Fig 2: Validation of Pum1 associations with RNA-binding proteins in mouse brain ARepresentative western blot of proteins pulled down by IP against Pum1 compared with IgG from wild-type mice brain without (left) and with (right) RNase treatment. In this panel, after IP-Pum1, we immunoblotted for Pum1 (positive control), Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10 (see Materials and Methods).BRepresentative western blots from WT and Pum1 -/- mouse brains, with and without RNase. Molecular weights at the right are in kilodaltons (kDa). All mice were sacrificed at 10 weeks of age. IP against IgG was used as a negative control, and Input (1% from the initial protein lysate; please see Materials and Methods) as a loading control.

Fig 3: Protein and mRNA quantification from WT, Pum1 +/- and Pum1 -/- mouse brains ARepresentative western blot with relative quantifications of Pum1, Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10 from whole brains of WT, Pum1 +/- and Pum1 -/- mice. All data were normalized to Gapdh protein levels. The numbers on the right are the respective molecular weights in kilodaltons (kDa).BmRNA level quantification by qPCR of Pum1, Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10 from whole brains of WT, Pum1 +/- and Pum1 -/- mice. Data information: All data were normalized to Gapdh mRNA levels. All the experiments were conducted with equal numbers of male (at least 3 per genotype) and female (at least 3 per genotype) mice at 10 weeks of age, for a total of at least 6 mice per genotype (data represent mean ± SEM). The p values were calculated by the Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Shared targets are upregulated in PADDAS but not in PRCA AmRNA level quantification of PUM1 neuronal targets in common with FMRP, PUM2, AGO2, and RBFOX3 (Fig 5E and Dataset EV5) in S1 (R1147W, blue bars) and S2 and 3 (T1035S, orange bars) cell lines compared with their respective healthy controls. Only genes expressed in both R1147W and T1035S cell lines are represented here, for a total of 54 genes.BRepresentative western blots (left panel) and relative quantifications (right panel) of PUM1 and its interactors (AGO2, CNOT1, FMRP, and MOV10) in R1147W patient-derived cell lines after Myc-PUM1-WT expression.CmRNA quantification of the top 15 shared target genes from panel (A) in R1147W patient-derived cell lines after Myc-PUM1-WT expression. Data information: All data were normalized to GAPDH mRNA or protein levels and experiments were performed at least three times. Data represent mean ± SEM. P values were calculated by two-tailed Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001. The full list of shared targets expressed in fibroblast and lymphoblast cell lines is reported in Dataset EV5. Source data are available online for this figure.

Fig 5: Pum1 loss affects Pum1 interactors and the microRNA machinery by brain region and sex A–CRepresentative western blots of Pum1, Pum2, Fmrp, Ago2, Rbfox3, Cnot1, and Mov10 in (A) cerebellum, (B) hippocampus, and (C) cortex in both male and female WT, Pum1 +/-, and Pum1 -/- mice. All the experiments were conducted with equal numbers of 10-week-old male and female mice per genotype, for a total of at least 6 mice per genotype (data represent mean ± SEM). To the right are molecular weights in kilodaltons (kDa). Graphs below show quantification for each protein by brain region, sex, and genotype. All data were normalized to Gapdh protein levels. P values were calculated by two-tailed Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001. P1 indicates Pum1. See Appendix Fig S3 for mRNA quantification for each interactor, brain region, and sex.DHeatmap showing 166 microRNAs from cerebella of Pum1 -/- male and female mice that were dysregulated (fold change -3 to +3) relative to wild-type cerebellum. The full list of miRNA names and fold changes are available in Dataset EV3. See Appendix Fig S4 for male and female miRNA scatter plots.EHeatmap showing mRNA quantification by qPCR for 49 targets co-bound by a minimum of eight dysregulated miRNAs (> 25% change) from panel (D). Statistical significance and magnitude of dysregulation are illustrated for both male and female in Appendix Fig S4. The entire list of targets predicted to be co-bound by at least two miRNAs is presented in Dataset EV4.