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Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA

View Article: PubMed Central - PubMed

ABSTRACT

Paraspeckles are sub-nuclear domains that are nucleated by long noncoding RNA Neat1. While interaction of protein components of paraspeckles and Neat1 is understood, there is limited information on the interaction of non-structural RNA components with paraspeckles. Here, by varying paraspeckle number and size, we investigate how paraspeckles influence the nuclear organization of their non-structural RNA component Ctn RNA. Our results show that Ctn RNA remains nuclear-retained in the absence of intact paraspeckles, suggesting that they do not regulate nuclear retention of Ctn RNA. In the absence of Neat1, Ctn RNA continues to interact with paraspeckle protein NonO to form residual nuclear foci. In addition, in the absence of Neat1-nucleated paraspeckles, a subset of Ctn RNA localizes to the perinucleolar regions. Concomitant with increase in number of paraspeckles, transcriptional reactivation resulted in increased number of paraspeckle-localized Ctn RNA foci. Similar to Neat1, proteasome inhibition altered the localization of Ctn RNA, where it formed enlarged paraspeckle-like foci. Super-resolution structured illumination microscopic analyses revealed that in paraspeckles, Ctn RNA partially co-localized with Neat1, and displayed a more heterogeneous intra-paraspeckle localization. Collectively, these results show that while paraspeckles do not influence nuclear retention of Ctn RNA, they modulate its intranuclear compartmentalization.

No MeSH data available.


Related in: MedlinePlus

Number of Ctn RNA positive foci and their association with paraspeckles is increased upon transcriptional reactivation.(A) Schematic showing the experimental design. (B) RNA-FISH to detect Ctn RNA (green) and Neat1 (red) in control (ethanol) and DRB-recovered transformed WT-MEFs. (C) RNA-FISH analysis of Ctn RNA and Neat1 localization in a single cell of control (ethanol-treated) and DRB-recovered transformed WT-MEF. (D) Graph showing average number of paraspeckles per cell in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. (E) Graph showing percentage of cells positive for Ctn RNA foci in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. (F) RT-qPCR analysis of Ctn RNA levels in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. Gapdh was used as the normalization control in RT-qPCR experiments. Scale bar indicates 10 μm. DNA is counterstained with DAPI (blue). Error bars in (D–F) represent mean ± SD of three independent experiments. *P < 0.05, ns: not significant using Student’s t test.
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f4: Number of Ctn RNA positive foci and their association with paraspeckles is increased upon transcriptional reactivation.(A) Schematic showing the experimental design. (B) RNA-FISH to detect Ctn RNA (green) and Neat1 (red) in control (ethanol) and DRB-recovered transformed WT-MEFs. (C) RNA-FISH analysis of Ctn RNA and Neat1 localization in a single cell of control (ethanol-treated) and DRB-recovered transformed WT-MEF. (D) Graph showing average number of paraspeckles per cell in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. (E) Graph showing percentage of cells positive for Ctn RNA foci in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. (F) RT-qPCR analysis of Ctn RNA levels in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. Gapdh was used as the normalization control in RT-qPCR experiments. Scale bar indicates 10 μm. DNA is counterstained with DAPI (blue). Error bars in (D–F) represent mean ± SD of three independent experiments. *P < 0.05, ns: not significant using Student’s t test.

Mentions: The adenosine analogue 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) inhibits RNA polymerase II-mediated transcriptional elongation by inactivating the activity of pTEFb kinase. Previous studies have shown that paraspeckle protein PSP1 re-localized to perinucleolar caps after 5 min of DRB treatment and remarkably, paraspeckles structure was disrupted ~40 min after DRB treatment1219. Interestingly, paraspeckles reformed upon removal of DRB from the culture medium indicating that paraspeckle maintenance is coupled with Neat1 transcription1219. We have previously reported that Ctn RNA foci were also disrupted upon RNA pol II transcription inhibition, including treatment of cells with DRB26. Therefore, we investigated the reassembly of Ctn RNA foci upon transcription reactivation. To this end, we treated transformed WT-MEFs with DRB for three hours. Next, we removed DRB by washing cells with medium, cultured the cells in fresh media for another 3 hours, performed co-RNA-FISH and counted the number of Ctn RNA foci in both untreated and DRB recovered cells (Fig. 4A). In agreement with previous studies that demonstrated increased paraspeckle number upon DRB recovery, we observed a two to three-fold increase in the number of paraspeckles/cell, as observed by Neat1 staining (~4/cell in control to ~8/cell in treated) (Fig. 4B–D)19. In addition, we also observed a concomitant two-fold increase in number of cells with Ctn RNA decorating Neat1 positive paraspeckles (Fig. 4B,C,E) (~30% of cells showed Ctn RNA foci in control cells whereas ~70% of transcription-reactivated cells showed Ctn RNA positive paraspeckles). Furthermore, Ctn RNA staining within the paraspeckles appeared more prominent in transcription-reactivated cells, indicating a possible increase in the number of Ctn RNA molecules per paraspeckle during DRB-recovery.


Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA
Number of Ctn RNA positive foci and their association with paraspeckles is increased upon transcriptional reactivation.(A) Schematic showing the experimental design. (B) RNA-FISH to detect Ctn RNA (green) and Neat1 (red) in control (ethanol) and DRB-recovered transformed WT-MEFs. (C) RNA-FISH analysis of Ctn RNA and Neat1 localization in a single cell of control (ethanol-treated) and DRB-recovered transformed WT-MEF. (D) Graph showing average number of paraspeckles per cell in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. (E) Graph showing percentage of cells positive for Ctn RNA foci in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. (F) RT-qPCR analysis of Ctn RNA levels in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. Gapdh was used as the normalization control in RT-qPCR experiments. Scale bar indicates 10 μm. DNA is counterstained with DAPI (blue). Error bars in (D–F) represent mean ± SD of three independent experiments. *P < 0.05, ns: not significant using Student’s t test.
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f4: Number of Ctn RNA positive foci and their association with paraspeckles is increased upon transcriptional reactivation.(A) Schematic showing the experimental design. (B) RNA-FISH to detect Ctn RNA (green) and Neat1 (red) in control (ethanol) and DRB-recovered transformed WT-MEFs. (C) RNA-FISH analysis of Ctn RNA and Neat1 localization in a single cell of control (ethanol-treated) and DRB-recovered transformed WT-MEF. (D) Graph showing average number of paraspeckles per cell in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. (E) Graph showing percentage of cells positive for Ctn RNA foci in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. (F) RT-qPCR analysis of Ctn RNA levels in control (ethanol-treated) and DRB-recovered transformed WT-MEFs. Gapdh was used as the normalization control in RT-qPCR experiments. Scale bar indicates 10 μm. DNA is counterstained with DAPI (blue). Error bars in (D–F) represent mean ± SD of three independent experiments. *P < 0.05, ns: not significant using Student’s t test.
Mentions: The adenosine analogue 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) inhibits RNA polymerase II-mediated transcriptional elongation by inactivating the activity of pTEFb kinase. Previous studies have shown that paraspeckle protein PSP1 re-localized to perinucleolar caps after 5 min of DRB treatment and remarkably, paraspeckles structure was disrupted ~40 min after DRB treatment1219. Interestingly, paraspeckles reformed upon removal of DRB from the culture medium indicating that paraspeckle maintenance is coupled with Neat1 transcription1219. We have previously reported that Ctn RNA foci were also disrupted upon RNA pol II transcription inhibition, including treatment of cells with DRB26. Therefore, we investigated the reassembly of Ctn RNA foci upon transcription reactivation. To this end, we treated transformed WT-MEFs with DRB for three hours. Next, we removed DRB by washing cells with medium, cultured the cells in fresh media for another 3 hours, performed co-RNA-FISH and counted the number of Ctn RNA foci in both untreated and DRB recovered cells (Fig. 4A). In agreement with previous studies that demonstrated increased paraspeckle number upon DRB recovery, we observed a two to three-fold increase in the number of paraspeckles/cell, as observed by Neat1 staining (~4/cell in control to ~8/cell in treated) (Fig. 4B–D)19. In addition, we also observed a concomitant two-fold increase in number of cells with Ctn RNA decorating Neat1 positive paraspeckles (Fig. 4B,C,E) (~30% of cells showed Ctn RNA foci in control cells whereas ~70% of transcription-reactivated cells showed Ctn RNA positive paraspeckles). Furthermore, Ctn RNA staining within the paraspeckles appeared more prominent in transcription-reactivated cells, indicating a possible increase in the number of Ctn RNA molecules per paraspeckle during DRB-recovery.

View Article: PubMed Central - PubMed

ABSTRACT

Paraspeckles are sub-nuclear domains that are nucleated by long noncoding RNA Neat1. While interaction of protein components of paraspeckles and Neat1 is understood, there is limited information on the interaction of non-structural RNA components with paraspeckles. Here, by varying paraspeckle number and size, we investigate how paraspeckles influence the nuclear organization of their non-structural RNA component Ctn RNA. Our results show that Ctn RNA remains nuclear-retained in the absence of intact paraspeckles, suggesting that they do not regulate nuclear retention of Ctn RNA. In the absence of Neat1, Ctn RNA continues to interact with paraspeckle protein NonO to form residual nuclear foci. In addition, in the absence of Neat1-nucleated paraspeckles, a subset of Ctn RNA localizes to the perinucleolar regions. Concomitant with increase in number of paraspeckles, transcriptional reactivation resulted in increased number of paraspeckle-localized Ctn RNA foci. Similar to Neat1, proteasome inhibition altered the localization of Ctn RNA, where it formed enlarged paraspeckle-like foci. Super-resolution structured illumination microscopic analyses revealed that in paraspeckles, Ctn RNA partially co-localized with Neat1, and displayed a more heterogeneous intra-paraspeckle localization. Collectively, these results show that while paraspeckles do not influence nuclear retention of Ctn RNA, they modulate its intranuclear compartmentalization.

No MeSH data available.


Related in: MedlinePlus