inhibitor run-off (Shah et al., 2020), FMRP-CLIPped mRNAs (Maurin et al., 2018; Darnell et al., 2011). P-values from each trait were calculated by performing student t-test between the mRNAs that matched to the published dataset and the ones that did not.

Comparison of RPF peaks in the RG of WT and FMR1-KO mice.

A) Representation of how peaks of RPFs are selected. B) Table of the number of peaks between replicates of WT RNA Granule (N = 3), FMR1-KO RNA Granule (N = 3) and combined (N = 6), and the percentage of peaks with FXS related motif. C) Example of RPF producing consensus peaks over the entire length of Tubb2b. Asterisks indicate consensus peaks (seen in all six samples with peaks within 6 bp).

RPM of hippocampal cultures derived from WT and FMR1-KO mice.

A) Summary of the protocol for puromycylation HHT-Runoff and DHPG Reactivation on WT and FMR1-KO hippocampal culture. B) Representative confocal images for puromycylated ribosomes with or without HHT runoff and DHPG reactivation. Circle denotes puromycin puncta. No visible staining was seen in the absence of puromycin. Scale bar shown below C) Quantification of RPM puncta density of puncta>50 microns from the cell body. Numbers are neurites/cultures. WT (42/5); WT DHPG (54/5), FMR1-KO (41/4), FMR1-KO DHPG (25/3). One way ANOVA (F158,3)= 5.32, p< 0.005) *, p<0.05 post-Hoc Tukey HSD test. D) Quantification of size of RPM puncta >50 microns from the cell body. WT 189/5; WTDHPG 5171/5, FMR1 KO (118/4), FMRP KO DHPG (48/3). One way ANOVA showed no significance (P>0.5).