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. 2021 May 21;12:3027. doi: 10.1038/s41467-021-23188-8

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Workflow of the RNA stability experiment. b Volcano plots showing the log2 fold change of each mRNA and the corresponding −log10 (FDR) of the differential gene expression analysis comparing Fus^ΔNLS/+ to Fus^+/+ samples at each time point with significance threshold 0.05 (horizontal line). Significantly decreased mRNAs are highlighted in green, increased in purple and all synaptic FUS targets identified in the CLIP-seq data in blue. c Selected GO terms enriched in the set of significantly increased mRNAs at 12 h. One-sided hypergeometric test, p-value unadjusted for multiple comparisons. d Hierarchical clustering of the log fold changes of the set of increased mRNAs to three groups. The x-axis shows the time points and the y-axis the logFC (Fus^ΔNLS/+/Fus^+/+); synaptic FUS targets are highlighted in blue. e Bar plot of different sets of synaptic FUS targets and the location of synapse-specific peaks in these genes. FUS targets with no stability change in Fus^ΔNLS/+ compared to Fus^+/+ (unchanged) and the sets of significantly changed mRNAs (up or down) at 8, 12, and 24 h after treatment. The three rightmost bars comprise the FUS targets with no change in synaptoneurosomes (SNS) at 6-months (unchanged), accumulated and decreased targets. f Bar plot of FUS targets grouped by binding location and the percentage of targets with a specific RNA level change. Targets with the majority of peaks in exons (exon), equal number of peaks in exons and UTR/intron (exon + other), majority of peaks in 3′UTR (3′UTR) and majority of peaks in introns (intron). Accumulated RNAs in 6-month-old SNS (up), RNAs with increases stability (increased), unchanged RNA levels (unchanged), less abundant RNAs in 6-month-old SNS (down) and less stable RNAs (decreased). g Log2 fold change (Fus^ΔNLS/+/Fus^+/+) of the stability experiment at 12 h (y-axis) and SNS of 6-month-old mice (x-axis) for all synaptic FUS targets with significant changes in stability at 12 h (blue points, n = 76 mRNAs). R = Pearson correlation coefficient, p = p-value (two-sided Pearson correlation test), linear fit (gray line) and 95% confidence interval (gray region). h Summary of the observed changes for selected synaptic FUS targets. RNAs that were accumulated in synaptoneurosomes of 6-month-old Fus^ΔNLS/+ mice are highlighted in magenta; RNAs with increased stability at any time point are indicated by a small arrow; the colored boxes indicate the location of the synaptoneurosome-specific FUS peaks.