Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1. Differential expression of mirnas from the pri-mir-17-92a locus.

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1 Supplementary Figure 1 Differential expression of mirnas from the pri-mir-17-92a locus. (a) The mir-17-92a expression unit in the third intron of the host mir-17hg transcript. (b,c) Impact of knockdown of Ago1-4 on mirna expression from the mir-17-92a locus, quantified by RT-qPCR (b) and the Ago knockdown efficiency was verified by Western blotting (c). (d,e) Impact of knockdown of Dicer on mirna expression from the mir-17-92a locus, quantified by RT-qPCR (d) and the Dicer knockdown efficiency verified by Western blotting (e). Uncropped images of Western blots in c and e are shown in Supplementary Data Set 1. Data in b,d are presented as mean ± SEM (n=3, technical replicates). *P < 0.05; **P < 0.01; ***P < 0.001, determined by two-tailed Student s t test. Source data are reported in Source Data for Supplementary Figure 1.

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3 Supplementary Figure 2 Evidence for the involvement of paraspeckle-associated proteins and NEAT1 in mirna biogenesis. (a) The knockdown efficiencies of PSF (left) and NONO (right) were verified by Western blotting. (b,c) Ablation of paraspeckle key components PSF (b) or NONO (c) down regulated representative mirnas as indicated. (d) Illustration of mirna sensor reporters: Individual antisense mirnas were cloned into the 3 UTR of the Renilla luciferase in psicheck2. Reduced mirna expression would stabilize the Renilla mrna, thus increasing the relative luciferase activity. (e) Levels of mirna sensor reporters in response to knockdown of individual paraspeckle-associated proteins or NEAT1. GFP and the RNA binding protein PTB1 served as controls. (f) Knockout of PSPC1 by CRISPR up-regulates representative mirnas as indicated. (g) The absence of PSPC1 was verified by Western blotting in two independent cell lines. Uncropped images of Western blots in a,g are shown in Supplementary Data Set 1. Data in b,c,e,f are presented as mean ± SEM (n=3, technical replicates for b,c,f; n=4, cell culture for e). *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant, determined by two-tailed Student s t test. Source data are reported in Source Data for Supplementary Figure 2.

4 Supplementary Figure 3 Interrelationship of paraspeckle associated proteins and RNA. (a) HeLa cells were immunostained with anti-nono (green) and anti-pspc1 (red) antibodies. DAPI was used to indicate nucleus. In wild-type cells (WT), NONO and PSPC1 co-localized in paraspeckles. In response to knockdown (KD) of PSF (second raw) or NONO (third raw), the paraspeckle marker PSCP1 no longer showed foci. In contrast, in PSPC1 knockout (KO) cells, NONO continued to show foci. Scale bars, 10 μm. (b) RT-qPCR was performed to confirm NEAT1 knockdown with a stealth sirna and the impact on mirna expression. (c-f) Western blot analysis of the Microprocessor Drosha/DGCR8 and a panel of other paraspeckle-associated RBPs upon knockdown of paraspeckle-associated proteins or NEAT1. Data in b are presented as mean ± SEM (n=3, technical replicates). *P < 0.05; **P < 0.01; ***P < 0.001, determined by two-tailed Student s t test. Source data are reported in Source Data for Supplementary Figure 3. Uncropped images of Western blots are shown in Supplementary Data Set 1.

5 Supplementary Figure 4 mirna profiling by small RNA-seq and validation by RT-qPCR. (a,b) Counts of reference sequences (trnas, snornas, spike-in RNA, rrnas) between duplicated small RNA-seq libraries from sigfp-treated cells (a) or between sipsf and sigfp-treated cells (b). (c,d) Correlation of mirna counts between duplicated libraries from sigfp-treated (c) or sipsf-treated HeLa cells (d). (e) A panel of mirnas validated by RT-qPCR. (f) Heatmap representation of validated mirna expression from the mir-17-92a locus (left) or other pri-mirnas (right) in response to knockdown of various paraspeckle-associated proteins or NEAT1 as indicated. Data in e are presented as mean ± SEM (n=3, technical replicates). **P < 0.01; ***P < 0.001; NS, not significant, determined by two-tailed Student s t test. Data source data are reported in Source Data for Supplementary Figure 4.

6 Supplementary Figure 5 CLIP-seq analysis of PSF/NONO interactions with RNA. (a) The anti-psf immunoprecipitated complex trimmed with two different concentrations of MNase (1: 1,000 or 1: 50,000 dilution) and analyzed by autoradiography (bottom panel) and Western blotting (upper panel). (b) Reproducibility between independently constructed CLIP-seq libraries for NONO and PSF. Global comparison was performed with 1 Kb-binned genome. (c) The genomic distribution of NONO and PSF CLIP-seq peaks. (d) Additional examples of NONO and PSF binding on a set of pri-mirnas in HeLa cell. Uncropped images of Western blots and autoradiography in a are shown in Supplementary Data Set 1.

7 Supplementary Figure 6 Exogenous DGCR8 interacts with endogenous NONO. (a) The pri-mirna-based reporter assay for pri-mir-612 processing relative to pri-mir-17-92a processing in response to knockdown of the Microprocessor. (b) Detection of FLAG-tagged DGCR8 in anti-nono immunoprecipitant. (c) Detection of NONO in FLAG-tagged DGCR8 immunoprecipitant. * indicates IgG heavy chain. Data are shown in a as mean ± SEM (n=3, cell culture). *P < 0.05; ***P < 0.001; NS, not significant, determined by two-tailed Student s t test. Data source are reported in Source Data for Supplementary Figure 6. Uncropped images of Western blots in b and c are shown in Supplementary Data Set 1.