Fig 2: Application of SuperStructure algorithm to SAF-A, hnRNP-C, and SC35 SMLM data. (A) Reconstructed dSTORM images by using the shifted histograms method with a pixel size of 10.6 nm. Insets of 4-μm2 size of reconstructed dSTORM images and spatial positions of the data. Palettes represent the cluster ID computed by running SuperStructure with Nmin=0 and ε at the start of the first super-cluster regime. (B) Identified clusters for increasing values of ε in the regimes where clusters merge. (C) Normalized average SuperStructure curves in the range [0:150] nm. The number of detected clusters has been normalized with the total number of localizations in the system. The average is calculated over six independent datasets (nuclei). Solid curves indicate that SuperStructure analysis was run on the entire nucleus, and the resulting curves for the six independent datasets were averaged (all-nucleus curves). Dashed curves indicate that SuperStructure analysis was run in five local ROIs for each of the six nuclei, and then the curves of each region (for each nucleus) were averaged (local curves). In hnRNP-C, these local regions were chosen within the nuclear mesh (to exclude nucleoli), and in SC35, they were chosen within speckles. Vertical dashed lines highlight different SuperStructure regimes: intra-cluster, first super-cluster, and second super-cluster regime. For SAF-A and hnRNP-C, the exponential regime of clusters merging (first super-cluster regime) is highlighted with a solid straight line. In case of SC35, two regimes are highlighted, the merging of clusters within speckles (first super-cluster regime) and the merging of speckles with isolated clusters (second super-cluster regime). (D) Normalized all-nucleus average SuperStructure curves in the range [0:200] nm for the three proteins. Average is computed over six nuclei. Shaded regions represent standard deviation from the average. Poisson fits (Eq. 1) for the intra-cluster regime at small ε are shown in the inset. (E) Intra-cluster density of emitters ρem as parameter of Poisson fit for six independent nuclei (Eq. 1). (F) Normalized decay length λ* for the super-cluster regimes highlighted in C for six independent nuclei. SuperStructure curves were fit with Eq. 4 to extract the decay length λ, and then the normalization λ*=λ/ρcl−1/2 was performed (where ρcl is the detected cluster density at the beginning of each regime of interest). Details are explained in Materials and methods and Fig. S3. P values were calculated using a Student’s t test: ns, P > 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3: Differential gene expression by 1,25(OH)2D or hnRNPC1/2 knockdown. Weighted Venn diagrams depicting overlap of differentially expressed genes: (A) Control versus hnRNPC1/C2 knockdown (Ctrl versus KD; maize), control versus 1,25 (10 nM, 6 h) (Ctrl versus 1,25; grey) or hnRNPC1/C2 knockdown vs 1,25(OH)2D combined with hnRNPC1/C2 knockdown (KD versus 1,25+KD; blue). (B) Control versus hnRNPC1/C2 knockdown (Ctrl versus KD, maize) or combined 1,25(OH)2D and hnRNPC1/C2 knockdown (Ctrl versus 1,25+KD, green). (C) Up- and down-regulated differentially expressed genes: (left) Control versus 1,25(OH)2D (Ctrl versus 1,25) or hnRNPC1/C2 knockdown versus 1,25(OH)2D combined hnRNPC1/C2 knockdown (KD versus 1,25+KD); (right) Control versus hnRNPC1/C2 knockdown (Ctrl versus KD). (D) Weighted Venn diagrams depicting overlap of differentially expressed genes between Ctrl versus 1,25(OH)2D and KD versus 1,25(OH)2D and hnRNPC1/C2 knockdown. Numbers of differentially expressed genes are shown within each segment. Heatmaps show patterns of down-regulated (blue) differentially expressed genes (upper heatmaps) and up-regulated (red) differentially expressed genes (lower heatmaps) for each treatment/knockdown comparison group and the overlapping differentially expressed genes common to those comparisons. * includes CYP24A1.

Fig 4: ADAR1 and hnRNP C suppress Alu exonization of SELENON mRNA.a Human SELENON gene with exons (boxes) and introns (lines). The Alu exon and SECIS are indicated by orange and magenta boxes, respectively. Sense and antisense strands of Alu elements are indicated by blue and red arrows, respectively. Int. and Term. represent initiation and termination codons. b Intronic region of SELENON including four Alu elements (green dashed box in a). Purple column represents A-to-I editing sites determined by ICE method. Pink column represents iCLIP sites for hnRNP C49. c RNA sequence of AS-Alu1 in SELENON. A-to-I editing sites are boxed in red, with site numbers shown in Supplementary Fig. 5. hnRNP C binding site identified by iCLIP is boxed. The Alu exon sequence is colored in orange. 3'SS and 5'SS of the Alu exon are indicated by underbars. PTC (UGA) is indicated in red. d Alu exonization of SELENON in HeLa cells upon knockdown of each target mRNA. Control siRNA targeted luciferase. Upper panel shows RT-PCR products of a part of SELENON mRNA spanning exons 2 and 3. Lower panel shows the inclusion ratio (%), determined by RT-qPCR. Data are presented as mean values ±S.D. Statistical significance was determined by an unpaired two-tailed t test; n = 3 biologically independent samples. e Alu exonization of SELENON in HeLa cells upon double-knockdown of target mRNAs. Upper panel shows the inclusion and skipping isoforms with a schematical illustration. Lower panel shows the inclusion ratio (%), as described in d, above. f cDNA sequences of the Alu exon 5'SS in SELENON pre-mRNAs from HeLa cells treated with siRNAs targeting luciferase (control) and ADAR1 (KD). The editing sites are indicated. Consensus sequence of 5'SS (underlined) base-pairs with a part of U1 snRNA. The electropherograms for A, G, T, and C are colored green, red, gray, and blue, respectively. ?, pseudouridine. g Minigene constructs bearing the Alu exon (WT) or its editing-mimic mutant (AG). Skipping and inclusion isoforms from the minigene were detected by RT-PCR. This experiment was done once. Source data and unprocessed gel images are provided in Source Data file.

Fig 5: hnRNP U, but not other RBPs, exhibits lost nuclear function following hyperosmotic stress. (A) Delta CT of TDP-43 mRNA targets in WT U2OS cells treated with the indicated stressors for 1 h (top) and 4 h (bottom). ZNF565 and GSTM4 mRNAs (left) are expected to increase in abundance with lost TDP-43 function, while DOCK1, PFKP, and Unc13A mRNAs (right) are expected to decrease in abundance with lost TDP-43 function. N = 3 biological replicates. Two-way ANOVA was used to calculate statistical significance. (B) Delta CT of hnRNP mRNA targets in WT U2OS cells treated with the indicated stressors for 1 h (i) and 4 h (ii). PTBP2 mRNA (hnRNP I target) is expected to increase in abundance with lost hnRNP I function. IER3 mRNA (hnRNP U target) is expected to decrease in abundance with lost hnRNP U function. N = 3 biological replicates. Two-way ANOVA was used to calculate statistical significance. (C) Representative images of WT U2OS cells stained for incorporated BrU (using a BrdU antibody) following co-treatment with the indicated stressors and 1 mM 5-bromo-uridine for 1 h. (D) Quantification of normalized nuclear BrU intensity from images represented in (C). Each data point represents the average nuclear intensity of BrU fluorescence from >50 cells, normalized to the average nuclear BrU intensity from the untreated cells in each biological replicate. N = 3 biological replicates. One-way ANOVA was used to calculate statistical significance. *p < 0.05, **p < 0.01, ****p < 0.001. GOI, gene of interest. The scale bar represents 5 µm.