Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Colocalization of endogenous CF I_m large subunits with nuclear markers.
  HeLa cells were double-labeled with a rabbit antiserum that recognizes CF I_m large subunits (green) and with mouse monoclonal antibodies that recognize different nuclear markers (red). a – e : anti-CF I_m; a’: anti-p80 coilin (5P10, (Almeida et al., 1998); b’: anti–PANA (Clevenger and Epstein, 1984); c’: anti-U2AF65 (MC3, (Gama-Carvalho et al. , 1997); d’: anti-SR (16H3); e’: anti-SC35; a’’- e’’: merges of the green and red channels. Bar: 10 μm.
• Supplemental Figure 3 - Colocalization of endogenous CF I_m large subunits with SC35 in the cell cycle.
  Three sequential planes of the cells shown in Figure 3B demonstrate the degree of overlap between different compartments. Arrowheads indicate colocalization in speckles. Broken arrows indicate CF I_m68 foci.
• Supplemental Figure 2A - Localization of pre-mRNA cleavage factors.
  A. Localization of CF I_m68 relative to the 100 kDa subunit of cleavage and polyadenylation specificity factor (CPSF100) or the 64 kDa of the cleavage specificity factor (CstF64). HeLa cells were double-labeled with a rabbit antiserum that recognizes CF I_m68 (panels a’ and b’, red) and with mouse monoclonal antibody to CPSF100 (panel a, green Jenny et al. , 1994), or with a chicken antibody to CstF64 (panel b, green). The images of the green fluorescence and the red fluorescence are merged in panels a’’ and b’’. All three cleavage factors are characterized by a diffuse nucleoplasmic staining. Colocalization with CF I_m68 occurs mainly in the nucleoplasm while neither the speckle-like enrichments nor the bright foci that are stained with the anti-CF I_m68 antiserum colocalize with the ’cleavage bodies’ previously described for CSPF100 (Schul et al. , 1996).
• Supplemental Figure 2B - Localization of pre-mRNA cleavage factors.
  B. Localization of PSP1 relative to CPSF100 or CstF64. HeLa cells were double-labeled with a rabbit antiserum that recognizes PSP1 (panels a’ and b’, red) and with mouse monoclonal antibody to CPSF100 (panel a, green, Jenny et al.), or with a chicken antibody to CstF64 (panel b, green). The images of the green fluorescence and the red fluorescence are merged in panels a’’ and b’’. Neither CPSF100 foci nor the CstF64 enrichments (broken arrows) colocalize with the paraspeckles stained by the PSP1 antibody (arrowheads).
• Supplemental Figure 4 - Confocal microscope section of HeLa cells that were incubated for 10 min with BrUTP (panel a, green) and immunostained with anti-CF I_m68 antiserum (panel b, red). The merged colocalization image (panel c) shows overlap between anti-CF I_m68 staining and BrRNA mainly in the nucleoplasm while the CF I_m68 foci are adjacent but do not overlap with the anti-BrUTP labeled spots. Arrowhead indicate CF I_m68 foci. Broken arrows indicate spot-like sites of BrUTP accumulation.