supplementary information Normalized Men ε/β ncrnas level 1.0 Ratio of Men ε/men β expression 0.5 DOI: /ncb2140

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1 DOI: /ncb Normalized Men ε/β ncrnas level DOX +DOX Ratio of Men ε/men β expression 5 0 -DOX +DOX Figure S1 Men ε/β reporter gene expression level by qrt-pcr. Left, after 18 h of DOX induction, Men ε/β ncrnas level was measured by qrt- PCR and there was a one-fold increase over the endogenous level (n=4, mean±s.e.m.). Right, the ratio of Men ε ncrna vs. Men β ncrna remained the same after 18 h of DOX induction (n=4, mean±s.e.m.). 1

2 ECFP-LacI EYFP-MS2 mcherry-psp1 Merge mcherry-p54nrb mcherry-psf Spector_Fig Figure S2 Simultaneous visualization of the Men ε/β ncrnas transcription site, nascent Men ε/β ncrnas transcripts, and protein recruitment in single living cells. After 12 h of DOX induction, Men ε/β reporter gene locus was labeled with ECFP-LacI, Men ε/β ncrnas were labeled with EYFP-MS2, and the recruitment of mcherry-psp1, p54nrb or PSF protein was visualized in the C2C12 nucleus. Scale bar, 5 mm. 2

3 ECFP-LacI EYFP-MS2 α-psp1 Merge α-psp1 α-p54nrb α-p54nrb Spector_Figu Figure S3 Transcription of Men ε/β ncrnas recruits endogenous paraspeckle proteins. After 12 h of DOX induction, Men ε/β reporter gene locus was labeled with ECFP-LacI, Men ε/β ncrnas were labeled with EYFP-MS2, and endogenous paraspeckles were labeled with antibodies against PSP1 or p54nrb in the C2C12 nucleus. Scale bar, 5 mm. 3

4 ECFP-LacI EYFP-MS2 mcherry-sc35 Merge mcherry-sf2/asf Spector_Fig Figure S4 Transcription of Men ε/β ncrnas does not recruit SC35 or SF2/ASF protein. After 12 h of DOX induction, Men ε/β reporter gene locus was labeled with ECFP-LacI, Men ε/β ncrnas were labeled with EYFP-MS2, and the localization of mcherry-sc35 or SF2/ASF protein was visualized in the C2C12 nucleus. Scale bar, 5 mm. 4

5 ECFP-LacI EYFP-LacI-PSF Ctn RNA Green+Red+DAPI Spector_Fig Figure S5 Immobilization of PSF fails to recruit Ctn RNA. Tethering PSF protein to a specific locus (arrow) in the C2C12 nucleus fails to recruit Ctn RNA, which is unlike the endogenous paraspeckles (arrowhead) that can retain Ctn RNA. Scale bar, 5 mm. 5

6 Men ε/β gene loci Paraspeckles Merge Ctn RNA gene loci Average distance between paraspeckles and gene loci (μm) Men ε/β Ctn RNA Spector_F Figure S6 Paraspeckles localize close to endogenous Men ε/β gene loci in NIH 3T3 cells. Left, paraspeckles, labeled by PSP1 antibody, were often found localized adjacent to Men ε/β gene loci, but not to Ctn RNA gene loci, in interphase nuclei of NIH 3T3 cells. Scale bar, 5 mm. Right, quantification of the distance between paraspeckles and Men ε/β gene and Ctn RNA gene loci (n=50 paraspeckles in 10 cells each, mean±s.e.m.). 6

7 Supplementary Movie Legends Movie S1-3 Accumulation of EYFP-MS2 and recruitment of mcherry-psp1 to Men ε/β reporter locus upon Men ε/β transcription. Movie S4 Accumulation of EYFP-MS2 and recruitment of mcherry-p54nrb to Men ε/β reporter locus upon Men ε/β transcription. Movie S5 Accumulation of EYFP-MS2 and recruitment of mcherry-psf to Men ε/β reporter locus upon Men ε/β transcription. Movie S6 Visualization of paraspeckles by mcherry-psp1 during DRB treatment and washout. DRB was added at T=5 min and washed out at T=90 min. Movie S7. De novo formed paraspeckle was disassembled upon DOX withdrawal. Green: EYFP-MS2; red: mcherry-psf. Movie S8 Dynamics of paraspeckle through the cell-cycle. Green: EYFP-MS2; red: mcherry-p54nrb. Movie S9-11 Budding and splitting of paraspeckle during interphase. Green: EYFP-MS2; red: mcherry-psf. 7

8 Supplementary Table S1 Table S1. Values of mobile fraction and t 1/2 of paraspeckle proteins and Men ε/β ncrnas. Localization Fm (%) t 1/2 (s) n PSP1 Nucleoplasm Endogenous paraspeckles De novo formed paraspeckles p54nrb Nucleoplasm Endogenous paraspeckles De novo formed paraspeckles PSF Nucleoplasm Endogenous paraspeckles De novo formed paraspeckles Men ε/β ncrnas De novo formed paraspeckles N/A >