Fig 1.
ASO-mediated depletion of NEAT1 partially rescued CX5461-induced transcriptional inhibition.
A. qRT-PCR of the levels of NEAT1 lncRNA in HeLa cells either mock-treated or transfected with NEAT1 or control ASOs. B. Dose-dependent reduction of 47S rRNA precursor by CX5461 was partially rescued in NEAT1-depeted cells. C. Nascent rRNA was visualized by IF staining of incorporated 5-FU. HeLa cells were transfected with 30 nM cy3-labled ASOs and treated with DMSO or CX5461 (120 nM) for 2.5 hrs. Cells were pulsed with 1 mM 5-FU for 10 mins.
Fig 2.
ASO-mediated depletion of NEAT1 alleviated the RNAP I inhibition induced nucleolar disruption.
A. IF showed the localization of fibrillarin to nucleoli (red arrows) in DMSO-treated HeLa cells. B. Fibrillarin redistributed to nucleoplasmic foci (the green box) or perinucleolar caps (red arrows) by CX5461. C. NEAT1 ASOs had no effect on the distribution of fibrillarin in DMSO treated cells. Cy3-labled NEAT1 ASOs (30 nM) were transfected for 2 hrs, followed by DMSO treatment for 2.5 hrs. D. Redistribution of fibrillarin to nucleoplasmic foci by CX5461 was prevented in cells containing NEAT1 ASOs (the red box). HeLa cells were transfected with 30 nM NEAT1 ASOs for 2 hrs, followed by CX5461 treatment (250 nM) for 2.5 hrs. E. Alleviated CX5461-induced nucleolar disruption was specific for NEAT1 depletion. HeLa cells were treated as described in Fig 2D with multiple NEAT1 ASOs or control ASOs. F and G. Combined NEAT1-FISH and IF of fibrillarin in HeLa cells. HeLa cells were treated as described in Fig 2D with either control ASO (F.) or NEAT1 ASO-1 (G.). Cells that contain enriched amount of ASOs were boxed in red. Cell that contain little or no ASOs were boxed in green.
Fig 3.
NEAT1 depletion rescued the clearance of RPL5 from nucleoli by CX5461.
HeLa cells were mock-transfected or transfected with 30 nM NEAT1 ASOs, followed by the treatment with DMSO or 125 nM CX5461 for 6 hrs.
Fig 4.
CX5461-induced nucleolar disruption was not attenuated upon disassembly of paraspeckles by depleting essential protein components.
A. Western analysis of protein reduction by corresponding siRNAs. B. Depletion of the selected paraspeckle proteins has no effect on nucleolar localization of fibrillarin in the presence of DMSO. C. Depletion of the selected paraspeckle proteins failed to rescue the CX5461-induced mislocalization of fibrillarin to nucleoplasm.
Fig 5.
Depletion of NEAT1 lncRNA allowed increased association between P54nrb/ PSF and c-Myc mRNA to facilitate the IRES-translation of c-Myc.
A. Western analysis of protein reduction by corresponding siRNAs. B. Simultaneous depletion of P54nrb and PSF by siRNAs caused proteolytic cleavage of PARP. C. qRT-PCR suggested the upregulation of p21Waf1/Cip1 and Gadd45a mRNAs upon depletion of PSF. D. Simultaneous depletion of P54nrb and PSF by siRNAs reduced levels of c-Myc protein in the presence of CX5461. HeLa cells were transfected with individual siRNAs at a final concentration of 3 nM for 48 hrs, followed by the treatment with DMSO or 125 nM CX5461 for 6 hrs. E. Depletion of NEAT1 lncRNA rescued levels of c-Myc protein in the presence of CX5461 in HeLa cells. HeLa cells were mock-transfected or transfected with 30 nM NEAT1 or control ASOs for 2hrs, followed by the treatment with DMSO or 125 nM CX5461 for 6 hrs. F. qRT-PCR showed levels of NEAT1 lncRNA and c-Myc mRNA in HeLa cells. G. qRT-PCR for relative levels of c-Myc and control LDLr mRNAs in pooled polysome (F12-F25) and mono-ribosome (80S) (F6-F10) fractions from CX5461-treated HeLa cells transfected with NEAT1 ASO-1 or control ASO (con.), as in S5 Fig). Error bars indicate s.d. of three independent experiments. P values were calculated based on unpaired t-test. NS, not significant. H. HeLa cells were mock-transfected or transfected with 30 nM NEAT1 for 2 hrs, followed by the treatment with DMSO, CX5461 (125 nM), or CX5461 (125 nM)/4E1Rcat (10 μM) for 6 hrs. I and J. qRT-PCR of levels of c-Myc RNA co-immunoprecipitated with anti-P54nrb (I.) or anti-PSF (J.) antibodies. Immunoprecipitation by anti-mouse IgG antibody serves as a background control. Results are presented as percent recovery from the input material. K. qRT-PCR showed levels of NEAT1 lncRNA in MCF7 cells. L. Depletion of NEAT1 lncRNA failed to rescue the levels of c-Myc protein in the presence of CX5461 in MCF7 cells. Experiments were performed as described in Fig 5E.