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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 is nuclear-retained in absence of intact paraspeckles.(A) RNA-FISH to detect Ctn RNA (green) and Neat1 (red) in DRB-recovered (3 hrs) WT and Neat1-KO MEFs. Arrow (a–f) indicates Ctn RNA foci and arrowhead (d,f) indicates perinucleolar localization of Ctn RNA. (B) Graph showing average number of Ctn RNA foci per cell in WT and Neat1-KO MEFs. (C) RNA-FISH to detect Ctn RNA and Neat1 in DRB-recovered control (Ctrl) and NonO-depleted transformed WT-MEFs. Please note that Ctn RNA shows increased paraspeckle association upon DRB recovery (please see Fig. 4). Arrow (a,c) marks Ctn RNA and Neat1 positive paraspeckle. Arrowhead (d,f) shows Ctn RNA positive but Neat1 negative paraspeckle-like nuclear body. (D) Graph showing average number of Ctn RNA foci per cell in (Ctrl) and NonO-depleted transformed WT-MEFs. (E,F) RT-qPCR to estimate Ctn RNA levels in nuclear and cytoplasmic fractions of (E) WT and Neat1-KO MEFs and (F) Ctrl and NonO-depleted MEFs. (G) RT-qPCR to detect total levels of Ctn RNA in WT and Neat1-KO MEFs. (H) Total levels of Ctn RNA in control and NonO siRNA treated MEFs. 3′UTR-1 primer pair has been used to measure Ctn RNA levels (Figure S2). Gapdh was used as the normalization control in RT-qPCR experiments. Scale bar indicates 10 μm. Error bars in (B,D,E–H) represent mean ± SD of three independent experiments. *P < 0.05, ns: not significant, using Student’s t test.
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f1: Ctn RNA is nuclear-retained in absence of intact paraspeckles.(A) RNA-FISH to detect Ctn RNA (green) and Neat1 (red) in DRB-recovered (3 hrs) WT and Neat1-KO MEFs. Arrow (a–f) indicates Ctn RNA foci and arrowhead (d,f) indicates perinucleolar localization of Ctn RNA. (B) Graph showing average number of Ctn RNA foci per cell in WT and Neat1-KO MEFs. (C) RNA-FISH to detect Ctn RNA and Neat1 in DRB-recovered control (Ctrl) and NonO-depleted transformed WT-MEFs. Please note that Ctn RNA shows increased paraspeckle association upon DRB recovery (please see Fig. 4). Arrow (a,c) marks Ctn RNA and Neat1 positive paraspeckle. Arrowhead (d,f) shows Ctn RNA positive but Neat1 negative paraspeckle-like nuclear body. (D) Graph showing average number of Ctn RNA foci per cell in (Ctrl) and NonO-depleted transformed WT-MEFs. (E,F) RT-qPCR to estimate Ctn RNA levels in nuclear and cytoplasmic fractions of (E) WT and Neat1-KO MEFs and (F) Ctrl and NonO-depleted MEFs. (G) RT-qPCR to detect total levels of Ctn RNA in WT and Neat1-KO MEFs. (H) Total levels of Ctn RNA in control and NonO siRNA treated MEFs. 3′UTR-1 primer pair has been used to measure Ctn RNA levels (Figure S2). Gapdh was used as the normalization control in RT-qPCR experiments. Scale bar indicates 10 μm. Error bars in (B,D,E–H) represent mean ± SD of three independent experiments. *P < 0.05, ns: not significant, using Student’s t test.

Mentions: Previous studies have speculated that paraspeckles are involved in the nuclear retention of A-to-I edited transcripts91026272829. Ctn RNA is a paraspeckle-associated transcript that is A-to-I edited within its long 3′UTR26. To investigate if paraspeckles regulate the nuclear retention of Ctn RNA, we determined the cellular localization of Ctn RNA in WT-MEFs (Mouse embryonic fibroblasts) and Neat1-KO (knockout) MEFs by RNA-FISH (RNA-Fluorescent in situ hybridization) analysis24. Neat1 lncRNA has been shown to nucleate paraspeckles and thus, in the absence of Neat1, paraspeckle structure is disrupted910121419. We observed that in WT-MEFs, Ctn RNA co-localized with Neat1 with in the intact paraspeckles. In addition, Ctn RNA also displayed homogenous nuclear distribution (Fig. 1Aa–c and S1A–C). In Neat1-KO MEFs too, where intact paraspeckles were absent, Ctn RNA continued to localize in the nucleus (Figs 1Ad–f and S1C). Since paraspeckle protein NonO has been shown to interact with, and influence the nuclear localization of hyper-edited RNAs, we ascertained if NonO regulates nuclear retention of A-to-I edited Ctn RNA9. We performed RNA-FISH to determine Ctn RNA and Neat1 co-localization in control and NonO-depleted WT-MEFs (Figs 1Ca–f, and S1D–E). Paraspeckle proteins NonO and SFPQ associate with, and stabilize the longer isoform of Neat1, thus, stabilizing paraspeckle structure12. Neat1 RNA-FISH analysis confirmed the reduction in paraspeckle number in NonO-depleted cells (Figs 1C and S1D,E). However, NonO-depleted cells continued to show nuclear and paraspeckle association of Ctn RNA, suggesting that NonO does not influence the nuclear retention of Ctn RNA (Fig. 1C).


Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA
Ctn RNA is nuclear-retained in absence of intact paraspeckles.(A) RNA-FISH to detect Ctn RNA (green) and Neat1 (red) in DRB-recovered (3 hrs) WT and Neat1-KO MEFs. Arrow (a–f) indicates Ctn RNA foci and arrowhead (d,f) indicates perinucleolar localization of Ctn RNA. (B) Graph showing average number of Ctn RNA foci per cell in WT and Neat1-KO MEFs. (C) RNA-FISH to detect Ctn RNA and Neat1 in DRB-recovered control (Ctrl) and NonO-depleted transformed WT-MEFs. Please note that Ctn RNA shows increased paraspeckle association upon DRB recovery (please see Fig. 4). Arrow (a,c) marks Ctn RNA and Neat1 positive paraspeckle. Arrowhead (d,f) shows Ctn RNA positive but Neat1 negative paraspeckle-like nuclear body. (D) Graph showing average number of Ctn RNA foci per cell in (Ctrl) and NonO-depleted transformed WT-MEFs. (E,F) RT-qPCR to estimate Ctn RNA levels in nuclear and cytoplasmic fractions of (E) WT and Neat1-KO MEFs and (F) Ctrl and NonO-depleted MEFs. (G) RT-qPCR to detect total levels of Ctn RNA in WT and Neat1-KO MEFs. (H) Total levels of Ctn RNA in control and NonO siRNA treated MEFs. 3′UTR-1 primer pair has been used to measure Ctn RNA levels (Figure S2). Gapdh was used as the normalization control in RT-qPCR experiments. Scale bar indicates 10 μm. Error bars in (B,D,E–H) represent mean ± SD of three independent experiments. *P < 0.05, ns: not significant, using Student’s t test.
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f1: Ctn RNA is nuclear-retained in absence of intact paraspeckles.(A) RNA-FISH to detect Ctn RNA (green) and Neat1 (red) in DRB-recovered (3 hrs) WT and Neat1-KO MEFs. Arrow (a–f) indicates Ctn RNA foci and arrowhead (d,f) indicates perinucleolar localization of Ctn RNA. (B) Graph showing average number of Ctn RNA foci per cell in WT and Neat1-KO MEFs. (C) RNA-FISH to detect Ctn RNA and Neat1 in DRB-recovered control (Ctrl) and NonO-depleted transformed WT-MEFs. Please note that Ctn RNA shows increased paraspeckle association upon DRB recovery (please see Fig. 4). Arrow (a,c) marks Ctn RNA and Neat1 positive paraspeckle. Arrowhead (d,f) shows Ctn RNA positive but Neat1 negative paraspeckle-like nuclear body. (D) Graph showing average number of Ctn RNA foci per cell in (Ctrl) and NonO-depleted transformed WT-MEFs. (E,F) RT-qPCR to estimate Ctn RNA levels in nuclear and cytoplasmic fractions of (E) WT and Neat1-KO MEFs and (F) Ctrl and NonO-depleted MEFs. (G) RT-qPCR to detect total levels of Ctn RNA in WT and Neat1-KO MEFs. (H) Total levels of Ctn RNA in control and NonO siRNA treated MEFs. 3′UTR-1 primer pair has been used to measure Ctn RNA levels (Figure S2). Gapdh was used as the normalization control in RT-qPCR experiments. Scale bar indicates 10 μm. Error bars in (B,D,E–H) represent mean ± SD of three independent experiments. *P < 0.05, ns: not significant, using Student’s t test.
Mentions: Previous studies have speculated that paraspeckles are involved in the nuclear retention of A-to-I edited transcripts91026272829. Ctn RNA is a paraspeckle-associated transcript that is A-to-I edited within its long 3′UTR26. To investigate if paraspeckles regulate the nuclear retention of Ctn RNA, we determined the cellular localization of Ctn RNA in WT-MEFs (Mouse embryonic fibroblasts) and Neat1-KO (knockout) MEFs by RNA-FISH (RNA-Fluorescent in situ hybridization) analysis24. Neat1 lncRNA has been shown to nucleate paraspeckles and thus, in the absence of Neat1, paraspeckle structure is disrupted910121419. We observed that in WT-MEFs, Ctn RNA co-localized with Neat1 with in the intact paraspeckles. In addition, Ctn RNA also displayed homogenous nuclear distribution (Fig. 1Aa–c and S1A–C). In Neat1-KO MEFs too, where intact paraspeckles were absent, Ctn RNA continued to localize in the nucleus (Figs 1Ad–f and S1C). Since paraspeckle protein NonO has been shown to interact with, and influence the nuclear localization of hyper-edited RNAs, we ascertained if NonO regulates nuclear retention of A-to-I edited Ctn RNA9. We performed RNA-FISH to determine Ctn RNA and Neat1 co-localization in control and NonO-depleted WT-MEFs (Figs 1Ca–f, and S1D–E). Paraspeckle proteins NonO and SFPQ associate with, and stabilize the longer isoform of Neat1, thus, stabilizing paraspeckle structure12. Neat1 RNA-FISH analysis confirmed the reduction in paraspeckle number in NonO-depleted cells (Figs 1C and S1D,E). However, NonO-depleted cells continued to show nuclear and paraspeckle association of Ctn RNA, suggesting that NonO does not influence the nuclear retention of Ctn RNA (Fig. 1C).

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