Fig 2: Features of the dsRNA regions Distributions of the lengths of the dsRNA regions identified in the dsRNA-enriched libraries (siHN_J2 and siNC_J2) or in the two types of control libraries (input and IgG controls).Percentages of the dsRNA regions identified in control or HNRNPC KD cells, which were divided into different categories according to the intra-genic locations of the dsRNAs.Percentages of the up-regulated dsRNA regions upon HNRNPC knock-down, which were divided into different categories according to the type of their host genes (left) or intra-genic locations (right).MA plot showing differential expression of the genes, upon HNRNPC knock-down. The x-axis represents the average read count of each gene (in logarithm scale), and the y-axis represents log2 fold change of the read count in siHNRNPC vs. siNC. The differentially expressed genes, identified by DESeq2 and shown in Fig 2A, were marked in blue. The host genes of the up-regulated dsRNA regions upon HNRNPC knock-down were marked in red.

Fig 3: Global SUMOylome Differences between MEFs and ESCs(A) Volcano plot displaying differentially modified SUMO2/3 sites in MEFs (blue dots) versus ESCs (red dots). n = 4 cell culture replicates, q < 0.05 with permutation-based FDR calculated at an s0 of 0.5. The black line in the graph represents the FDR cut-off threshold.(B) Venn diagrams showing the numbers of SUMO2/3 sites (left) and SUMO2/3 target proteins (right) preferentially SUMOylated (log2 fold change [FC] > 1) in MEFs, ESCs, or common to both cell types.(C) Correlation between protein abundance (x axis) and the SUMO2/3 abundance for the corresponding proteins (y axis) in MEFs (top) and ESCs (bottom). The dotted line corresponds to the regression line.(D) Immunoblots for Hnrnpc and SUMO2/3 in MEFs and ESCs after 2 days continuous treatment with DMSO or 2 µM ML-792. Histone H3 was used as a loading control given the comparable histone/chromatin content per cell, regardless of cell type. Signals were obtained from the same blot. Two different exposures of the same blot are shown for Hnrnpc. Representative example, n = 2.(E) Scheme of the SUMOylation cycle (left) and immunoblots (right) for the indicated SUMO enzymes in MEFs and ESCs. Whole-cell lysates from the same number of cells were loaded each time for the two cell types. Fourteen independent blots are shown. Histone H3 was used as a loading control given the comparable histone/chromatin content per cell, regardless of cell type. Loading control for Senp3 is shown; see also Figure S1H. The asterisk indicates a non-specific band. Representative example, n = 2.(F) Relative protein abundance of members of the SUMOylation machinery identified in the total proteome of MEFs and ESCs, normalized to histone H3 levels. The ratio was fixed to 100% for the cell type with the highest expression for each factor. Error bars indicate mean ± SD.See also Figures S1 and Tables S1, S2, and S3.

Fig 4: RBP interaction network based on RBP-associated RNA editing sites. A Numbers of RBP pairs (Y-axis) sharing the same groups of RNA editing events (X-axis). B RBP interaction network showing the overlapping RNA editing events associated with each pair of RBPs in K562 cells. The thickness of the edges indicates the number of shared editing sites. C RBP interaction network based on the lengths of the overlapping RBP-binding RNA regions. D EMSA assay showing the shifted RNA bands upon preincubation with the two proteins HNRNPC and ILF3 pooled together

Fig 5: Repression of HNRNPC resulted in elevation of endogenous dsRNA Immunofluorescence analysis of the dsRNA in MCF7 cells after knock-down of HNRNPC, with 4',6-diamidino-2-phenylindole (DAPI) staining (blue) and anti-dsRNA antibody J2 (green). Cells transfected with poly I:C was included as a positive control of dsRNA, and the cells treated with RNase III was used as a negative control. siNC: non-targeting siRNA as a negative control, siHN-1: siRNA sequence 1 for HNRNPC, siHN-2: siRNA sequence 2 for HNRNPC. The size of scale bar is 10 µm.Counts of dsRNA regions in the siNC control cells or in the cells with siHNRNPC, identified in the dsRNA-enriched libraries with anti-dsRNA J2 pull-down or in the control libraries including the IgG control and the input control for dsRNA pull-down. Each bar represents the overlapping dsRNA species from two replicate experiments. Among each set of the dsRNA regions, the dsRNAs from Alus were marked in gray.MA plot showing all the dsRNA regions identified in the dsRNA-enriched libraries. For each dsRNA region, mean of the read counts (log2) for two replicates of siHNRNPC and siNC cells was shown on the x-axis, and the average fold change (log2) in siHNRNPC vs. siNC on the y-axis. The up-regulated dsRNA regions were marked in red and down-regulated regions in black.An example of the up-regulated dsRNA regions upon HNRNPC knock-down. On top of the figure are plots of the read densities along the dsRNA region, in the dsRNA-enriched libraries (J2) and the two control libraries (IgG and input), from the siNC and siHNRNPC cells. This dsRNA region was originated from intron 4 of DDX21 transcript ENSG00000165732.12-001, which is shown at the bottom of the figure. The Alu DNA elements and the HNRNPC binding regions in this intron were also marked.From the results of RepeatMasker, percentage of the up-regulated dsRNA regions (928 regions highlighted in panel C) was counted that contain or largely overlap with the Alu elements. For comparison, repeated for 10 times, the same number of intra-genic regions was randomly selected and the percentage of Alu was counted with RepeatMasker. The results, i.e., percentages of Alu in these 10 random sets, were summarized as box plot.Percentage of the introns harboring the up-regulated dsRNA regions (698 introns in total, accounting for 864 of the 928 regions shown in panel C) that contain the previously identified HNRNPC binding regions. For comparison, 10 sets of randomly selected introns, which have the same length distributions as the 698 introns, were also queried. The results, i.e., percentages of the introns containing the HNRNPC binding regions in these 10 random sets, were summarized as box plot.