Circadian RNA expression elicited by 3’-UTR IRAlu-paraspeckle associated elements
- Aix Marseille Université, CNRS, CRN2M-UMR7286, France
Figures
Figure 1
Localization by confocal microscopy of paraspeckle proteins in GH4C1 pituitary cells.
Cells are grown on coverslips and labeled for immunofluorescence with antibody to SFPQ (green A, C), NONO (green B, D) PSPC1 (red A, B) or RBM14 (red C, D). SFPQ or NONO staining is merged with …
Figure 2
Neat1 RNA in paraspeckle nuclear bodies: association with paraspeckle proteins and visualization by FISH.
(A) Association of paraspeckle proteins with Neat1 RNA RNA Immuno-Precipitation (RIP) experiments (n=4 for each antibody) using antibodies directed against NONO, SFPQ, RBM14 or PSPC1 show the …
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Figure 2—source data 1
Sequences of qPCR primers and oligonucleotides.
- https://doi.org/10.7554/eLife.14837.005
Figure 3 with 1 supplement
Rhythmic expression and association of paraspeckle components.
(A) Rhythmic expression of two paraspeckle proteins in pituitary GH4C1 cells The expression of PSPC1 and RBM14 is determined by Western Blot analysis over a 30 hr time period. Each data point (mean …
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Figure 3—source data 1
Cosinor analysis of the rhythmic expression pattern of paraspeckle components in GH4C1 cells.
- https://doi.org/10.7554/eLife.14837.007
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Figure 3—source data 2
Cosinor analysis of the rhythmic binding of the four paraspeckle-associated proteins on Neat1 RNA in GH4C1 cells.
- https://doi.org/10.7554/eLife.14837.008
Figure 3—figure supplement 1
Rhythmic expression of the long noncoding Neat1 RNA in diverse circadian oscillators.
Daily pattern of expression of the long noncoding Neat1 RNA in different tissue of mice maintained in a 12h/12h light-dark cycle and sacrificed at different Zeitgeber Time (ZT). ZT0 corresponds to …
Figure 4 with 2 supplements
Influence of IRAlu elements inserted in 3'-UTR egfp mRNA.
(A–C) Decrease in EGFP expression by insertion of IRAlus in the 3’ -UTR of egfp mRNA. IRAlus and Alu were PCR-amplified from the 3’-UTR of Nicn1 and then inserted separately into the 3’-UTR of egfp …
Figure 4—figure supplement 1
Secondary structure of the first 2500 nucleotides of the Neat1 RNA as predicted by MaxExpect software.
(A) The structure is colored according to a base-pairing probability code (zoom to view base colors). (B) Enlargement of the red box in A. The two anti-sens oligonucleotide probes designed (S oligo …
Figure 4—figure supplement 2
Enrichment of Neat1 RNA relative to input after RNA pull-down.
Enrichment obtained after using the two anti-sens specific (S oligo 1 and S oligo 2) or a non-specific (NS) oligonucleotide probes. **p<0.01 ***p<0.001vs non-specific oligonucleotide probe.
Figure 5 with 1 supplement
IRAlu element induced egfp mRNA circadian nuclear retention and EGFP circadian cytoplasmic expression.
(A) Rhythmic ratio of nuclear versus cytoplasmic egfp mRNA levels in Alu-egfp and IRAlu-egfp cell lines. Experimental values (n=3 for each cell line) can be adequately fitted (R2>0.55) with a …
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Figure 5—source data 1
Cosinor analysis of the rhythmic ratio of nuclear versus cytoplasmic egfp mRNA levels in Alu-egfp and IRAlu-egfp cell lines.
- https://doi.org/10.7554/eLife.14837.014
Figure 5—figure supplement 1
Effects of knockdown of Neat1 by Neat1 siRNA or Neat1 ASO.
(A) On Neat1 RNA levels: Neat1 RNA levels are reduced by about 40% as a result of siRNA or ASO treatment. ****p<0.0001 (B–D) On the Neat1 RNA circadian pattern: The pattern of Neat1 RNA expression …
Figure 6 with 2 supplements
Characterization of paraspeckle-associated mRNA.
(A) Venn diagram representation of the overlap between transcripts linked to the paraspeckle (Neat1 RNA targets) with transcripts known as rhythmic genes in the mouse pituitary gland (Hughes et al., …
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Figure 6—source data 1
List of Neat1 RNA target genes.
- https://doi.org/10.7554/eLife.14837.018
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Figure 6—source data 2
Cosinor analysis of the relative ratio nuclear/cytoplasmic mRNA of seven Neat1 RNA targets in GH4C1 cells.
- https://doi.org/10.7554/eLife.14837.019
Figure 6—figure supplement 1
Enrichment relative to input in seven selected mRNA after Neat1 RNA pull-down.
Enrichment relative to input obtained after using the two anti-sens specific (S oligo 1 and S oligo 2) or a non-specific (NS) oligonucleotide probes. ***p<0.001vs non-specific oligonucleotide probe.
Figure 6—figure supplement 2
Rhythmic ratio of nuclear versus cytoplasmic mRNA levels of four NEAT1 RNA target genes.
(A) Experimental values (n=2) of the ratio nuclear versus cytoplasmic mRNA levels can be adequately fitted (R2>0.55) with a non-linear cosinor equation in which the period value is set to 24 hr (see …
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