SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2985544862> ?p ?o ?g. }

Showing items 1 to 67 of 67 with 100 items per page.

W2985544862 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3’-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3’-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level. https://doi.org/10.7554/eLife.14837.001 eLife digest Many biological features of animals, including body temperature and hormone levels, follow daily rhythms that repeat every 24 hours. These so-called circadian rhythms are driven by an internal body clock and are essential for the organism to adapt to the daily cycle of light and dark. Circadian rhythms also take place inside individual cells – for example, the amount of a given protein in a cell often rises and falls over each 24-hour period. To generate these daily fluctuations, the processes used to make proteins based on the instructions encoded within a gene must be carefully controlled. Genes are first copied or ‘transcribed' into intermediate molecules called messenger RNAs (mRNAs). These mRNA molecules must then travel out of the cell’s nucleus before they can be de-coded to produce proteins. This means that daily fluctuations in mRNA and protein levels could occur because the rate at which the DNA is transcribed fluctuates or because controlling the steps that occur after transcription. However it is not clear how much these post-transcriptional steps contribute to circadian rhythms inside cells. Recently, structures called paraspeckles were seen inside the nucleus. These structures are made from a long RNA molecule that does not code for a protein, and a number of proteins that can bind mRNA molecules. Paraspeckles are thought to prevent certain mRNAs from leaving the nucleus and therefore stop them from being decoded to make proteins. Torres et al. have now investigated whether paraspeckles may play a role in circadian rhythms. Torres et al. looked at the long non-coding RNA and several proteins that are known to be components of paraspeckles in cells taken from the pituitary glands of rats using a variety of techniques. These cells were chosen because they were known to have a working circadian clock. The analysis showed that the levels of these components, as well as the number of paraspeckles within the nucleus, changed over the course of a daily cycle. Torres et al. then confirmed that mRNAs containing a sequence that is known to recruit mRNAs to paraspeckes (the IRAlu sequence) could be also retained in the nucleus or released with a circadian rhythm. This pattern was lost when the paraspeckles were disrupted. These findings suggest that daily fluctuations in protein levels can be post-transcriptionally controlled by paraspeckles rhythmically retaining mRNAs in the nucleus. Future studies could explore whether it may be possible to control circadian rhythms by targeting the paraspeckles, which could help to improve conditions where the internal body clock goes wrong. https://doi.org/10.7554/eLife.14837.002 Introduction The circadian clock orchestrates daily rhythms in metabolism, physiology and behavior that allow organisms to anticipate regular changes in their environment, increasing their adaptation (Asher and Schibler, 2011). Circadian rhythms are underpinned by daily rhythms of gene expression. The transcriptional component of these rhythms is well understood (Zhang and Kay, 2010). The circadian variation in abundance of the positive (Clock and Bmal1) and the negative (Per1, Per2 and Cry1, Cry2) components of these loops drive the circadian transcription of both direct targets genome-wide and a cascade of circadian output transcription factors, which together mediate the circadian transcriptional profile of a cell type or tissue (Asher and Schibler, 2011; Rey et al., 2011). Though the core circadian system has concentrated on transcriptional control, it has been apparent that substantial regulation is achieved after transcription so that post-transcriptional controls are emerging as crucial modulators of circadian clocks (Lim and Allada, 2013; Wang et al., 2013; Menet et al., 2012; Koike et al., 2012; Hurley et al., 2014). While in eukaryotes, approximately 1%-10% genes are subjected to circadian control directly or indirectly only ~1/5 of the mRNAs that display rhythmic expression are directly driven by transcription, which suggests that post-transcriptional mechanisms including RNA splicing, polyadenylation, mRNA stability, mRNA cytoplasmic export and RNAs nuclear retention are essential layers for generation of gene expression rhythmicity (Partch et al., 2014; Menet et al., 2012; Koike et al., 2012; Hurley et al., 2014). Paraspeckles are recently identified nuclear bodies that have been shown to retain RNAs in the nucleus. Paraspeckles contain proteins PSPC1, RBM14, NONO, and SFPQ (Prasanth et al., 2005) and are usually detected as a variable number of discrete dots found in close proximity to nuclear speckles (Bond and Fox, 2009). One long noncoding RNA, nuclear-enriched abundant transcript one (Neat1), exclusively localized to paraspeckles serves as a structural component (Hutchinson et al., 2007; Chen and Carmichael, 2009; Clemson et al., 2009; Sasaki and Hirose, 2009; Sunwoo et al., 2009). The locus generates short and long transcripts from the same promoter, which have previously been identified as MENε (Neat1-1) and MENβ (Neat1-2), respectively (Guru et al., 1997; Sasaki and Hirose, 2009). Because specific depletion of Neat1-2 leads to disruption of paraspeckles (Sasaki and Hirose, 2009) Neat1-1 alone cannot induce paraspeckle formation. Paraspeckles have been shown to retain in the nucleus RNAs containing duplex structures (Chen and Carmichael, 2008). This is the case for the mouse cationic amino acid transporter 2 (Cat2) transcribed nuclear RNA, Ctn-RNA, an alternatively spliced form of the Cat2 mRNA, which contains a dsRNA structure resulting from inverted short inter-spersed nuclear elements (SINEs) in its 3’-UTR (Prasanth et al., 2005). In human cells, hundreds of genes contain inverted repeated SINEs (mainly Alu elements) in their 3’-UTRs. Alu elements are unique to primates and account for almost all of the human SINEs and >10% of the genome. Their abundance leads to the frequent occurrence of inverted repeat structures (inverted repeated Alu elements [IRAlus]) in gene regions (Chen et al., 2008). It has been reported previously that mRNAs containing IRAlus in their 3’-UTRs like Nicolin 1 (NICN1) or Lin 28 are retained in the nucleus in paraspeckles (Chen et al., 2008; Chen and Carmichael, 2008). Therefore, this nuclear retention pathway of IRAlus in 3’-UTRs of genes provides an additional layer of gene regulation by sequestering mature mRNAs within the nucleus. We recently reported that two protein components of paraspeckles, namely NONO and SFPQ, display a circadian expression pattern in primary cultures of pituitary cells as well as in a rat pituitary cell line, the GH4C1 cells (Becquet et al., 2014; Guillaumond et al., 2011). We used this cell line to determine whether one of the posttranscriptional mechanisms allowing circadian gene expression in pituitary cells could involve the circadian nuclear mRNA retention by paraspeckle bodies. To this end, we first characterized the presence of paraspeckles and we showed that these nuclear bodies were rhythmically expressed in the rat GH4C1 pituitary cell line. We then made a series of EGFP-fused IRAlu or Alu constructs and transfected them into GH4C1 cells to investigate the EGFP expression and the fates of the egfp-IRAlu or egfp-Alu RNA. We showed that an IRAlu element in the 3’-UTR of the egfp mRNA strongly repressed EGFP expression. Further, this reduction was accompanied by significant nuclear retention of the mRNAs, likely by paraspeckle bodies. We showed also that insertion of IRAlus in the 3’-UTR of EGFP reporter gene allowed rhythmic nuclear egfp-IRAlu RNA retention and rhythmic EGFP protein expression. Finally, this rhythmic nuclear egfp-IRAlu RNA retention as well as the rhythmic nuclear retention of some known cycling transcripts that were shown here associated with paraspeckles was lost when paraspeckles were disrupted. Results Characterization of paraspeckle nuclear bodies in pituitary GH4C1 cells Visualization of paraspeckle protein components by immunofluorescence and biochemical evidence for their association with the long noncoding RNA Neat1 The presence of paraspeckles in GH4C1 cells was anatomically evidenced by confocal microscopy on the basis of the overlap of their protein components using antibodies directed against SFPQ, NONO, PSPC1 and RBM14. SFPQ (green Figure 1A,C), NONO (green Figure 1B,D), PSPC1 (red Figure 1A,B) and RBM14 (red Figure 1C,D) staining appeared as punctate clusters located exclusively within the boundaries of the nucleus as delimitated by Hoechst labeling (Figure 1A,B,C,D, 4th column). When SFPQ or NONO staining was merged with PSPC1 (Figure 1A,B, 3rd and 4th columns) on the one hand or RBM14 (Figure 1C,D, 3rd and 4th columns) on the other hand, some punctate clusters were found to overlap with each other in subnuclear structures reminiscent of paraspeckles. The protein components of paraspeckles were further shown to be associated with the long noncoding RNA Neat1lncRNA Neat1, using RNA-immunoprecipitation (RIP) experiments with antibodies directed against NONO, SFPQ, RBM14 or PSPC1. Primers used in RT-qPCR detected both long (Neat1-2) and short (Neat1-1) isoforms of Neat1 RNA (Figure 2—source data 1). A 20- to 80-fold enrichment in Neat1 RNA was obtained with these antibodies as compared to an irrelevant non-specific antibody (Figure 2A). These results showed that the four major protein components of paraspeckles bound the structural lncRNA Neat1, confirming the presence of paraspeckle nuclear bodies in GH4C1 cells. Figure 1 Download asset Open asset 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 PSPC1 (A, B 3th column) or RBM14 (C, D 3th column). Nuclear staining by Hoechst for the same samples is added in 4th column. Arrows indicate punctate clusters in which two paraspeckle proteins overlap. Scale bars: 5 µm. https://doi.org/10.7554/eLife.14837.003 Figure 2 Download asset Open asset 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 enrichment in Neat1 RNA obtained as compared to an irrelevant non-specific antibody (see Figure 2—source data 1 for primer sequences). **p<0.01 vs non-specific antibody. B. Visualization of Neat1 RNA by Fluorescence in situ Hybridization and confocal laser scanning microscope Left Panel: RNA-FISH shows the distribution of Neat1 RNA in a few distinct foci (arrows). The round aspect of the foci under confocal laser scanning microscope is shown in the insert in which assigned foci is enlarged. Scale bars: 1 µm Right Panel: The extent of the nucleus is shown with Hoechst staining. Foci containing Neat1 RNA localize within the nucleus sometimes in the close vicinity of nucleus boundaries. Scale bars: 5 µm. (C) Visualization of Neat1 RNA by Fluorescence in situ Hybridization and super resolution Left Panel: Conventional fluorescence microscopy of Neat1 RNA-FISH. The nucleus is outlined with hand-drawn dashed lines to indicate the nuclear periphery. Scale bars: 5 µm. Right Upper Panel: Enlargement of the foci assigned in left panel allows to show the poor resolution of paraspeckle under conventional fluorescence microscopy (in red). In white is the superimposed high-resolution image obtained after STORM analysis. Note that the size of paraspeckle after STORM analysis is strongly reduced. Scale bars: 0.5 µm. Right Bottom Panel: Enlargement of the STORM analysis shown in the upper panel with measurements of width (Wstorm=0.14 µm) and height (hstorm=0.17 µm). Note the elliptical shape of the foci analyzed. Scale bars: 0.1 µm. https://doi.org/10.7554/eLife.14837.004 Figure 2—source data 1 Sequences of qPCR primers and oligonucleotides. https://doi.org/10.7554/eLife.14837.005 Download elife-14837-fig2-data1-v2.docx Visualization of paraspeckle nuclear bodies by fluorescence in situ hybridization (FISH) of long noncoding RNA Neat1 Paraspeckle nuclear bodies were then visualized using Neat1 RNA staining by FISH. Under confocal laser scanning microscope, Neat1 RNA FISH staining appeared as regular punctates within the boundaries of the nucleus (Figure 2B). Number of foci was low and variable from one cell to the other. To gain resolution in the intranuclear spatial arrangement of Neat1 RNA, we combined RNA FISH with Stochastic Optical Reconstruction Microscopy (STORM). Under conventional fluorescence microscopy, resolution of paraspeckle bodies was very low as can be seen in the left part and in upper panel of the right part (red staining) in Figure 2C. The increase in resolution from conventional to super-resolution microscopy was apparent in the upper panel of the right part of Figure 2C in which the high-resolution image obtained after STORM analysis (white staining) was superimposed to the conventional fluorescence image (red staining). Due to the poor resolution, the size of the paraspeckles was imprecise and could not be measured under conventional fluorescence microscope (red staining). By contrast, dimensions could be precisely measured under STORM (bottom panel of the right part in Figure 2C). In addition to the precision in the real size of paraspeckle bodies, super-resolution analysis allowed also to show that Neat1 labeling of paraspeckle bodies was not round in shape, being merely elliptical. Indeed using an imaging software (NIS-Elements, Nikon France S.A, Champigny sur Marne, France) we showed that the averaged ratio of Height to Width (n=10) was >1 (1.53 ± 0.1) and the mean surface area was 24,960 ± 4831 nm2 (Figure 2C). Rhythmic expression of paraspeckle components in GH4C1 cells Rhythmic expression of protein components To determine whether paraspeckle nuclear bodies displayed a circadian expression pattern we looked for rhythmic expression of their protein components as determined by Western blot analysis in nuclear protein extracts. We previously reported that the two proteins NONO and SFPQ followed a rhythmic expression pattern in GH4C1 cells (Guillaumond et al., 2011). It holds also true for two other paraspeckle-associated proteins, RBM14 and PSPC1, as reported in Figure 3A. Indeed, RBM14 and PSPC1 proteins displayed a rhythmic pattern in GH4C1 cells over the T2-T30 time period that could be fitted with a non-linear cosinor fit in which the period value (2pi/Frequency) was constrained to the circadian period value 24 hr (equation values given in Figure 3—source data 1). It may be noticed that the rhythmic expression pattern of RBM14 and PSPC1 proteins (Figure 3A) matched quite correctly with that of NONO and SFPQ proteins we previously reported (see Figure 6A in Guillaumond et al., 2011). Figure 3 with 1 supplement see all Download asset Open asset 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 ± SD of three independent samples) represents the ratio of the depicted proteins to ATF2 and is expressed relative to the value obtained at ZT 30. Experimental values can be adequately fitted (R2>0.55) with a non-linear cosinor equation in which the period value is set to 24 hr (see also Figure 3—source data 1). (B) Rhythmic expression of the long noncoding Neat1 RNA. The expression of the lncRNA Neat1 is determined by RT-qPCR over a 40 hr time period. Primers used to allow the detection of both Neat1-1 and Neat1-2. Experimental values (n=4) expressed as a percent of the initial value obtained at ZT 0 can be adequately fitted (R2>0.55) with a non-linear cosinor equation in which the period value is set to 24 hr (see also Figure 3—source data 1). (C) Rhythmic association of paraspeckle proteins with Neat1 RNA RNA Immuno-Precipitation (RIP) experiments (n=4 for each antibody) are performed over a 30h time period. At each time point, the levels of Neat1 RNA determined after immuno-precipitation by the antibodies directed against NONO, SFPQ, RBM14 and PSPC1 were normalized relative to Neat1 RNA input levels and expressed as a percent of the value obtained at T0. Experimental values can be adequately fitted (R2>0.55) with a non-linear cosinor equation in which the period value is set to 24 hr (see also Figure 3—source data 2). (D–E) Rhythmic fluctuations of paraspeckle number Cells were arrested at four different times after the medium change and processed for FISH of Neat1 RNA. At each time point, 20 to 35 images from four wells of 100 000 cells obtained in two different experiments were acquired under a confocal microscope with a 40X objective. At each time point, the total number of paraspeckles per well and the mean number of paraspeckles per cell were calculated. **p<0.001 ****p<0.0001. https://doi.org/10.7554/eLife.14837.006 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 Download elife-14837-fig3-data1-v2.docx 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 Download elife-14837-fig3-data2-v2.docx Rhythmic expression of Neat1 RNA We further looked for a circadian expression pattern of the structural element of paraspeckles, namely the lncRNA Neat1. As shown in Figure 3B, Neat1 RNA levels (Neat1-1 + Neat1-2) displayed a rhythmic pattern in GH4C1 cells over the T2-T38 time period (cosinor fit values given in Figure 3—source data 1). Neat1 RNA levels (Neat1-1 + Neat1-2) also displayed a circadian expression pattern in several mouse tissues including the pituitary gland but also other peripheral oscillators such as the spleen or the adrenal gland (Figure 3—figure supplement 1). This was also the case in the central clock, namely the suprachiasmatic nuclei (Figure 3—figure supplement 1). Rhythmic binding of paraspeckle proteins on Neat1 RNA We used RIP experiments to examine whether binding of NONO, SFPQ, RBM14 and PSPC1 on Neat1 RNA in GH4C1 cells was rhythmic. As shown in Figure 3C, binding of the four paraspeckle-associated proteins on Neat1 RNA displayed a rhythmic pattern (cosinor fit values given in Figure 3—source data 2). Maximum binding on Neat1 RNA was reached between 6 and 10 hr after the medium change for the four proteins (Figure 3C). Rhythmic number of paraspeckles In cells arrested at four different times after the medium change, the total number of paraspeckles per well was shown to fluctuate, being significantly lower 15 hr after the medium change i.e. at a time when Neat1 RNA levels were around the lowest levels (Figure 3D, F3,103=9.531 p<0.0001). Furthermore at this time point, the mean number of paraspeckles per cell reached a minimum value (Figure 3D, F3447= 5.456, p<0.001). Taken together, these results showed that the rhythm of Neat1 and its associated proteins reported above translate into a rhythm in the number of paraspeckles inside the cells. Influence of IRAlu elements inserted in 3’-UTR EGFP mRNA IRAlu elements reduced EGFP protein expression Paraspeckles have been shown to retain in the nucleus RNAs containing duplex structures from inverted repeats of the conserved Alu sequences (IRAlu elements) within their 3’-UTR (Chen and Carmichael, 2008). This has been shown to be the case for Nicolin 1 (NICN1) gene (Chen and Carmichael, 2008). We utilized EGFP expression reporter system to investigate the effects of IRAlu from the 3’-UTR of NICN1 gene. The single antisense Alu, or the IRAlu elements cloned from the 3’-UTR of NICN1 were inserted each between the EGFP cDNA 3’-UTR region and the SV40 polyadenylation signal of the expression vector pEGFP-C1 to generate constructs that were then stably transfected into GH4C1 cells. In agreement with previous results by Chen et al (Chen and Carmichael, 2008), the IRAlu elements derived from NICN1 significantly reduced EGFP expression when compared with the Alu element (Figure 4A–C). This is here evidenced both by the significant reduction in the number of fluorescent cells (Figure 4B) and by the significant decrease in relative EGFP levels measured by western blotting in IRAlu-egfp cell line compared to both Alu–egfp cell line (Figure 4C). By contrast, we have not observed any difference in gene expression between the parent plasmid pEGFP and derivatives that contained a single Alu element (data not shown). Figure 4 with 2 supplements see all Download asset Open asset 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 mRNA. GH4C1 cell lines, Alu-egfp and IRAlu-egfp cell lines were established by transfection of the indicated plasmids. (A) Representative example of fluorescence and corresponding bright field pictures taken 48 hr after platting of each cell line. Scale bars equal 50 µm. (B) Quantitative analysis of the percent of fluorescent cells in each cell line. Data are means ± SEM of 18 measures performed in 3 independent experiments. (C) Quantification of relative levels of eGFP investigated by western blotting with anti-GFP antibody in total protein extracts from the two cell lines. Tubulin was used as the loading control. Data are mean ± SEM of values obtained from three experiments in IRAlu-egfp cell line and are expressed as a percent of the corresponding value obtained in Alu-egfp cell line. (D) Nuclear and cytoplasmic egfp mRNA were quantified by qPCR in each cell line and normalized to the relative amount of gapdh mRNA (n=8 for each cell line). Ratio of nuclear versus cytoplasmic egfp mRNA levels are compared between IRAlu-egfp and Alu-egfp cell lines. (E) Enrichment in lncRNA Neat1 after RNA Immuno-Precipitation (RIP) with an antibody directed against PSPC1 relative to an irrelevant antibody (left panel) or after RNA pull-down with two different specific biotinylated oligonucleotides (S oligo 1 and S oligo 2) relative to a non-specific oligonucleotide (two right panels). The relative enrichment in lncRNA Neat1 obtained after either RIP (n=3 for each cell line) or RNA pull-down (n=6 for each cell line) is not statistically different in Alu-egfp versus IRAlu-egfp cell lines F. Enrichment in egfp mRNA after RNA Immuno-Precipitation (RIP) with an antibody directed against PSPC1 relative to an irrelevant antibody (left panel) or after RNA pull-down with two different specific biotinylated oligonucleotides (S oligo 1 and S oligo 2) relative to a non-specific oligonucleotide (two right panels). The relative enrichment in egfp mRNA obtained after either RIP (n=3 for each cell line) or RNA pull-down (n=6 for each cell line) is statistically higher in IRAlu-egfp versus Alu-egfp cell lines. *p<0.05 **p<0.01***p<0.001****p<0.0001. https://doi.org/10.7554/eLife.14837.010 IRAlu element induced egfp mRNA nuclear retention The reduced EGFP protein expression in IRAlu-egfp cell line suggested that IRAlu can induce a stronger nuclear retention of egfp mRNA than Alu did, as previously shown by Chen et al (Chen and Carmichael, 2008). Given that egfp mRNA cytoplasmic localization correlates strongly with EGFP expression, we confirmed in our cell lines that after fractionating cytoplasmic and nuclear RNAs, IRAlu-containing egfp mRNA appeared to be preferentially retained in the nucleus in comparison with Alu-containing egfp mRNA. As shown in Figure 4D, the IRAlu from Nicn1 caused a more than two-fold greater nuclear retention of the egfp mRNA when compared with the corresponding Alu element. On the other hand, there is no significant difference in nuclear/cytoplasmic distribution of egfp-Alu mRNA compared with control (data not shown), consistent with our finding that a single Alu element in the 3’-UTR does not affect EGFP gene expression. It then appears that nuclear retention of IRAlu-containing egfp mRNA correlates with silencing of EGFP protein expression (Figure 4A–C). IRAlu element associated with paraspeckle components IRAlu element associated with PSPC1 protein We also asked whether IRAlu-containing egfp mRNA is associated with paraspeckle protein PSPC1. To answer this question, we performed RIP experiments on Alu-egfp and IRAlu-egfp cell lines using PSPC1 antibody compared to irrelevant antibody. While the enrichment in lncRNA Neat1 obtained after use of PSPC1 antibody was comparable in the two cell lines (Figure 4E), enrichment in egfp mRNA was significant only in IRAlu-egfp cell line (Figure 4F), attesting that PSPC1 protein was associated to IRAlu-egfp and not to Alu-egfp mRNA. IRAlu element associated with lncRNA Neat1 We next asked whether IRAlu-containing egfp mRNA is associated with endogenous lncRNA Neat1. To answer this question, we adapted a pull-down Neat1 technology from two published approaches (RIA (RNA-interactome analysis) and CHART (Capture Hybridization Analysis of RNA Targets) (Kretz et al., 2013; Simon, 2013; West et al., 2014). This technology was based on an affinity purification of Neat1 RNA together with its protein partners and mRNA targets by using oligonucleotides that are complementary to Neat1 RNA sequences. In our adapted technology, anti-sense oligonucleotides were designed in stretches of Neat1 RNA available for hybridization and not occluded by in silico predicted secondary structure (Figure 4—figure supplement 1). With this strategy, we found that two anti-sense oligonucleotides allowed a 30 to 40-fold enrichment in Neat1 RNA compared to Neat1 RNA input from cross-linked GH4C1 cellular extracts (Figure 4—figure supplement 2). We performed Neat1 RNA pull-down using these two specific biotinylated complementary oligonucleotides that target Neat1 and one biotynylated irrelevant probe in cell lines stably expressing Alu-containing egfp mRNA or IRAlu-containing egfp mRNA. While one of the specific oligonucleotide (S oligo 2) is more efficient than the other (S oligo 1) to pull-down Neat1, the enrichment in Neat1 was not statistically different in the two Alu-egfp and IRAlu-egfp cell lines (Figure 4E). By contrast, the amounts of egfp mRNA retrieved after Neat1 RNA pull-down by the two specific probes compared to the non-specific probe were significantly higher in IRAlu-egfp compared to Alu-egfp cell line (Figure 4F). This result clearly showed that IRAlu-egfp mRNA was preferentially associated with Neat1-containing paraspeckles compared to Alu-egfp mRNA. IRAlu element induced egfp mRNA circadian nuclear retention Since we showed that paraspeckles displayed a circadian expression in GH4C1 cells, we asked whether IRAlu-egfp mRNA shown here to be associated with paraspeckles could be rhythmically retained in the nucleus. To answer this question, we fractionated cytoplasmic and nuclear RNAs in IRAlu-egfp and Alu-egfp cells harvested every 4 hr during 44 hr. We showed that in addition to the previously described higher ratio of nuclear versus cytoplasmic egfp mRNA in IRAlu-egfp cell line compared to Alu-egfp cell lines (Figure 4D) we also found high amplitude circadian variations in this ratio in IRAlu-egfp cell line (Figure 5A). Whereas it was observed that the nuclear/cytoplasmic ratio of egfp mRNA could also be fitted by a cosinor equation in Alu-egfp cell line, the magnitude of this rhythmic pattern was highly reduced (Figure 5A) as compared to IRAlu-egfp cell line (Figure 5—source data 1). Figure 5 with 1 supplement see all Download asset Open asset IRAlu element induced egfp mRNA circadian nuclear retention and EGFP circadian cytoplasmic expression. (A) Rhythmic ratio of nuclear versus cyt" @default.
W2985544862 created "2019-11-22" @default.
W2985544862 creator A5011835851 @default.
W2985544862 creator A5019272527 @default.
W2985544862 creator A5019554731 @default.
W2985544862 creator A5042694739 @default.
W2985544862 creator A5043395233 @default.
W2985544862 creator A5047359278 @default.
W2985544862 creator A5080184992 @default.
W2985544862 creator A5081238657 @default.
W2985544862 date "2016-05-31" @default.
W2985544862 modified "2023-09-24" @default.
W2985544862 title "Author response: Circadian RNA expression elicited by 3’-UTR IRAlu-paraspeckle associated elements" @default.
W2985544862 doi "https://doi.org/10.7554/elife.14837.027" @default.
W2985544862 hasPublicationYear "2016" @default.
W2985544862 type Work @default.
W2985544862 sameAs 2985544862 @default.
W2985544862 citedByCount "0" @default.
W2985544862 crossrefType "peer-review" @default.
W2985544862 hasAuthorship W2985544862A5011835851 @default.
W2985544862 hasAuthorship W2985544862A5019272527 @default.
W2985544862 hasAuthorship W2985544862A5019554731 @default.
W2985544862 hasAuthorship W2985544862A5042694739 @default.
W2985544862 hasAuthorship W2985544862A5043395233 @default.
W2985544862 hasAuthorship W2985544862A5047359278 @default.
W2985544862 hasAuthorship W2985544862A5080184992 @default.
W2985544862 hasAuthorship W2985544862A5081238657 @default.
W2985544862 hasBestOaLocation W29855448621 @default.
W2985544862 hasConcept C104317684 @default.
W2985544862 hasConcept C121446783 @default.
W2985544862 hasConcept C169760540 @default.
W2985544862 hasConcept C22667442 @default.
W2985544862 hasConcept C38606739 @default.
W2985544862 hasConcept C54355233 @default.
W2985544862 hasConcept C67705224 @default.
W2985544862 hasConcept C70721500 @default.
W2985544862 hasConcept C86803240 @default.
W2985544862 hasConcept C89604277 @default.
W2985544862 hasConcept C95444343 @default.
W2985544862 hasConceptScore W2985544862C104317684 @default.
W2985544862 hasConceptScore W2985544862C121446783 @default.
W2985544862 hasConceptScore W2985544862C169760540 @default.
W2985544862 hasConceptScore W2985544862C22667442 @default.
W2985544862 hasConceptScore W2985544862C38606739 @default.
W2985544862 hasConceptScore W2985544862C54355233 @default.
W2985544862 hasConceptScore W2985544862C67705224 @default.
W2985544862 hasConceptScore W2985544862C70721500 @default.
W2985544862 hasConceptScore W2985544862C86803240 @default.
W2985544862 hasConceptScore W2985544862C89604277 @default.
W2985544862 hasConceptScore W2985544862C95444343 @default.
W2985544862 hasLocation W29855448621 @default.
W2985544862 hasOpenAccess W2985544862 @default.
W2985544862 hasPrimaryLocation W29855448621 @default.
W2985544862 hasRelatedWork W2007884363 @default.
W2985544862 hasRelatedWork W2014486057 @default.
W2985544862 hasRelatedWork W2043536313 @default.
W2985544862 hasRelatedWork W2059595766 @default.
W2985544862 hasRelatedWork W2155492035 @default.
W2985544862 hasRelatedWork W2377012683 @default.
W2985544862 hasRelatedWork W2381171195 @default.
W2985544862 hasRelatedWork W2500689306 @default.
W2985544862 hasRelatedWork W3089303308 @default.
W2985544862 hasRelatedWork W4379141838 @default.
W2985544862 isParatext "false" @default.
W2985544862 isRetracted "false" @default.
W2985544862 magId "2985544862" @default.
W2985544862 workType "peer-review" @default.