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1 DOI: /ncb2157 Figure S1 Immobilization of histone pre-mrna to chromatin leads to formation of histone locus body with associated Cajal body. Endogenous histone H2b(e) pre-mrna is processed with high efficiency (A). Northern blot analysis using the specific oligoprobe against the coding region of histone H2b(e) sequence indicates that endogenous histone H2b transcripts are processed more efficiently than exogenous histone H2b-MS2 pre-mrnas. De novo HLBs formed by tethering H2b-MS2 RNA contain FLASH, a key histone pre-mrna processing factor (B). Microscopy analysis of de novo HLB and CB formation on the LacO array in HeLa cells coexpressing H2b-MS2 RNA with GFP-LacI-NLS-MS2 coat protein and stained with anti-flash and anti-coilin antibodies, and DAPI. All H2b-MS2 RNA mutants were ectopically expressed to similar levels as endogenous H2b (C). Quantitative real-time PCR analysis on transcription levels of H2b-MS2 constructs transiently expressed in HeLa cells. Results are averages from three experiments. Scale bar, 2 μm. 1
2 Figure S2 The kinetics of de novo HLB and CB formation by histone H2b-MS2 transcripts and de novo formation of HLB and CB by factors involved in histone gene expression and the histone 3 -end processing. Microscopy analysis of de novo HLB and CB formation on the LacO array at the indicated times after IPTG withdrawal in HeLa cells transiently cotransfected with histone H2b-MS2 and GFP-LacI-NLS-coat protein. Cells were treated with IPTG for 16 hr, which prevents binding of LacI-fusion proteins to the LacO array (A). IPTG was then washed out to determine rebinding kinetics of GFP-LacI-NLS-coat protein to the LacO array. Thirty minutes after IPTG washout GFP-LacI-NLS-coat protein is detectable on the LacO array (not shown). However, two hours after IPTG washout de novo HLB with associated de novo CB are formed with immobilized H2b-MS2 transcripts detected by specific antibodies against NPAT and coilin (B). Factor specifically associated with the 3 -end of histone pre-mrna is capable of forming HLB and CB de novo. Microscopy analysis of HeLa cells transiently transfected with LacI-fusion proteins (green). Immobilization of Lsm11, a unique component of U7 snrnp (C), leads to de novo CB formation detected by anti-coilin antibody. Tethering of CPSF73 (D) and CPSF100 (E), components of the cleavage complex involved in the 3 -end processing of histone pre-mrna trigger de novo formation of HLB detected by anti-flash or anti-npat antibody and CB detected by anti-gemin2 or anti-coilin antibody (D, E). Scale bar, 2 μm. 2
3 Figure S3 The kinetics of de novo HLB and CB formation by immobilized SLBP, a protein selectively associated with the 3 -end of histone pre-mrna, on chromatin. Microscopy analysis of de novo HLB and CB formation on the LacO array at the indicated times after IPTG withdrawal in HeLa cells transiently transfected with GFP-LacI-SLBP and stained with antibodies against NPAT and coilin. Cells were treated with IPTG for 16 hr, which prevents binding of GFP-LacI-SLBP to the LacO array (A). However, HLB and CB are not formed on the LacO array tethering SLBP 1 hour after IPTG washout (B). In agreement with tethering histone H2b-MS2 on chromatin, two hours after IPTG withdrawal de novo HLB and CB are formed on the LacO array with tethered SLBP (C). Scale bar, 2 μm. 3
4 Figure S4 Immobilized β-globin-ms2 wild-type transcripts on the LacO array contained a mixture of spliced and unspliced RNAs. RNA FISH using a fluorescently labeled probe against intron 1 of β-globin minigene (red) combined with immunofluorescence staining using the antibody against SR splicing factor SC35 (white) in HeLa cells transiently transfected with β-globin-ms2 minigene constructs and GFP-LacI-NLS-MS2 coat protein (A to C). As a negative control, intron 1 is undetectable on the LacO array tethering β-globin-ms2 intron-less RNAs (C). Scale bar, 2 μm. Immobilization of SR splicing factor SC35 triggers accumulation of speckles around the LacO array (D). Microscopy analysis of HeLa cells transiently transfected with GFP-LacI-SC35 (green) tethered on chromatin (red) and immunocolocalized with anti-coilin antibody (white). Scale bar, 2 μm. 4
5 Figure S5 The kinetics of de novo paraspeckle formation by NEAT1-MS2 ncrna. Microscopy analysis of de novo paraspeckle formation on the LacO array after IPTG withdrawal in HeLa cells transiently cotransfected with NEAT1-MS2 ncrna and GFP-LacI-NLS-MS2 coat protein by RNA FISH using a fluorescently labeled probe against NEAT1 (red) and stained with anti-psp1 antibody (white). Scale bar, 2 μm. 5
6 Figure S6 Immobilization of the non-coding satiii RNAs on the LacO array form nsbs without heat shock induction. RNA FISH using the fluorescently labeled probe against satiii transcript (red) combined with immunofluorescence microscopy on HeLa cells transiently cotransfected with satiii-ms2 RNAs and GFP-LacI-NLS-MS2 coat protein and stained with antibody against SR splicing factor SF2/ASF (white). Scale bar, 2 μm. 6
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p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
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File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables File Name: Peer Review File Description: Primer Name Sequence (5'-3') AT ( C) RT-PCR USP21 F 5'-TTCCCATGGCTCCTTCCACATGAT-3'