Circadian RNA expression elicited by 3’-UTR IRAlu-paraspeckle associated elements
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 .
Paraspeckles are found in almost all of the cultured cell lines and primary cultures from tissues ( Bond and Fox , 2009 ) , except for embryonic stem cells ( Chen and Carmichael , 2009 ) . Four RNA-binding proteins , including three members of the Drosophila melanogaster behavior human splicing ( DBHS ) family proteins ( NONO , PSPC1 and SFPQ ) and RNA-binding motif protein 14 ( RBM14 ) are classified as 'classical' paraspeckle protein components ( Bond and Fox , 2009; Prasanth et al . , 2005; Fox et al . , 2002; Nakagawa and Hirose , 2012 ) . Both endogenous and tagged forms of these proteins are found localized within the nucleoplasm as well as paraspeckles in mammalian cells . With a confocal fluorescent study , we report in a rat pituitary cell line that all four paraspeckle protein components are distributed both in the nucleoplasm and in subnuclear foci . Among these later , we identify paraspeckle nuclear bodies by overlapping side-by-side the staining of two paraspeckle protein components . This allows not only to provide evidence for the existence of paraspeckles in rat pituitary cell nuclei but also to show that as already reported in other cell types , paraspeckles are small , irregularly sized and unevenly distributed subnuclear bodies . Using RIP experiments , we further show that all four paraspeckle protein components are associated with the specific paraspeckle localized lncRNA Neat1 . This later is further detected by RNA FISH . Confirming previous reports in other cell types , when identified by a combination of Neat1 RNA FISH and confocal microscopy , paraspeckles appear as round foci in rat pituitary cells . The diameter of such round foci is in the range of that estimated in previous studies ( around 360 nm ) . However after use of a combination of Neat1 RNA FISH and STORM analysis , paraspeckles appear more likely as oblong structures with smaller dimensions reminiscent of the interchromatin granule-associated zone ( IGAZ ) that had been observed under electron microscope and had been reported to correspond to paraspeckles ( Cardinale et al . , 2007; Bond and Fox , 2009; Souquere et al . , 2010 ) . Width and height measurements obtained after STORM analysis of Neat1 RNA FISH staining are smaller than the dimensions of IGAZ nuclear bodies established by an electron microscopy analysis in human and mouse cells ( Souquere et al . , 2010 ) suggesting that either IGAZ/paraspeckle nuclear bodies are smaller in rat than in mouse cells or alternatively and more likely , Neat1 RNA staining does not fill in all the nuclear bodies as already proposed ( Souquere et al . , 2010 ) . In a previous study , we reported that two paraspeckle proteins , namely NONO and SFPQ , display a circadian expression pattern in rat pituitary cells ( Guillaumond et al . , 2011 ) and we find here that it is also the case for the two other 'classical' paraspeckle proteins , PSPC1 and RBM14 . Furthermore , all four proteins bind rhythmically to Neat1 , suggesting that the pools of proteins involved in paraspeckle composition vary with time . In addition , the lncRNA Neat1 itself displays a circadian expression pattern and we further found using a pair of primers allowing the specific detection of the long form of Neat1 RNA , Neat1-2 ( Figure 2—source data 1 ) that the expression of this form known to be sufficient for the formation of paraspeckle displays a circadian pattern ( data not shown ) . The circadian expression pattern of Neat1 RNA is not the privilege of 'in vitro' pituitary cells since it is also found 'ex vivo' in several mouse tissues including the pituitary gland but also numerous other peripheral oscillators such as the spleen or the adrenal gland . This holds also true for the central clock , namely the suprachiasmatic nuclei . Finally , in GH4C1 cells , the circadian variations in Neat1 RNA levels are shown here to reflect rhythmic variations in paraspeckle number within the cells . It may then be assumed that the circadian expression pattern of Neat1 RNA described here in a number of mouse tissue also reflects variations in paraspeckle number although it cannot be excluded that paraspeckle size could also oscillate over the circadian period both in pituitary cells and in other oscillators . As previously reported by Chen and Carmichael ( Chen et al . , 2008 ) , we found that a pair of IRAlus in the 3’-UTR of egfp can strongly silence EGFP expression in GH4C1 pituitary cell line and after fractionating cytoplasmic and nuclear RNAs from our two cell lines , we observed that IRAlus-containing egfp mRNA appear to be preferentially retained in the nucleus in comparison with those having a single Alu element . It then appeared that silencing EGFP expression mechanism relied on egfp mRNA nuclear retention . To obtain evidence that nuclear retained IRAlus are associated with paraspeckle bodies , we immunoprecipitated RNA complexes containing paraspeckle protein PSPC1 and characterized the associated egfp mRNA . We found IRAlu-containing egfp mRNA to be associated with PSPC1 whereas Alu-containing egfp mRNA was not . While PSPC1 as well as NONO and SFPQ accumulate in paraspeckles , these proteins are also found elsewhere throughout the nucleoplasm and thus may have functions apart from paraspeckle retention . In contrast to paraspeckle-associated proteins that are multifunctional , NEAT1 may have only one function . We then developed a technique to characterize nuclear mRNA Neat1 targets using a Neat1 RNA pull-down approach that allowed us to show the association of IRAlu egfp mRNA with Neat1 providing a much more convincing evidence for a nuclear retention of IRAlu egfp mRNA by paraspeckles . Given their presumed functions in gene expression through corresponding mRNA nuclear retention and since they display a circadian expression pattern , it may be assumed that paraspeckle bodies can rhythmically retain RNAs in the nucleus leading to a rhythmic expression of the corresponding gene . In keeping with this view , we showed that IRAlu elements inserted in 3’-UTR of egfp reporter mRNA allow for its circadian retention within the nucleus and consequently for EGFP protein circadian expression . However , it may be noticed that egfp mRNA displayed also a circadian nuclear retention when a single antisens Alu element was inserted in its 3’-UTR but in this case the variations of the ratio over the circadian period was highly reduced in contrast to the high amplitude circadian variations of the nuclear versus cytoplasmic ratio observed in IRAlu-egfp cell line . These small amplitude variations of the ratio between nuclear and cytoplasmic mRNA contents in Alu-egfp cell line were further accompanied by a weak number of cells expressing a circadian pattern of EGFP expression . Since antisens Alu elements within the 3’-UTR of mRNAs can be the target of several RNA-binding proteins ( Kelley et al . , 2014; Zarnack et al . , 2013 ) it may be speculated that some of these RNA-binding proteins display a rhythmic expression pattern accounting for the weak EGFP circadian expression observed in Alu-egfp cell line . Among the identified RNA-binding proteins that target antisens Alu elements within the 3’-UTR of mRNAs , HNRNPC was shown to display a circadian expression pattern in several tissues including the mouse pituitary ( see CIRCA: http://circadb . hogeneschlab . org/ ) . This is not the case for HNRNPU and STAUFEN 1 ( STAU1 ) -mediated messenger RNA decay we did not find to display a circadian expression pattern in GH4C1 cells ( Guillaumond et al . , 2011 ) and ( data not shown ) and are then unlikely involved in circadian EGFP expression in Alu-egfp cell line . The mechanism involved in the circadian nuclear retention of Alu-containing egfp mRNA remains anyway to be determined . Nuclear retention of egfp mRNA in IRAlu-egfp cells is no more cycling when paraspeckles were disrupted assuming that the nuclear retention of the reporter gene by paraspeckles contributes to cycling . To determine whether this paraspeckle impact on rhythmic gene expression occurs 'in vivo' on a much larger scale , we characterized in our pituitary cells the list of the putative Neat1 RNA target genes and we analyzed in publicly available datasets the known cycling transcripts that are included in this Neat1 RNA target list . Whereas it is well known that rhythmic circadian genes are tissue specific and that it is unfavorable to compare datasets obtained in two different species we found a robust overlap of our gene list with circadian genes in the mouse pituitary ( Hughes et al . , 2007 ) and a closer overlap with post-transcriptional circadian genes in the mouse liver ( Menet et al . , 2012 ) . Furthermore , on a few circadian genes common to either of these lists or both , we showed a loss of rhythmic pattern when paraspeckles were disrupted assuming that paraspeckle retention plays a relevant role in circadian gene expression . It is now widely admitted that post-transcriptional circadian regulation plays a major role in determining oscillations at the mRNA level . In agreement with this view , the new mechanism described here suggests that circadian mRNA oscillations could be post-transcriptionally controlled through rhythmic nuclear retention by paraspeckle nuclear bodies .
GH4C1 cells , a rat pituitary somatolactotroph line , were obtained in 2012 from ATCC ( CCL-82 . 2 , lot number: 58945448 , Molsheim , France ) with certificate analysis and were confirmed to be free of mycoplasma ( MycoAlert , Lonza , Levallois-Perret , France ) . They were grown in HamF10 medium supplemented with 15% horse serum and 2% fetal calf serum . To synchronize cells between themselves , GH4C1 cells were transferred to fresh medium and were harvested after this fresh medium replacement ( T0 ) every 4 hr from T2 to T30-38 . For the generation of stable GH4C1 cell lines , cells were transfected with the plasmid constructs expressing a neomycin resistance gene by Lipofectamine Plus ( Invitrogen , Cergy Pontoise , F ) . Cells were selected with 250 μg/ml G418 ( Invitrogen ) beginning 48 hr after transfection . Alu and IRAlus elements from Nicn1 were cloned by PCR from genomic DNA obtained from Hela cells . Sequences were then inserted in the pEGFP-C1 ( Clontech , Mountain View , CA ) at the BglII and HindIII sites as described by Chen et al . ( Chen and Carmichael , 2008 ) . Predesigned siRNAs targeting rat Neat1 were purchased from GeneCust ( siRNA Set , GeneCust , Dudelange , Luxembourg ) . IRAlu-egfp cells plated in 6-well dishes were transfected with 500 pmol of a pool of four siRNA ( 125 pmol each , see Figure 2—source data 1 for the sequences ) using Lipofectamine 2000 ( Life Technologies , Saint Aubin , France ) . As controls , 500 pmol of non-targeting siRNA from GeneCust ( negative control siRNA , see Figure 2—source data 1 for the sequences ) were used . Cells were harvested 48 hr after transfection . The antisense oligonucleotides ( ASO ) used for Neat1 knockdown experiments were phosphorothioate modified at their backbone to increase their stability . Two Neat1 ASO were pooled that were targeted to regions common to NEAT1_1 and NEAT1_2 isoforms ( see Figure 2—source data 1 for the sequences ) . IRAlu-egfp cells plated in 6-well dishes were transfected with 300 pmol of the pool of the two ASO ( 150 pmol each , see Figure 2—source data 1 for the sequences ) using Lipofectamine 2000 ( Life Technologies ) . Cells were harvested 48 hr after transfection . Nuclear , cytoplasmic or total RNA was prepared from GH4C1 cells using an RNA XS kit ( Macherey Nagel , Hoerdt , France ) . Total RNA purification was performed on 24-well cell dishes . Nuclear and cytoplasmic RNA isolation was performed using 10 cm cell dishes that were rinsed twice with ice-cold PBS , incubated in 1 ml of ice-cold cell lysis buffer A ( 10 mmol/L Tris pH 7 . 4 , 3 mmol/L MgCl2 , 10 mmol/L NaCl and 0 . 5% NP-40 ) . Nuclei and cytoplasma were separated by centrifugation ( 500 x g for 5 min ) . One-sixth of the supernatant was used to prepare cytoplasmic RNA . To obtain pure nuclear RNA , the nuclear pellets were subjected to two additional washes with 1 ml lysis buffer A and were then extracted with XS kit reagent . Total RNA ( 500 ng ) was then used for cDNA synthesis performed with a High Capacity RNA to cDNA kit ( Applied Biosystem , Courtaboeuf , France ) . Real-time PCR was performed on a 7500 fast Real-Time qPCR system ( Applied Biosystems ) using Fast SYBR Green mix ( Applied Biosystems ) . The sequences of the primers used in qPCR are given in Figure 2—source data 1 . mRNA accumulation was normalized relative to Gapdh mRNA levels . Total or nuclear protein extracts prepared as previously described ( Becquet et al . , 2001 ) from confluent GH4C1 cells grown in 10 cm dishes , were submitted to Western-blot analysis as previously described ( Guillaumond et al . , 2011 ) with polyclonal primary antibodies raised against PSPC1 ( 1:1000 , sc-84576 , Santa Cruz Biotechnology , Heidelberg , Germany ) , RBM14 ( 1 µg/ml , Abcam , Paris , France ) , ATF2 ( 1/1000 , sc-187 , Santa Cruz Biotechnology ) , GFP ( 1/1000 , A6455 , Molecular Probes , Paisley , UK ) and monoclonal primary antibody raised against α-Tubulin ( 1/1000 , T 6199 , Sigma-Aldrich , Saint-Louis , USA ) . The ratio of PSPC1 and RBM14 to ATF2 , a constitutive transcription factor , and the ratio of GFP to tubulin were determined by densitometry using ImageJ software ( National Institutes of Health , USA ) . GH4C1 cells grown in 10 cm dishes were rinsed two times with 5 ml cold phosphate buffer saline ( PBS ) . Cells were then harvested by scraping in ice-cold PBS and transferred to a centrifuge tube . After centrifugation ( 2500 x g for 5 min ) cells were pelleted and suspended in 100 µl of Polysome lysis buffer ( PLB; 10 mM HEPES , pH 7 . 0 , 0 . 1M KCl , 5 mM MgCl2 , 25 mM EDTA , 0 . 5% NP40 , 1 mM DTT , 100 U/ml RNAse OUT and complete protease inhibitor cocktail ) . After mixing by pipetting up and down , cells were kept on ice for 5 min to allow the hypotonic PLB buffer to swell the cells . The cell lysate was then aliquoted and stored at −80°C . Cell lysate was centrifuged at 14 , 000 x g for 10 min at 4°C and diluted 1/100 in NET2 buffer ( NET2 buffer corresponded to NT2 buffer: 50 mM Tris-HCl , pH 7 . 4 , 150 mM NaCl , 1 mM MgCl2 and 0 . 05% NP40 added with 1 mM DTT , 20 mM EDTA , 200 U/ml RNAse Out ) . An aliquot of diluted cell lysate was removed and represented the starting material or 'input' which was processed alongside the immunoprecipitation to compare with immunoprecipitated mRNAs at the end . RIP experiments were performed overnight at 4°C on diluted cell lysate with antibodies to NONO ( ab45359 , Abcam ) , SFPQ ( ab38148 , Abcam ) , PSPC1 ( SAB4200068 , Sigma-Aldrich , Saint-Quentin Fallavier , France ) and RBM14 ( ab70636 , Abcam ) or non-specific rabbit polyclonal antibody ( anti-Furin , sc-20801 , Santa-Cruz Biotechnology ) . After incubation was completed , 15 µl of Magna ChIP protein A magnetic beads ( 16–661 , Millipore , Molsheim , France ) were added for 1h at 4°C . Beads were washed 6 times with cold NT2 buffer and treated by proteinase K for 30 min at 55°C . RNA eluted from beads was purified using Nucleospin RNA XS ( Macherey-Nagel ) and processed for cDNA synthesis using a High Capacity RNA to cDNA kit ( Applied Biosystems ) . Neat1 RNA pull-down is a hybridization-based strategy that uses complementary oligonucleotides to purify Neat1 RNA together with its targets from reversibly cross-linked extracts . In cross-linked extracts , it is expected that some regions of the RNA will be more accessible for hybridization than others due in particular to secondary structure . To design oligonucleotides that target these regions and then can hybridize specifically to Neat1 RNA , we modeled the secondary structure of Neat1 RNA using bioinformatics ( MaxExpect software [Lu et al . , 2009] ) . Two antisense DNA oligonucleotide probes that target accessible regions of the lncRNA Neat1were designed and used for Neat1 RNA specific pull-down ( Figure 4—figure supplement 1 ) . One biotynylated irrelevant probe was used for Neat1 RNA non-specific pull-down . All three probes were biotinylated at the 3′ end ( Figure 2—source data 1 ) . Briefly , 10 cm cell dishes were incubated in 1 ml of ice-cold cell lysis buffer A as described above . Nuclei were scraped and separated by centrifugation ( 500 x g for 5 min ) . The nuclear pellets were then fixed with 1% paraformaldehyde in PBS for 10 min at room temperature . Crosslinking was then quenched with 1 . 25 M glycine for 5 min . Cross-linked nuclei were rinsed two times again with PBS and pelleted at 500 x g . Nuclear pellets were stored at −80°C . To prepare lysates , nuclear pellets were suspended in lysis buffer ( 50 mM Tris , pH 7 . 0 , 10 mM EDTA , 1% SDS added with a protease inhibitor cocktail and RNAse-Out ) , and sonicated using BioruptorPlus ( Diagenode , Seraing , Belgium ) by 2 pulses of 30 s allowing complete lysate solubilization . RNA was in the size range of 400 to 2000 nucleotides . Nuclear lysates were diluted V/V in hybridization buffer ( 750 mM NaCl , 1% SDS , 50 mM Tris , pH 7 . 0 , 1 mM EDTA , 15% formamide ) . The two specific or the non-specific probes ( 100 pmol ) were added to 1 . 2 ml of diluted lysate , which was mixed by end-to-end rotation at room temperature 4 to 6 hr . Streptavidin-magnetic C1 beads ( Dynabeads My OneStreptavidin C1– Invitrogen Life Technologies ) were added to hybridization reaction ( 50 μl per 100 pmol of probes ) and the whole reaction was mixed overnight at room temperature . Beads–biotin-probes–RNA adducts were captured by magnets ( Invitrogen ) and washed five times with a wash buffer ( 2×SSC , 0 . 5% SDS ) . After the last wash , buffer was removed carefully . For RNA elution , beads were suspended in 100 μl RNA proteinase K buffer ( 100 mM NaCl , 10 mM Tris , pH 7 . 0 , 1 mM EDTA , 0 . 5% SDS ) and 100 μg proteinase K ( Ambion ) . After incubation at 45°C for 45 min , RNA was isolated using NucleoSpinRNA XS ( Macherey-Nagel ) . Eluted RNA was subject to RNA-sequencing or RT–qPCR for the detection of enriched transcripts . The construction of Illumina DNA libraries from RNA pools obtained with each of the two specific oligonucleotides and the sequencing were performed by Helixio ( Clermont-Ferrand , France ) . Libraries were prepared with Illumina TruSeq Stranded mRNA Sample Preparation kit . Sequencing was done on Illumina NextSeq 500 ( 60 million reads per sample on average ) . Analyses were performed on a local instance of Galaxy ( Afgan et al . , 2016 ) . Paired-end reads ( 75bp ) were aligned to the Rat reference genome ( Rnor_6 . 0 . 80 , Ensembl ) using TopHat2 v2 . 209 with default parameters ( Kim et al . , 2013 ) . We further used Cuffflinks v2 . 2 . 1 ( Trapnell et al . , 2012 ) to quantify mRNA level and fragment per kilobase per million of mapped reads ( FPKM ) were calculated . Consistent with prior studies ( Nagaraj et al . , 2011; Hart et al . , 2013 ) , the log2 ( FPKM ) distribution shows a primary peak of high expression genes , with a long left shoulder of low-expression transcripts . Therefore , only transcripts with FPKM values higher than 1 were taken into account . In order to assess the specificity of the NEAT1 RNA pull-down we crossed the results obtain with the two oligonucleotides ( see Neat1 RNA pull-down ) to generate the list of transcripts associated to the paraspeckles . All the RNA sequencing data are available at Gene Expression Omnibus ( GEO ) ( accession no . GSE81972 ) . GH4C1 cells were grown on glass coverslips coated with poly-ornithine . After incubation in 30% normal blocking serum containing 0 . 2% Triton X-100 ( 30 min ) , coverslips were transferred to primary antibody or a mixture of primary antibodies ( overnight , 4°C ) and , after rinsing , to fluorescent secondary antibody or a mixture of fluorescent secondary antibodies ( 2 hr , 22°C ) . Nuclei were stained with Hoechst dye ( 1 µmol/ml , 5 min ) . All dilutions and rinsing between each step of the procedures were made in Tris buffer added with 1% normal serum . NONO , SFPQ , PSPC1 , RBM14 were detected using the respective polyclonal rabbit antibodies: ab45359 ( 1: 1000 ) Abcam , ab38148 ( 1: 1000 ) Abcam , HPA038904 ( 1: 1000 ) Sigma-Aldrich , ab70636 ( 1: 1000 ) Abcam . After mounting with PBS containing 50% glycerol , the reacted sections were kept in darkness at 4°C until examination . To detect Neat1 RNA , GH4C1 cells grown on glass coverslips coated with poly-ornithine were fixed in 3 . 6% formaldehyde . Hybridization was carried out using Custom Stellaris FISH Probes ( Biosearch Technologies , Novato , USA ) . Probes used are Neat1 probes labeled with Quasar 570 Dye or Neat1 probes labeled with Quasar 670 for STORM analysis . Nuclei of the cells were counterstained by Hoechst solution ( 5 ng/ml ) . The confocal image acquisition was performed on a Zeiss LSM780 confocal microscope equipped with a 63X 1 . 4 N . A . oil immersion objective . Dyes used in either immunocytochemistry ( ICC ) or RNA-Fish were imaged using the appropriate wavelength for optimal dye excitation , i . e . 405 nm for Hoechst , 561 nm for Cy3 and Quasar 570 , and 633 nm for both Cy5 and Quasar 670 . The spectral detection of emitted fluorescence were set as follows: 420–480 nm for Hoechst , 560–670 nm for Cy3 or Quasar 570 and 640–750 nm for Cy5 or Quasar 670 . Three dimensional z-stacks were collected automatically as frame by frame sequential image series . To enhance resolution , the ICC images were deconvolved based on a theoretical point spread function ( PSF ) using AutoQuantX ( Media Cybernetics , Rockville , USA ) . Prior to observation , samples were mounted in imaging buffer ( 50 mM Tris pH 8 . 0 , 20 mM NaCl , 0 . 5 mg/ml glucose oxydase , 40 µg/ml catalase , 10% w/v glucose , 10 mM mercaptoethylamine ) . Imaging was performed with a Nikon Storm System ( N-Storm , Nikon France S . A , Champigny sur Marne , France ) equipped with a 100X 1 . 49 N . A . oil immersion objective . A reference conventional fluorescence image of the cell of interest was acquired before recording storm sequence of images . For super resolution image acquisition , the sample stained with Quasar 670 dye was excited by a 647 nm imaging laser . Illumination with 405 nm light was gradually increased along recording to reactivate Quasar 670 blinking . The fluorescence emission was separated from the excitation light by adapted dichroic mirror ( 660 nm ) and filters ( 692/4 nm ) and collected on an IXon EMCCD camera ( Andor Technology , Belfast , UK ) . Typically , dSTORM images were reconstructed from 40000 frames using NIS-software ( Nikon ) . For real-time monitoring of intracellular fluorescence circadian oscillations , 4 × 104 Alu-egfp or IRAlu-egfp GH4C1 cells were seeded on glass-bottom 24 well plates ( coated with poly- ornithine ) . The next day , the medium was changed with a growth medium containing 100 nM forskolin to synchronize the cellular circadian oscillators . 20 min later , the medium was changed again with a DMEM without phenol red medium containing 100 mM 4- ( 2-hydroxyethyl ) -1-piperazineethanesulfonic acid ( HEPES ) and 100 mM pyruvate . The next day , plates were transferred to an inverted microscope ( Axiovert-200 , Zeiss ) equipped with an environmental chamber , the temperature of which was set to 37°C and CO2 regulated to 5% . Egfp was excited through a 475/40 band-pass filter . An EMCCD camera ( Rolera EM-C2 , Q-Imaging ) was used to collect emitted fluorescence at 530/50 nm . Time-lapse recordings of multiple regions in each well were realized over 96 hr with 1 picture every 30 min ( Metamorph , Roper Scientific ) , using a 40X objective . The data were processed for tracking and measuring fluorescence fluctuations of individual cells with the CGE ( Circadian Gene Expression ) Plugging of ImageJ ( Sage et al . , 2010 ) . Cells retained for analysis fulfilled two criteria: they were detected for at least 48 hr and their mean fluorescent level was 10% higher than mean level of the background . After processing of the cells for FISH of Neat1 RNA , quantification of paraspeckle bodies was performed on confocal images using a 40X objective . At each time point analyzed , 20 to 35 fields acquired in four wells from two different experiments were counted to estimate the total paraspeckle number per well of 100000 cells and to estimate among cells which expressed paraspeckles the mean number per cell . Data from real time monitored cells or mean experimental values ( ± SEM ) expressed as a percent of initial value were fitted ( Prism4 software , GraphPad Software , Inc . ) by a non-linear sine wave equation to find the set of parameters that gives the least-squared distance between the data and the equation: y ( t ) = B + A*exp ( -t ) *sin ( 2*pi*t/T + P ) where B is the Baseline , A is the Amplitude , T is the period and P is the Phase-shift . Goodness-of-fit was quantified using R squared , experimental values being considered well fitted by cosinor regression when the R squared was higher than 0 . 55 . A statistically significant circadian oscillation was considered if the 95% confidence interval for the amplitude did not include the zero value ( zero-amplitude test ) ( Yang et al . , 2009 ) ( Kavcic et al . , 2011 ) . Significant differences between groups were determined using either ordinary one-way ANOVA or unpaired t test ( Prism4 software ) . Values were considered significantly different for p<0 . 05 ( * ) .
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 .
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 .