Characterization of Sucrose Gradient Sedimentation.

A) A summary of the protocol for isolating the pellet and fraction 5/6 from mouse and rat 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 mouse and rat brains. Equal volumes of resuspended ethanol precipitates (fractions 1-10) and resuspended pellet were used. C) Average UV absorbance from fractions 1-10 and pellet from WT (Red) and FMR1- (blue) preparations (Error bars are S.D., N=3). D) Representative electron microscopy of WT and FMR1- ribosome clusters from fraction 5/6 and pellet. Scale bar is 200 nm). E) Representative immunoblots of fractions (as defined in A) for WT and FMR1- mice. For each blot, 1/100th of the Starter, 1/10th of Fraction 5/6 and 1/20th of the pellet fraction were loaded. F) Quantification of differences in enrichment between WT and FMR1-. Levels of proteins were determined from scans of immunoblots (see methods). Enrichment is defined by the ratio of level of protein (see Methods) between Fraction 5/6 and Pellet. For S6, the ratio of WT enrichment to FMR1- enrichment was calculated for each blot and the average calculated (N=8). For all the other RBPs, the WT and FMR1- enrichment were normalized to the S6 enrichment for that preparation and the normalized values were used to calculate the difference in enrichment between WT and FMR1- (UPF1, n=8, Stau2, n=8, PurA, n=3, G3BP1 (n=3), ZBP1 (N=3), hnRNPA2/B1 (N=3). Error bars are S.D. A one sample T-test against 1 was used to test significance with Bonferroni correction for multiple tests. No value reached p< 0.05.

Loss of FMR1 does not affect anisomycin-puromycin competition.

A) A summary of protocol for isolating the pellet and treating with puromycin. B) Representative immunoblot stained with antibodies to puromycin (anti-puro) (Top) to showcase the inhibition of puromycylation (100 uM) by anisomycin (100 uM) in liver polyribosomes (left) or comparing the pellet from Rat, Mouse WT and Mouse FMR1- (Right). Bottom: Corresponding membrane stained with Ponceau before immunoblotting. The liver experiment was replicated twice with similar results. C) Quantification of the amount of puromycylation resistant to anisomycin (puro +aniso/ puro) in Rat (N = 3), WT mouse (N = 4), FMR1- (N = 4). All groups are insignificant from each other (one way ANOVA, p > 0.05).

Higher Nuclease Reduces Size of RPFs in WT Pellet.

A) A summary of the protocol for the RPF procedure. B) Size distribution of normalized footprint reads from the WT Pellet fraction under standard or low nuclease treatment. C) Representative image for read coverage for WT Pellet fraction either with standard or low nuclease treatment) UTR, untranslated region; CDS, coding sequence for both RPFs and RNA-seq libraries. 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 the WT pellet fraction with either standard or low nuclease treatment. E) Representative image for the periodicity statistics for RPFs in different regions of the mRNA for standard or low nuclease treatment. Although the representative images above included only one replicate, similar results were observed across all three replicates.

High Magnesium Buffer does not Affect Ribosome structure

Composite cryo-EM maps of class 1 (A) and class 2 (B) 80S ribosomes found in the pellet 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 to facilitate visualization of the positions of the tRNA molecules in each class.

GSEA analysis WT and FMR1- RNA-seq and RPFs from pellet fraction

A) Description of RPF isolation and mapping. Results of Selected GSEA sets significantly affected by the loss of FMRP from analysis of B) RNA-seq, C) Abundance and D) Occupancy. Increases in FMR1- vs WT are to the left and decreases to the right.

Comparison of Putative Stalled mRNAs vs Total mRNAs in RPFs from the Pellet of WT and FMR1- mice.

Comparison of mRNAs associated with ribosome resistant to initiation inhibitor mediated run-off (Shah et al., 2020) and FMRP-CLIPped mRNAs (Maurin et al., 2018; Darnell et al., 2011) to all other mRNAs. A) WT Pellet abundance, B) FMR1- Pellet abundance, C) WT Pellet Occupancy, D) FMR1-Pellet Occupancy, E) WT Enrichment, and F) FMR1- Enrichment. As the N of the All mRNAs (13079) were much larger than for the selected groups: Shah (185), Maurin (264), Darnell (757) (Extended Data Table S6-1) a random set from the total RNA group that matched the number in the selected group was generated for a two-tailed Welch’s T-test with Bonferroni correction for multiple T-tests. The median P value from 10 random sets was used (Extended Data Table S6-1). (****, p<0.0001, ***, p<0.001, **, p<0.01, *, p<0.05).

Comparison of Putative Stalled mRNAs between WT and FMR1- mice.

Comparison of mRNAs associated with ribosome resistance 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. The fold change between WT and FMR1-was calculated using DEG (see Methods). These fold changes were then compared between the selected groups and All mRNas for A) Pellet abundance, B) Pellet occupancy, C) Pellet enrichment. As the N of the All mRNAs (13079) were much larger than for the selected groups: Shah (185), Maurin (264), Darnell (757) (Extended Data Table S7-1) a random set from the total RNA group that matched the number in the selected group was generated for a two-tailed Welch’s T-test with Bonferroni correction for multiple T-tests. The median P value from 10 random sets was used (Extended Data Table S7-1). (****, p<0.0001, ***, p<0.001, **, p<0.01, *, p<0.05).

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

A) Representation of how peaks of RPFs are selected. B) Table of the number of peaks between replicates of WT Pellet (N = 3), FMR1- Pellet (N = 3) and combined (N = 6), the percentage of peaks with FXS related motif and the enrichment of Aspartate (Asp) and Glutamate (Glu) in the peaks compared to non-peaks (Extended Table S8-2). C) RPF coverage of Tubb2b for the three replicates of WT and FMR1-. Shaded region is the open reading frame. Asterisks indicate consensus peaks (seen in all six samples with peaks within 6 bp).

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

A) Summary of the protocol for puromycylation HHT-Runoff and DHPG Reactivation on WT and FMR1- 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. Numbers are neurites/cultures. WT (42/5); WT DHPG (54/5), FMR1- (41/4), FMR1- DHPG (25/3). One way ANOVA F(3,158)= 5.32, p< 0.005) *, p<0.05 post-Hoc Tukey HSD test. Box and Whisker plot with line representing the median. D) Quantification of size of RPM puncta. WT 189/5; WT DHPG 171/5, FMR1- (118/4), FMR1-DHPG (48/3). Box and Whisker plot with line representing the median. One way ANOVA showed no significance (P>0.05).

Higher Nuclease reduces size of RPFs in FMR1- pellet .

A) Size distribution of normalized footprint reads from the FMR1- Pellet fraction under standard or low nuclease treatment. B) Representative image for read coverage for the FMR1- Pellet fraction either with standard or low nuclease treatment, UTR, untranslated region; CDS, coding sequence for both RPFs and RNA-seq libraries. C) 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 the FMR1- pellet fraction with either standard or low nuclease treatment. E) Representative image for the periodicity statistics for RPFs in different regions of the mRNA for standard or low nuclease treatment. Although the representative images above included only one replicate, similar results were observed across all three replicates.

Higher Nuclease reduces size of RPFs in FMR1- Fraction 5/6

A) Size distribution of normalized footprint reads from the FMR1- fraction 5/6 under standard or low nuclease treatment. B) Representative image for read coverage for the FMR1- fraction 5/6 either with standard or low nuclease treatment, UTR, untranslated region; CDS, coding sequence for both RPFs and RNA-seq libraries. C) 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 the FMR1- fraction 5/6 with either standard or low nuclease treatment. E) Representative image for the periodicity statistics for RPFs in different regions of the mRNA for standard or low nuclease treatment. Although the representative images above included only one replicate, similar results were observed across all three replicates.

Higher Nuclease reduces size of RPFs in WT fraction 5/6.

A) Size distribution of normalized footprint reads from the WT fraction 5/6 under standard or low nuclease treatment. B) Representative image for read coverage for the WT fraction 5/6 either with standard or low nuclease treatment, UTR, untranslated region; CDS, coding sequence for both RPFs and RNA-seq libraries. C) 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 the WT fraction 5/6 with either standard or low nuclease treatment. E) Representative image for the periodicity statistics for RPFs in different regions of the mRNA for standard or low nuclease treatment. Although the representative images above included only one replicate, similar results were observed across all three replicates.

Single particle analysis image processing workflow.

The cryo-EM dataset obtained from the pellet fraction after purification in high-magnesium buffer and RNase I treatment was subjected to the image-processing workflow shown in the figure. The diagram shows the main image processing steps performed on this dataset and the two main ribosome populations identified by the image classification approaches. The resolutions for the consensus maps and for each subunit in the maps obtained through local refinement are also indicated.

Resolution analysis of the two major classes of 80S ribosomes in the Granule Fraction purified under high magnesium conditions.

Consensus cryo-EM maps (left panels) were initially calculated for class 1 (A) and class 2 (B) from the RNase I-treated pellet Fraction purified under high magnesium buffer conditions. These maps were subsequently refined by local refinement by dividing the 80S ribosome into two major bodies, the 60S and the 40S particles. The top panels in (A) and (B) show the local resolution analysis of the 80S consensus and composite maps obtained using local refinement. Maps are colored according to their local resolution using the color coding indicated in the scale bars. The Fourier shell correlation (FSC) curves for the consensus maps and each one of the subunits after the maps were subjected to local refinement are shown for class 1 and 2. For each class, we show the following FSC plots: ‘No mask’, ‘Loose’, ‘Tight’, and ‘Corrected’. These plots were calculated as described in the Methods section. We used a FSC threshold of 0.143 to report the overall resolution of the maps. The ‘Viewing direction distribution’ plot in panels (A) and (B) show the orientation distribution of the particles contributing to the cryo-EM map for each one of the classes. These plots are 2D histograms that show the number of particles with a viewing direction at a particular elevation/azimuth bin. The number of particles can be inferred by the color code scale bar to the right of each plot.

PCA Analysis.

A) PCA analysis of Pellet RPFs from WT and FMR1-replicates. B) PCA analysis of Pellet RPFs and RNA-SEQ samples from WT and FMR1-replicates.

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

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

Occupancy analysis with Subsets of FMRP Targets.and RNAs resistant to Run-Off.

The mRNAs found to be resistant to ribosome run-off (Shah et al, 2020) were divided into two sets consisting of mRNAs that are also FMRP targets (identified in Maurin et al, 2018 or Darnell et al., 2011) or not. The FMRP targets were divided into two sets consisting of mRNAs identified as one of the 200 most abundant mRNAs resistant to run-ff (Shah et al, 2020) or not. Box and Whisker plots are shown with line for mean. Different from the total RNA group using two-tailed Welch’s t-test with Bonferroni correction for multiple T-tests. Differences between the two sets was also evaluated with a two-tailed Welch’s t-test. A) Occupancy in WT pellet B) Occupancy in FMR1- pellet C) Difference in Occupancy between WT and FMR1-

Effect of Anisomycin and HHT on RPM of hippocampal cultures derived from WT and FMR1-mice.

A) Representative confocal images for puromycylated ribosomes treated either with puromycin and anisomycin together or pre-treated with HHT for 15 minutes before treating with puromycin. Circle denotes puromycin puncta. No visible staining was seen in the absence of puromycin. Quantification of RPM puncta density of puncta>50 microns from the cell body compared to WT (see Fig. 9) shown as box and whisker plits. There is no effect of adding anisomycin with puromycin or preincubation with HHT on the puncta density (B,D) or puncta size (C, E) in WT (B,C) or FMR1- (D,E). WT numbers are the same as Figure 9. Numbers are neurites/cultures. Puncta Density WT (42/5); WT anisomycin (A) (27/4); WT homoharringtonin (H) (36/5), FMR1- (41/4), FMR1-KO anisomycin (A) (23/3), FMRP homoharringtonin (45,5). One way ANOVA showed no significance for density or size (P>0.05). F) For clarity the data is also presented as mean +/- S.D.

Data acquisition, reconstruction and refinement parameters and data deposition codes for the cryo-EM dataset.