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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 interacts with paraspeckle component NonO in absence of intact paraspeckles.(A) RNA-FISH to detect Ctn RNA (green) and NonO (red) in DRB-recovered WT and Neat1-KO MEFs. Scale bar indicates 10 μm. Arrow (a–h) indicates Ctn RNA and NonO positive nuclear foci. DNA is counterstained with DAPI (blue). (B) Graph showing percentage co-localization in WT and Neat1-KO MEFs. (C) NonO-RIP (RNA immunoprecipitation) followed by RT-qPCR analysis of Ctn RNA to determine interaction of NonO and Ctn RNA in WT and Neat1-KO MEFs. (D) Western blot showing NonO levels in WT and Neat1-KO MEFs. Tubulin was used as a loading control. Gapdh was used as the normalization control in RT-qPCR experiments. Error bars in (B,C) represent mean ± SD of three independent experiments. **P < 0.01 using Student’s t test.
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f2: Ctn RNA interacts with paraspeckle component NonO in absence of intact paraspeckles.(A) RNA-FISH to detect Ctn RNA (green) and NonO (red) in DRB-recovered WT and Neat1-KO MEFs. Scale bar indicates 10 μm. Arrow (a–h) indicates Ctn RNA and NonO positive nuclear foci. DNA is counterstained with DAPI (blue). (B) Graph showing percentage co-localization in WT and Neat1-KO MEFs. (C) NonO-RIP (RNA immunoprecipitation) followed by RT-qPCR analysis of Ctn RNA to determine interaction of NonO and Ctn RNA in WT and Neat1-KO MEFs. (D) Western blot showing NonO levels in WT and Neat1-KO MEFs. Tubulin was used as a loading control. Gapdh was used as the normalization control in RT-qPCR experiments. Error bars in (B,C) represent mean ± SD of three independent experiments. **P < 0.01 using Student’s t test.

Mentions: Next, we wanted to determine if disruption of paraspeckle structure affects the interaction of Ctn RNA with other paraspeckle components. Ctn RNA has been shown to interact with paraspeckle proteins such as NonO and PSP126. In the current study, we ascertained if the Ctn RNA continues to associate with paraspeckle proteins in the absence of Neat1. To this end, we determined the localization of Ctn RNA and paraspeckle protein NonO by performing RNA-FISH followed by immunostaining for NonO in WT and Neat1-KO MEFs. We observed that as shown in previous studies, Ctn RNA and NonO co-localized in WT-MEFs (Fig. 2Aa–d)26. Surprisingly, in the Neat1 KO cells, a few but not all of the bright Ctn RNA foci co-localized with NonO-stained foci (Fig. 2Ae–h,B; please see the arrows).


Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA
Ctn RNA interacts with paraspeckle component NonO in absence of intact paraspeckles.(A) RNA-FISH to detect Ctn RNA (green) and NonO (red) in DRB-recovered WT and Neat1-KO MEFs. Scale bar indicates 10 μm. Arrow (a–h) indicates Ctn RNA and NonO positive nuclear foci. DNA is counterstained with DAPI (blue). (B) Graph showing percentage co-localization in WT and Neat1-KO MEFs. (C) NonO-RIP (RNA immunoprecipitation) followed by RT-qPCR analysis of Ctn RNA to determine interaction of NonO and Ctn RNA in WT and Neat1-KO MEFs. (D) Western blot showing NonO levels in WT and Neat1-KO MEFs. Tubulin was used as a loading control. Gapdh was used as the normalization control in RT-qPCR experiments. Error bars in (B,C) represent mean ± SD of three independent experiments. **P < 0.01 using Student’s t test.
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f2: Ctn RNA interacts with paraspeckle component NonO in absence of intact paraspeckles.(A) RNA-FISH to detect Ctn RNA (green) and NonO (red) in DRB-recovered WT and Neat1-KO MEFs. Scale bar indicates 10 μm. Arrow (a–h) indicates Ctn RNA and NonO positive nuclear foci. DNA is counterstained with DAPI (blue). (B) Graph showing percentage co-localization in WT and Neat1-KO MEFs. (C) NonO-RIP (RNA immunoprecipitation) followed by RT-qPCR analysis of Ctn RNA to determine interaction of NonO and Ctn RNA in WT and Neat1-KO MEFs. (D) Western blot showing NonO levels in WT and Neat1-KO MEFs. Tubulin was used as a loading control. Gapdh was used as the normalization control in RT-qPCR experiments. Error bars in (B,C) represent mean ± SD of three independent experiments. **P < 0.01 using Student’s t test.
Mentions: Next, we wanted to determine if disruption of paraspeckle structure affects the interaction of Ctn RNA with other paraspeckle components. Ctn RNA has been shown to interact with paraspeckle proteins such as NonO and PSP126. In the current study, we ascertained if the Ctn RNA continues to associate with paraspeckle proteins in the absence of Neat1. To this end, we determined the localization of Ctn RNA and paraspeckle protein NonO by performing RNA-FISH followed by immunostaining for NonO in WT and Neat1-KO MEFs. We observed that as shown in previous studies, Ctn RNA and NonO co-localized in WT-MEFs (Fig. 2Aa–d)26. Surprisingly, in the Neat1 KO cells, a few but not all of the bright Ctn RNA foci co-localized with NonO-stained foci (Fig. 2Ae–h,B; please see the arrows).

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