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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.


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Ctn RNA forms enlarged foci in proteasome-inhibited cells.(A) Schematic showing the experimental design. (B) RNA-FISH analysis of Ctn RNA (green) and Neat1 (red) localization in control (DMSO-treated) and MG132-treated transformed WT-MEFs. Scale bar indicates 10 μm. DNA is counterstained with DAPI (blue). (C) RT-qPCR analysis of Neat1 RNA levels in control (DMSO-treated) and MG132-treated transformed WT-MEFs. (D) RT-qPCR analysis of Ctn RNA levels in control (DMSO-treated) and MG132-treated transformed WT-MEFs. Gapdh was used as the normalization control in RT-qPCR experiments. Error bars in (C,D) represent mean ± SD of three independent experiments. ***P < 0.001, *P < 0.05 using Student’s t test.
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f5: Ctn RNA forms enlarged foci in proteasome-inhibited cells.(A) Schematic showing the experimental design. (B) RNA-FISH analysis of Ctn RNA (green) and Neat1 (red) localization in control (DMSO-treated) and MG132-treated transformed WT-MEFs. Scale bar indicates 10 μm. DNA is counterstained with DAPI (blue). (C) RT-qPCR analysis of Neat1 RNA levels in control (DMSO-treated) and MG132-treated transformed WT-MEFs. (D) RT-qPCR analysis of Ctn RNA levels in control (DMSO-treated) and MG132-treated transformed WT-MEFs. Gapdh was used as the normalization control in RT-qPCR experiments. Error bars in (C,D) represent mean ± SD of three independent experiments. ***P < 0.001, *P < 0.05 using Student’s t test.

Mentions: Recently, it was demonstrated that paraspeckles become dramatically enlarged upon proteasome inhibition23. Surprisingly, this enlargement in paraspeckle size was shown to be a result of Neat1 transcription activation, and not because of the accumulation of undegraded PSPs. In fact, upon proteasome inhibition, PSPs were sequestered into paraspeckles as evidenced by 50% depletion of these proteins from the nucleoplasm23. To determine if paraspeckle RNA component, Ctn RNA showed any changes upon proteasome inhibition, we treated transformed WT-MEFs with the proteasome inhibitor MG132 for 17 h and compared the Ctn RNA foci with control (DMSO-treated) cells (Fig. 5A). We observed that Ctn RNA also formed enlarged nuclear foci upon MG132-treatment (Figs 5B and S4).


Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA
Ctn RNA forms enlarged foci in proteasome-inhibited cells.(A) Schematic showing the experimental design. (B) RNA-FISH analysis of Ctn RNA (green) and Neat1 (red) localization in control (DMSO-treated) and MG132-treated transformed WT-MEFs. Scale bar indicates 10 μm. DNA is counterstained with DAPI (blue). (C) RT-qPCR analysis of Neat1 RNA levels in control (DMSO-treated) and MG132-treated transformed WT-MEFs. (D) RT-qPCR analysis of Ctn RNA levels in control (DMSO-treated) and MG132-treated transformed WT-MEFs. Gapdh was used as the normalization control in RT-qPCR experiments. Error bars in (C,D) represent mean ± SD of three independent experiments. ***P < 0.001, *P < 0.05 using Student’s t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036046&req=5

f5: Ctn RNA forms enlarged foci in proteasome-inhibited cells.(A) Schematic showing the experimental design. (B) RNA-FISH analysis of Ctn RNA (green) and Neat1 (red) localization in control (DMSO-treated) and MG132-treated transformed WT-MEFs. Scale bar indicates 10 μm. DNA is counterstained with DAPI (blue). (C) RT-qPCR analysis of Neat1 RNA levels in control (DMSO-treated) and MG132-treated transformed WT-MEFs. (D) RT-qPCR analysis of Ctn RNA levels in control (DMSO-treated) and MG132-treated transformed WT-MEFs. Gapdh was used as the normalization control in RT-qPCR experiments. Error bars in (C,D) represent mean ± SD of three independent experiments. ***P < 0.001, *P < 0.05 using Student’s t test.
Mentions: Recently, it was demonstrated that paraspeckles become dramatically enlarged upon proteasome inhibition23. Surprisingly, this enlargement in paraspeckle size was shown to be a result of Neat1 transcription activation, and not because of the accumulation of undegraded PSPs. In fact, upon proteasome inhibition, PSPs were sequestered into paraspeckles as evidenced by 50% depletion of these proteins from the nucleoplasm23. To determine if paraspeckle RNA component, Ctn RNA showed any changes upon proteasome inhibition, we treated transformed WT-MEFs with the proteasome inhibitor MG132 for 17 h and compared the Ctn RNA foci with control (DMSO-treated) cells (Fig. 5A). We observed that Ctn RNA also formed enlarged nuclear foci upon MG132-treatment (Figs 5B and S4).

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