Fig 2: Subcerebral projection neurons’ (SCPNs’) increase colocalizes with Sox5 and Bcl11b in Pum2 and TDP-43 mutants.(a) Coronal sections from neonatal (P0) brains of controls (Ctrl), Pum2 cKO, or Prnp-TARDBPA315T (TDP43A315T) mice were stained with antibodies recognizing Sox5 and Bcl11b in the prospective somatosensory area (pS). Quantification of Sox5 and Bcl11b colocalization from n = 3 mice of each genotype is shown to the right across six equal-sized bins. (b, c) Coronal sections from primary somatosensory cortex (S1) of controls, Pum2 cKO, and TDP43A315T mice at P7 traced for SCPNs combined with Sox5 (b) or Bcl11b (c) staining. White arrows in (a–c) indicate colocalization. Data are shown as means ± standard error of the mean (SEM), n = 3 for each genotype. *p=0.05, **p=0.01, two-tailed t-test. Pum2 cKO: Pum2fl/fl; Emx1Cre; V, VI: layers V and VI. Scale bars: 100 µm.

Fig 3: miR-346 Induces DNA Damage in part Independently of NORAD/PUM2, NORAD Promotes Target-Directed miR-346 Decay. A Western blot analysis of phospho-Ser139-?H2AX protein levels in LNCaP cells transfected with 20 nM miR-346 for indicated duration. ß-actin was used as a loading control. B, C qRT-PCR analysis of B) NORAD and C) miR-346 in cytosolic, soluble nuclear and chromatin fractions of 22RV1 cells. SNORD48 was used as a fractionation control and identified almost exclusively complexed with chromatin (Fig. S12B). D Immunofluorescent microscopy analysis of i,iii) phospho-Ser139-?H2AX protein levels in C42 cells transfected with miR-346 or NC (20 nM) ± siPUM2 (20 nM) for 72 h. E Illustration of NORAD:miR-346 putative interaction sites with features consistent with target-directed microRNA decay. F qRT-PCR quantification of number of miR-346 copies and NORAD TDMD sites in unperturbed C42B cells. Ten-fold serial dilutions of a miR-346 mimic and NORAD qPCR amplicon were prepared, reverse transcribed and analysed by qRT-PCR in parallel to C42B samples for absolute quantification. G qRT-PCR analysis of miR-346, - 221-3p and 222-3p levels in C42 cells transfected with siNORAD or siNC for 72 h. miR levels were normalised to U6, H qRT-PCR analysis of miR-346 levels in 22RV1 cells transfected with pcDNA3.1-NORAD WT or TDMD mutant for 72 h. miR levels were normalised to U6. Columns: mean ± SEM for three independent experiments performed in triplicate. J Western blot analysis of phospho-Ser139-?H2AX protein levels in C42 cells transfected with miR-346 (10 nM) ± WT or TDMD-mutant NORAD. ß-actin was used as a loading control. A representative image of four independent experiments is shown. Densitometry was performed using ImageJ. * P = 0.05, ** P = 0.005, *** P = 0.0001. See also Fig. S12, Table S7

Fig 4: Dendritic branches of Pum2–/– and WT mice via Golgi stainingRepresentative pyramidal neurons in the CA1 area of the hippocampus (A: WT, l B:Pum2–/–) and the cortex (C:WT and D: Pum2–/–). There is a significant increase in primary dendritic branching in the Pum2–/– mice as compared to WT in the hippocampus (E) but not in the cortex (F). Additional measurements of the dendritic branch intersections using Sholl analysis in consecutive 30, 60, 90, and 120 µm concentric bands radiating from the center of the soma indicated a significant difference between Pum2–/– and WT mice (G and H). *means p < 0.05 and **means p < 0.01. Data represent as mean ± SEM.

Fig 5: Repression by TRIM71 is independent of mammalian PUM1 and PUM2. (a) The PUM consensus motif is not enriched in 3'UTRs of TRIM71 targets. TRIM71 versus parental mRNA expression changes for mRNAs (Supplementary Table S2) with no (black), low scoring (blue) and high scoring (red) PUM sites in the respective 3'UTRs, depicted as cumulative densities. To control for motif enrichment potentially caused by 3'UTR length differences in regulated versus non-regulated genes we included a low scoring PUM site category. These are mRNAs that contain highly degenerate PUM sites unlikely to bind PUM1/2. (b) Knock-down of PUM1 and PUM2 does not affect TRIM71-mediated repression. HEK293 cells were co-transfected with indicated FL-3'UTR reporters, plasmids expressing TRIM71 or LacZ and a combination of siRNAs against PUM1 and PUM2, or a non-targeting control siRNA as indicated. FL was normalized to RL and values of normalized FL produced from samples expressing LacZ and the non-targeting control siRNA were set to 1. *P-value < 0.05; **P-value < 0.01.