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Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA

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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.

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Intra-paraspeckle localization of Ctn RNA.(A) Super-resolution structured illumination microscopy (SR-SIM) of Ctn RNA (green) and Neat1 (red) localization in DRB recovered and MG132-treated transformed WT-MEFs. Scale bar indicates 10 μm. Arrow (a–h) indicates paraspeckle where Ctn RNA does not show co-localization with Neat1. (B,C) Co-localization of a single paraspeckle in (B) DRB recovered and (C) MG132-treated transformed WT-MEF. (D,E) Quantitation of Ctn RNA and Neat1 co-localization in (Da–c) DRB recovered and (Ea–c) MG132 treated transformed WT-MEFs (performed using ZEN 2012). Numbers in image indicate the specific paraspeckle analyzed and corresponds to the number mentioned in the graph. For example, “1” in image refers to “paraspeckle 1” in graph.
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f6: Intra-paraspeckle localization of Ctn RNA.(A) Super-resolution structured illumination microscopy (SR-SIM) of Ctn RNA (green) and Neat1 (red) localization in DRB recovered and MG132-treated transformed WT-MEFs. Scale bar indicates 10 μm. Arrow (a–h) indicates paraspeckle where Ctn RNA does not show co-localization with Neat1. (B,C) Co-localization of a single paraspeckle in (B) DRB recovered and (C) MG132-treated transformed WT-MEF. (D,E) Quantitation of Ctn RNA and Neat1 co-localization in (Da–c) DRB recovered and (Ea–c) MG132 treated transformed WT-MEFs (performed using ZEN 2012). Numbers in image indicate the specific paraspeckle analyzed and corresponds to the number mentioned in the graph. For example, “1” in image refers to “paraspeckle 1” in graph.

Mentions: Ctn RNA and Neat1 localization studies, especially in the proteasome-inhibited cells, using conventional fluorescent microscopy indicated that only a fraction of the paraspeckle-associated Neat1 and Ctn RNA displayed complete co-localization (Fig. 5Be–h). To achieve a better understanding of the localization of these RNA molecules in paraspeckles, we used Super-resolution structured illumination (SR-SIM) microscopy to determine the molecular organization of Ctn RNA and Neat1 in MG132-treated and DRB-recovered transformed WT-MEFs (Fig. 6). We observed that under both conditions, Ctn RNA did not completely overlap with Neat1 positive paraspeckles (Fig. 6B–E). This suggests that Ctn RNA decorated only a part of Neat1-nucleated paraspeckles (Fig. 6A–C). Furthermore, we observed that not all Neat1-nucleated paraspeckles contained Ctn RNA (Fig. 6A; see arrow). We further quantitated the ratio and degree of overlap of Neat1 and Ctn RNA in paraspeckles (Fig. 6Da–c,Ea–c). The results showed that in DRB recovered cells, both Neat1 and Ctn RNA foci display a more homogenous intraparaspeckle distribution and largely overlap with each other (Fig. 6Da–c). However, upon MG132 treatment, Ctn RNA and Neat1 showed altered peak ratios and degree of overlap suggestive of a more heterogeneous intraparaspeckle distribution (Fig. 6Ea–c).


Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA
Intra-paraspeckle localization of Ctn RNA.(A) Super-resolution structured illumination microscopy (SR-SIM) of Ctn RNA (green) and Neat1 (red) localization in DRB recovered and MG132-treated transformed WT-MEFs. Scale bar indicates 10 μm. Arrow (a–h) indicates paraspeckle where Ctn RNA does not show co-localization with Neat1. (B,C) Co-localization of a single paraspeckle in (B) DRB recovered and (C) MG132-treated transformed WT-MEF. (D,E) Quantitation of Ctn RNA and Neat1 co-localization in (Da–c) DRB recovered and (Ea–c) MG132 treated transformed WT-MEFs (performed using ZEN 2012). Numbers in image indicate the specific paraspeckle analyzed and corresponds to the number mentioned in the graph. For example, “1” in image refers to “paraspeckle 1” in graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Intra-paraspeckle localization of Ctn RNA.(A) Super-resolution structured illumination microscopy (SR-SIM) of Ctn RNA (green) and Neat1 (red) localization in DRB recovered and MG132-treated transformed WT-MEFs. Scale bar indicates 10 μm. Arrow (a–h) indicates paraspeckle where Ctn RNA does not show co-localization with Neat1. (B,C) Co-localization of a single paraspeckle in (B) DRB recovered and (C) MG132-treated transformed WT-MEF. (D,E) Quantitation of Ctn RNA and Neat1 co-localization in (Da–c) DRB recovered and (Ea–c) MG132 treated transformed WT-MEFs (performed using ZEN 2012). Numbers in image indicate the specific paraspeckle analyzed and corresponds to the number mentioned in the graph. For example, “1” in image refers to “paraspeckle 1” in graph.
Mentions: Ctn RNA and Neat1 localization studies, especially in the proteasome-inhibited cells, using conventional fluorescent microscopy indicated that only a fraction of the paraspeckle-associated Neat1 and Ctn RNA displayed complete co-localization (Fig. 5Be–h). To achieve a better understanding of the localization of these RNA molecules in paraspeckles, we used Super-resolution structured illumination (SR-SIM) microscopy to determine the molecular organization of Ctn RNA and Neat1 in MG132-treated and DRB-recovered transformed WT-MEFs (Fig. 6). We observed that under both conditions, Ctn RNA did not completely overlap with Neat1 positive paraspeckles (Fig. 6B–E). This suggests that Ctn RNA decorated only a part of Neat1-nucleated paraspeckles (Fig. 6A–C). Furthermore, we observed that not all Neat1-nucleated paraspeckles contained Ctn RNA (Fig. 6A; see arrow). We further quantitated the ratio and degree of overlap of Neat1 and Ctn RNA in paraspeckles (Fig. 6Da–c,Ea–c). The results showed that in DRB recovered cells, both Neat1 and Ctn RNA foci display a more homogenous intraparaspeckle distribution and largely overlap with each other (Fig. 6Da–c). However, upon MG132 treatment, Ctn RNA and Neat1 showed altered peak ratios and degree of overlap suggestive of a more heterogeneous intraparaspeckle distribution (Fig. 6Ea–c).

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