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Vol. 18, Issue 8, 2817-2827, August 2007
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*Laboratoire de Biologie Moléculaire Eucaryote-Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5095, Institut d'Exploration Fonctionnelle des Génomes 109, 31062 Cedex Toulouse, France; and Institut Génétique Moléculaire Montpellier-Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5535, Université Montpellier II, 34293 Montpellier Cedex 5, France
Submitted October 16, 2006;
Revised April 27, 2007;
Accepted May 4, 2007
Monitoring Editor: A. Gregory Matera
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100-kb-long gene cluster comprising 86 homologous RBII-36 C/D RNA gene copies, all of them intron-encoded within the ncRNA gene Bsr. Here, we demonstrate that the Bsr gene is monoallelically expressed in primary rat embryonic fibroblasts as well as in hypothalamic neurons and yields a large amount of unspliced and spliced RNAs at the transcription site, mostly as elongated RNA signals. Surprisingly, spliced Bsr RNAs released from the transcription site mainly concentrate as numerous, stable nuclear foci that do not colocalize with any known subnuclear structures. On drug treatments, a fraction of Bsr RNA relocalizes to the cytoplasm and associates with stress granules (SGs), but not with P-bodies, pointing to a potential link between SGs and the metabolism of ncRNA. Thus, Bsr might represent a novel type of nuclear-retained transcript. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Cheng et al., 2005 and references therein). Among these ncRNAs, a large fraction belongs to the so-called "mRNA-like" family that includes spliced, poly(A) RNAs without any conserved and statistically significant open reading frame (ORF). These enigmatic RNAs can be transcribed from intronic sequences, intergenic regions, or even from pseudogenes, and a substantial fraction of them is antisense to protein-coding genes or to other ncRNA genes (Yelin et al., 2003; Katayama et al., 2005). It has been hypothesized that ncRNAs might exert a key role in the regulation of eukaryotic gene expression (Mattick, 2004). However, their roles are a matter of debate, as it is still unclear whether they are all functional or whether they simply reflect spurious transcription.
Of particular interest is a growing list of recent results demonstrating the involvement of ncRNAs in a broad range of epigenetic regulatory pathways (Bernstein and Allis, 2005), including in mammalian systems X-chromosome inactivation and genomic imprinting. X-chromosome inactivation is a developmentally regulated process that silences nearly all the genes residing on one X-chromosome (Xi) in mammalian females. It is controlled by the X-inactivation center (Xic), from which the spliced, 17-kb-long Xist ncRNA is produced (Brockdorff et al., 1992; Brown et al., 1992). Remarkably, Xist "coats" the future inactive X chromosome (Clemson et al., 1996) and initiates transcriptional gene silencing of nearly all the genes residing on it (Chow et al., 2005). Genomic imprinting is an epigenetic regulation that leads to preferential expression of one of the two alleles according to its parental origin. Most of the imprinted genes are clustered in large chromosomal domains spreading over megabases, and their monoallelic expression, from the paternal or the maternal allele, is tightly coordinated by an intricate network of epigenetic features, including allele-specific DNA methylation and histone-tail modifications, differential timing of DNA replication, or subnuclear localization, as well as expression of large ncRNA genes (Reik and Walter, 2001; Gribnau et al., 2003). Indeed, many imprinted ncRNA genes have been identified, with most of them expressed from the parental chromosome carrying neighboring, silent alleles of protein-coding genes (Sleutels and Barlow, 2002; O'Neill, 2005). Although their modes of action remain largely unknown, transcription of two of them, Air and Kcn1q0t1, and/or the ncRNAs per se are believed to be important for gene silencing (Sleutels et al., 2002; Mancini-Dinardo et al., 2006; Seidl et al., 2006).
The Dlk1-Gtl2 is an evolutionary conserved, 1-Mb imprinted chromosomal region lying on the distal arm of chromosome 12 in the mouse (corresponding to human 14q32 and rat 6q32; Figure 1A). It contains three protein-coding genes (Dlk1, Rtl1, and Dio3) that are only expressed from the paternal allele and multiple ncRNA genes that are only transcribed from the maternally inherited allele: 1) Gtl2, a large spliced and poly(A) RNA with multiple spliced isoforms (Schuster-Gossler et al., 1998; Miyoshi et al., 2000), 2) a poorly characterized antisense transcript to the Rtl1 gene (Seitz et al., 2003; Davis et al., 2005), and 3) numerous small regulatory RNAs belonging to the C/D RNA and microRNAs (miRNAs) gene families known to direct site-specific RNA 2'O-methylations and silence gene expression at the posttranscriptional level, respectively (Kiss, 2002; Zamore and Haley, 2005). Many of these small RNA genes, whose functions are highly elusive (Davis et al., 2005; Schratt et al., 2006), are organized into clusters of repeated, homologous gene copies, with most of them embedded in and processed from introns of huge noncoding transcripts extending over several hundred kilobases (Cavaille et al., 2002; Seitz et al., 2004a). All the ncRNA genes are transcribed in the same orientation, with an apparently coordinated spatial-temporal expression pattern (Cavaille et al., 2002; Seitz et al., 2004a; Tierling et al., 2006) and with an imprinted expression regulated by an intergenic, germline-derived, differentially methylated region (IG-DMR) located between Dlk1 and Gtl2 genes (Lin et al., 2003). Thus, it is not formally known whether they are synthesized from their own promoters or whether they are processed from a large, single transcription unit starting at the Gtl2 promoter. Interestingly, the genomic organization of the C/D RNA gene cluster at the Dlk1-Gtl2 domain, resembles the one at the imprinted Prader-Willi syndrome (PWS) chromosomal region, suggesting a functional and/or evolutionary link between repeated ncRNA genes and epigenetic imprinting processes (Cavaille et al., 2000; reviewed in Seitz et al., 2004b; Royo et al., 2006).
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; Lin et al., 2003). Several microRNAs from processed anti-RTL1 RNA direct RNA interference–mediated cleavages within the paternally expressed RTL1 mRNA, whereas imprinted miR-134 inhibits the localized translation of limk1 mRNA at the synapto-dendritic compartment of hippocampal neurons, highlighting the involvement of these small RNAs in the regulation of fetal growth and higher brain function, respectively (Davis et al., 2005; Schratt et al., 2006). Imprinted miRNAs are also suspected to be key players in polar overdominance phenomena, an unusual nonmendelian mode of inheritance described in sheep (Davis et al., 2005). Finally, the lack of the expression of all the ncRNA genes on the maternal chromosome leads to the expression of genes that are normally maternally repressed (Lin et al., 2003), raising the possibility that ncRNAs might function to silence in cis the neighboring protein-coding genes. To further understand these imprinted ncRNAs, we developed cell imaging approaches, to address yet unresolved issues regarding the metabolism of the large, spliced, C/D RNA host transcripts and their potential involvement in epigenetic regulation. We concentrated on the Bsr locus, a highly expressed transcription unit that encompasses a huge array of tandemly repeated C/D RNA genes (RBII-36) at the rat Dlk1-Gtl2 domain. Several questions were specifically addressed: What is the intracellular fate of these large mRNA-like transcripts? Are they exported to the cytoplasm to be rapidly degraded by the nonsense-mediated RNA decay system (NMD)? Do they remain associated with their own parental locus like the chromosomal Xist RNA? Can any other roles be envisioned?
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These primary cultures were routinely analyzed between days 8 and 14 after plating. For in situ hybridization, cells were plated on gelatin-coated (1%) 12-mm-diameter glass coverslips. REFs were either transiently transfected by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) or electroporated (Gene Pulser, Bio-Rad, Richmond, CA). Gene expression was routinely assayed 36–48 h after transfection. The mammalian expression vectors of GFP-H3 and YFP-PSP1
were generous gifts from Dr. P. Cook (University of Oxford) and from Dr. A. Lamond (University of Dundee), respectively, and the GFP-DCP1 and the GFP-G3BP plasmids were kindly provided by Dr. B. Séraphin (University of Paris 6, CNRS UPR 2167) and Dr. J. Tazi (IGMM, UMR 5535CNRS Montpellier), respectively. The GFP-MLBN1 plasmid and DMPK minigene were kindly provided by Dr. T. A. Cooper (Baylor College of Medicine, Houston, TX).
Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) with DNA oligonucleotide probes was performed as described in http://singerlab.aecom.yu.edu/protocols/. In the case of DNA/RNA detection, DNA was heat-denaturated (7 min at 85°C in 70% formamide, 2 x SSPE) before detection. The slides were then postfixed (10 min) before proceeding to RNA detection. Coverslips were mounted in Moviol DAPI (0.1 µg/ml). For in situ hybridization on rat brain sections, 10-µm cryosections were carried out. Fixation and hybridization on the sections were performed in the same conditions as those described above. Approximately 40–50 mer DNA oligonucleotide probes (Supplementary Data S5) were labeled with fluorolink Cy3 (Amersham Biosciences, Piscataway, NJ), Cy5 (Amersham Biosciences), or Oregon green (Molecular Probes, Eugene, OR). Images were captured with a CoolSnap ES camera (Roper Scientific, Tucson, AZ) mounted on a microscope (model DMRA, Leica, Deerfield, IL) with Leica 100 x plan Apo 1.4 and using the Metavue software (Universal Imaging, West Chester, PA). 3D deconvolution was performed with Metamorph (Universal Imaging). The observations were confirmed by at least two of the authors.
Cell Fractionation and Ribonuclease A/T1 Protection Assay
Trypsinized REFs were suspended in nuclei buffer [0.25 M sucrose, 10 mM Tris, pH 7.4, 2.5 mM MgCl2, 100 µg/ml collagenase (Sigma, St. Louis, MO), 2% Cemulsol NP10 (Rhône-Poulenc, a gift from J.-P. Zalta)] and disrupted with an Ultra-Turrax T25 basic (IKA-Werke, Staufen, Germany) (setting 2.7, 45 s). After 5 min of centrifugation (750 x g at 4°C), the supernatant was collected (= cytoplasmic fraction). The pellet was resuspended in nuclei buffer and disrupted again with Ultra-Turrax (setting 1.2, 30 s). Nuclei were then pelleted by centrifugation (5 min, 750 x g at 4°C), and RNAs were extracted with Trizol reagent (Invitrogen), whereas the cytoplasmic fraction was extracted twice with phenol/chloroform (saturated with water). Ribonuclease A/T1 protection assay (RPA) was performed according to standard protocol, with an internally 32P-labeled 85-nt-long riboprobe complementary to the exon–exon junction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Cavaille et al., 2001) as well as in the placenta, the trunk of embryos (embryonic day E15.5), and REFs (not shown). This locus is remarkable as it gives rise, not only to the mature RBII-36 C/D RNA, but also to a large amount of spliced, poly(A) transcripts, as well as poorly characterized, unprocessed intron-containing RNA species (Figure 1A; Cavaille et al., 2001). The Bsr locus has been predicted to be specific to the Rattus genus of rodents (Komine et al., 1999; Cavaille et al., 2001). The recent completion of the draft mouse and rat genomes allowed us to perform a more detailed sequence analysis. We now demonstrate unambiguously that the rat Dlk1-Gtl2 locus contains an 100-kb-long piece of DNA found neither in the mouse (Figure 1B), nor in any other available vertebrate genome and that this piece of DNA consists of at least 86 direct tandem repetitions of a 0.9-kb-long Bsr unit, spanning the entire C/D RNA-containing intron and one flanking 80-nt-long, noncoding exon (Figure 1A). Given that the whole Bsr locus is almost devoid of common interspersed repeats, these observations point to recent gene amplification events that occurred probably after the divergence between mouse and rat. Thus, Bsr gene might represent another example of a brain-specific noncoding RNA that has evolved only in a specific lineage (Pollard et al., 2006).
Monoallelic Expression at the Bsr Locus Is Resistant to TSA Treatment
We first checked whether Bsr gene is monoallelically expressed in REFs, by detecting simultaneously the Bsr locus with a mixture of four DNA oligonucleotide probes, designed to detect the template strand of Bsr repeated units (the DNA probes) and the nascent Bsr transcripts, the latter with an antisense DNA oligonucleotide probe designed to detect unspliced Bsr RNAs (the intronic probe). In 95.6% of the Bsr-expressing nuclei (n = 413), the intronic probe revealed only a single nuclear RNA signal overlapping one of the two DNA signals, thus demonstrating the monoallelic expression of the Bsr locus (Figure 2A). The remaining cells with two RNA signals mostly arise from large, tetra(poly)ploid nuclei (not shown), suggesting that Bsr monoallelic expression is unlikely to be regulated by a counting process. Monoallelic expression of the Bsr gene was also visualized in cultured primary neurons (prepared from the hypothalamus of E17 rat embryos) in which a single, nascent RNA signal was systematically detected in each nucleus of Tuj-positive neurons (Figure 2B), while only 5% of Tuj1-negative cells (mostly astrocytes as judged by their immunoreactivity with anti-GFAP antibodies, not shown), gave rise to weaker RNA signals at the transcription sites. The parental origin of the transcripts in REFs or neurons cannot be formally determined by our FISH protocol; however, we favor the hypothesis that the Bsr gene is expressed from the maternally inherited chromosome, as previously shown for the other surrounding ncRNA genes in the homologous imprinted mouse domain (Lin et al., 2003).
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-amanitin (20 µg/ml) strongly suggests that RNA polymerase II is transcribing this large locus (Figure 2C).
Inhibitors of histone deacetylases, e.g., trichostatin A (TSA), can induce the reactivation of the normally silent allele at the Igf2r locus (Hu et al., 2000). However, despite different experimental conditions (time courses ranging from 6 to 48 h and drug concentrations ranging from 0.3 to 3 µM), TSA treatments did not lead to any significant de-repression of the silent Bsr allele (Figure 2D). Rather, the only significant change we reproducibly observed was an increase in the proportion of monoallelic expression in REFs or Tuj1-negative cells (from 53 to 75% and from 5 to 50%, respectively; Figure 2D). Thus, the maintenance of monoallelic regulation at the Bsr locus is resistant to a drug that can affect the epigenetic state of chromatin.
Tracking the Intranuclear Fates of Noncoding RNAs Processed from the Bsr Locus
We next investigated the intracellular fates of the Bsr-associated transcripts by multicolor RNA FISH at the single nucleus level, by hybridizing simultaneously three fluorescent oligonucleotide probes: 1) an intronic-probe designed to detect unprocessed Bsr RNAs; 2) an RBII-36 probe designed to detect the fully processed RBII-36 RNA and any other RBII-36–containing RNA precursor; and 3) a spliced-probe designed to recognize specifically the exon–exon junctions, thus allowing the specific detection of spliced Bsr RNA species. In agreement with data obtained in the adult rat brain sections (Supplementary Data S1), RBII-36 probe reveals the nucleolus as well as a strong and single nucleoplasmic signal that merges perfectly with that detected by the intronic probe, which never stains the nucleolus (Figure 3A, a, b, and e). Thus, the extranucleolar RBII-36 signal represents the nascent Bsr transcripts at the site of transcription. Interestingly, in many cases the intronic RNA signals exhibit a characteristic "comet-like" shape with a strong and compact signal ("the head") followed by weaker and more dispersed signals (one or two "tail(s)") as exemplified in Figure 3, A (right) and B (left). Surprisingly, in cells expressing high amounts of Bsr, the RBII-36 probe also detects dot-like signals relatively far away from the tail, as illustrated in Figure 3A (right). Although most of them seem to be organized in a nonrandom manner, with an apparent linear axis, no clear vectorial intranuclear trafficking from the tails of the comet toward the nucleolus, the nuclear interior, or the nuclear envelope was noticed. Because these dot-like signals are also detected with the intronic-probe, they represent RBII-36 RNA precursors rather than fully mature RBII-36 RNAs traveling from their transcription site to the nucleolus.
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Spatial Relationships of Bsr-derived Transcripts in Relation to Their Locus and to Nuclear Architecture
Dual FISH to DNA and RNA was performed to address the question of whether there is a specific spatial relationship between the Bsr gene and these elongated RNA structures. Although heat denaturation affects to some extent the shape and the size of the RNA signals (not shown), in 83% of the examined nuclei (n = 58) the elongated nuclear RNA tracks and the comet-like signals extend beyond the side of the active Bsr allele, with the DNA signal positioned at one extremity within or even at the periphery of "the head" of the comet-like signals (Figure 4A). Remarkably, spliced Bsr RNA signals do not perfectly superimpose with the intronic Bsr RNA signals. Indeed, whereas a gradient of decreasing intensity of the unspliced signals, from the head to the tail, is frequently observed, the intensity of the spliced Bsr signals is relatively equal all along the track-like signals or even increased in "the tails." These data are consistent with intronic RNA tracks belonging to nascent transcripts and/or partially processed Bsr RNA intermediates, whereas spliced RNA signals at the transcription site more likely reflect Bsr RNA molecules that are subjected to cotranscriptional RNA splicing and/or are recently released from DNA.
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; Verschure et al., 1999; Politz and Pederson, 2000; Cremer et al., 2004).
Bsr RNA Is a Nuclear-retained RNA That Does Not Colocalize with Known Nuclear Bodies
To unambiguously demonstrate that Bsr RNA species are mainly present in the nucleus, cell fractionation was carried out, and the relative amounts of spliced Bsr RNA species in the nucleus and in the cytoplasm fractions were examined by using a sensitive ribonuclease protection assay. As shown in Figure 5A, spliced Bsr RNAs were only recovered in the nuclear fraction. Thus, we conclude that spliced Bsr RNAs are unlikely to be exported significantly to the cytoplasm.
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). Because these RNAs remain associated with speckles even in cells treated with transcriptional inhibitors, nucleus-restricted, poly(A) RNAs might contribute to organizing speckles (Huang et al., 1994). We have consequently investigated the intranuclear distribution of the nuclear Bsr dots relative to the nuclear speckle domains revealed by GFP-SC35 staining. As shown in Figure 5B (top), Bsr dot-like do not accumulate preferentially within SC35 domains although 70% are found at the edges of the speckles (n = 2439 dot-like analyzed). Similar nuclear staining patterns were also observed with speckles defined by U2 snRNPs (data not shown).
Paraspeckle domains are recently discovered nuclear bodies, usually found adjacent to speckles, that likely play a role in RNA synthesis and processing (Fox et al., 2002). Interestingly, a nuclear-retained transcript—the CTN-RNA—has recently been shown to localize to paraspeckle domains (Prasanth et al., 2005). As shown in Figure 5B (bottom), spliced Bsr RNA species are totally excluded from these nuclear structures as none of them overlap with the staining of a transiently transfected YFP-PSP1 or YFP-PSF (not shown), RNA-binding proteins enriched in paraspeckles. We thus conclude that Bsr RNA is neither a major component of the speckle, nor of the paraspeckle domains.
Transcripts from the mutant DMPK allele with expanded CUG repeats are retained in the nucleus and form multiple discrete nuclear foci that recruit the muscleblind-like (MBNL1) proteins (Davis et al., 1997; Ho et al., 2005). The highly repeated exonic structure of spliced Bsr transcripts prompted to us to test the possibility that they might enter the same intranuclear pathway that prevents efficient export of mutant DMPK transcripts. To test this hypothesis, we analyzed the distribution of Bsr foci relative to those of CUG repeats foci. The intranuclear location of CUG repeat foci was visualized by cotransfecting REFs with a GFP-MNLB1 expression plasmid and with a DMPK minigene containing 960 CUG repeats. Consistent with previous results (Ho et al., 2005), transcripts with CUG repeats recruit MNLB1 and induce the formation of punctate GFP-labeled CUG repeats (Figure 5C). Importantly, only a minority of those completely merge with foci containing spliced Bsr RNAs. We conclude from these observations that Bsr and CUG repeats foci do not occupy the same nuclear regions.
To gain further insights into the organization of these Bsr ribonuclear foci, a careful quantification analysis of the total fluorescence in individual dots was carried out. This analysis revealed that most of the Bsr nuclear dots contain a limited number of hybridized probes and supports the notion that they might correspond to single (or a few) RNA molecule(s) rather than clusters of multiple spliced Bsr RNAs attached to a putative nuclear structure (Supplementary Data S3).
Spliced Bsr RNAs Are Metabolically Stable Transcripts
Although we provide compelling evidence that spliced Bsr RNAs are mainly present in the nucleus, one could argue that Bsr RNAs are exported to the cytoplasm and then rapidly degraded by the NMD system, a quality control mechanism that eliminates transcripts carrying nonsense mutations. This process, which requires ongoing translation, is inhibited by protein synthesis inhibitors. Therefore, if Bsr RNAs enter the NMD pathway, their level of expression should increase when translation is halted. To test this hypothesis, REFs were treated with the translation elongation inhibitor cycloheximide, and the level of Bsr RNAs was analyzed. As shown in Figure 6A, no change in the steady state of Bsr RNAs was observed upon treatment with cycloheximide, whereas the steady state of two noncoding C/D RNA host transcripts used as positive controls, UHG and gas5 RNAs, was dramatically increased in agreement with previous reports (Tycowski et al., 1996; Smith and Steitz, 1998). As expected, no change was seen with the P0 ribosomal protein-coding mRNA. These data are consistent with spliced Bsr RNA being immune to NMD, most likely because it mainly remains within the nucleus.
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-amanitin (data not shown). The use of the RBII-36 probes also shows that nucleolar RNA signals are nearly unaltered, consistent with RBII-36 being incorporated into metabolically stable RNPs (Figure 6B, left). From these data, we conclude that nuclear spliced Bsr RNAs are relatively stable transcripts and that Bsr foci are unlikely to represent nuclear sites of rapid degradation.
Cytoplasmic Bsr RNAs Associate with Stress Granules But Not with P-Bodies
The vast majority of spliced Bsr RNA signals are detected in the nucleus. However limited but significant dot-like signals were observed in the cytoplasm of REFs and also in the dendritic compartments of hypothalamic neurons (Supplementary Data S5A). In REFs, their detection was highly variable, either in individual cells (partial or nearly total cytoplasmic relocation) or within the cell population (ranging from 1 to 12% of the cell population). Remarkably, a 2–10-fold increase in the proportion of REFs with cytoplasmic Bsr RNA signals was noticed during the course of actinomycin D treatment or after various drug treatments, as well as to some extent after electroporation or liposome-mediated transfections (Supplementary Data S4). These observations argue in favor of a mechanism occurring in normal conditions that retains Bsr RNAs in the nucleus. We reasoned that this leak of Bsr from the nucleus to the cytoplasm might result from a global cellular stress.
In response to environmental stresses, 40S ribosome-associated poly(A)+ mRNA accumulate into translationally silent mRNP complexes within discrete cytoplasmic structures, the so-called stress granules (SGs; Kedersha and Anderson, 2002; Kedersha et al., 2005). We therefore asked whether Bsr cytoplasmic RNAs accumulate in SGs by analyzing their distribution in REFs transiently transfected by a plasmid expressing the GFP-tagged G3BP endoribonuclease, a protein recruited to SGs under stress conditions (Tourriere et al., 2003). As shown in Figure 7A (top), 67% of cytoplasmic Bsr signals were found associated with arsenite-induced SGs, many of them being detected at their close periphery (n = 384 cytoplasmic foci analyzed). To avoid any bias or artifacts, only unambiguous cytoplasmic Bsr dots within cells displaying a moderate level of GFP signals were scored. Indeed, very large SGs, probably due to G3BP overexpression, frequently contain multiple Bsr RNA dots (up to 18, as illustrated in Figure 7A, bottom). A preferential association of Bsr RNAs within SGs was also observed in untransfected, arsenite-treated hypothalamic neurons, thus excluding the possibility that relocation of Bsr in SGs is simply due to GFP-G3BP expression (Supplementary Data S5B). To our knowledge, this is the first evidence that untranslated mRNAs can be present in SGs.
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). Because SGs are often juxtaposed with PBs, mRNAs destined for degradation might be sorted in SGs and then routed to P-bodies as proposed earlier (Kedersha et al., 2005). We addressed this hypothesis by simultaneously revealing PBs and SGs by GFP-DCP1 staining and hybridization to a Cy5-labeled oligo-dT probe to detect poly(A+) RNA species, respectively. As shown in Figure 7B, Bsr RNA species do not significantly colocalize with PBs (6% overlap) in arsenite-treated REFs (n = 1340 cytoplasmic dot-like signals analyzed). Reinforcing this notion, even in the case of Bsr-containing SGs positioned adjacent to PBs, no obvious overlap between Bsr foci and GFP-DCP1 signals was observed, again indicating that Bsr RNAs are unlikely to be targeted to PBs. The lack of detection of Bsr RNA signals within PBs is unlikely to reflect their rapid 5' to 3' degradation in these bodies, because cytoplasmic Bsr RNAs are still detected after actinomycin D treatment (Supplementary Data S4). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Thakur et al., 2004; O'Neill, 2005) and except for Xist (Clemson et al., 1996), their intranuclear trafficking has not been extensively documented so far. In this report, the imprinted Dlk1-Gtl2 domain was chosen as a model to further characterize the metabolism of large spliced C/D RNA host-gene transcripts synthesized and processed from the rat Bsr locus (Figure 1). Indeed, ncRNAs at the Dlk1-Gtl2 domain are believed to play an important role during development (Georgiades et al., 2000; Lin et al., 2003; Davis et al., 2005; Schratt et al., 2006). In addition, its highly repeated gene organization and its high level of expression make the Bsr locus an appropriate cellular model with which to address, through cell imaging approaches, key questions regarding the intracellular fate of imprinted ncRNAs.
By using a FISH-based protocol with specific oligonucleotide probes conjugated to fluorochromes, we visualized the monoallelic expression of the Bsr gene at the single-cell level (Figure 2) and successfully tracked Bsr-derived transcripts, from the transcription site to the interchromatin space (Figures 3
–5) and also to some extent in the cytoplasm including within dendrites of hypothalamic neurons (Figure 7, Supplementary Data S5). We showed that an unexpected large amount of spliced (or partially spliced) Bsr RNA signals accumulate in close proximity to its own locus (Figures 3 and 4). Bsr and Xist RNAs share some similar features in that they are monoallelically expressed genes that give rise to nuclear spliced, polyadenylated ncRNAs with many repeated sequence motifs. Repetitive sequences are known to attract gene silencing and the lack of expression of maternally expressed ncRNA genes at the Dlk1-Gtl2 domain is associated with the reactivation of the neighboring, silent protein-coding genes (Lin et al., 2003). Thus, and even though Bsr RNA species do not stably remain associated with their own locus (Figure 6) and they disappear during mitosis (not shown), these large nuclear RNA tracks around the transcription site might be the counterpart of the Xist RNA coating (Chow et al., 2005). This notion is reinforced because we have found that two other imprinted ncRNA gene loci also generate large RNA accumulation around their transcription sites (Royo and Cavaillé, unpublished data).
Alternatively, spliced Bsr RNAs at the transcription site might reflect cotranscriptional RNA splicing and/or RNA splicing taking place immediately thereafter, as suggested by the spatially organized RNA splicing along the tracks (Figure 4). Although it is not well understood why many Bsr RNA signals display an elongated shape with a polar orientation relative to their gene, our observations strongly recall linear RNA signals previously observed for a few viruses and cellular protein-coding transcripts (Lawrence et al., 1989; Xing et al., 1993; Dirks et al., 1995; Melcak et al., 2000). One can intuit that a large amount of RNAs at the transcription site might result from any rate-limiting step between transcription and the subsequent intranuclear RNA trafficking. A correlation between the extent of RNA splicing and the presence of tracks has been noticed (Dirks et al., 1995), and transcripts that are deficient in RNA processing are retained near the transcription site (Custodio et al., 1999). Thus, inefficient splicing of Bsr pre-RNAs might account for its localized nuclear accumulation. It should be emphasized, however, that the cotranscriptional hypothesis is not mutually exclusive of an involvement of Bsr RNAs in gene silencing by still unknown mechanisms, either in the maintenance and/or the establishment of imprinted regulation at the Dlk1-Gtl2 domain. Indeed, no stable accumulation of Air or Kcnq1ot1 RNAs at their parental chromosome has been noticed so far (Sleutels et al., 2002; Mancini-Dinardo et al., 2006; Seidl et al., 2006).
We have also made the surprising finding that Bsr RNAs released from the transcription site concentrate mostly in the interchromatin space as multiple, metabolically stable nuclear foci, rather than being rapidly exported to the cytoplasm as expected for spliced, polyadenylated RNAs. To our knowledge, nuclear RNA foci have only been well documented for two endogenously expressed, mammalian cellular transcripts: 1) the mutant DMPK alleles with expanded CUG trinucleotide repeats (Davis et al., 1997) and 2) the recently discovered CTN-RNA that localizes to paraspeckles (Prasanth et al., 2005). Remarkably, the intranuclear fate of Bsr transcripts differs considerably from that of these two nuclear-restricted transcripts (Figure 5) and above all dramatically contrasts with the other noncoding C/D RNA host gene transcripts, like Gas5 or UHG, which are short-lived transcripts associated with polysomes (Tycowski et al., 1996; Smith and Steitz, 1998). These observations strongly argue against the possibility that Bsr transcripts simply correspond to "RNA remnants" of the spliced host transcripts that are undergoing nuclear RNA degradation. The mechanisms underlying the nuclear retention of spliced Bsr RNAs and their potential functions have not yet been identified.
Although our study unambiguously demonstrates that Bsr RNA represents a novel nuclear-retained RNA, a cytoplasmic function could also be envisioned. First, a small subfraction of Bsr RNAs can escape the nucleus and are targeted to the dendritic compartments of hypothalamic neurons (Supplementary Data S5A). Remarkably, miR-134, whose gene maps downstream from the Bsr locus, also localizes to the synapto-dendritic compartment of rat neurons wherein it controls the growth of dendritic spines (Schratt et al., 2006). Whether the dendritic location of Bsr RNA reflects its involvement in the same cellular regulatory pathway is an intriguing question. Second, stress stimuli favor a relocation of Bsr RNAs in the cytoplasm, many of the transcripts being found within or in close proximity to SGs but not within PBs (Figure 7). SGs are thought to represent the sites of accumulation of stalled translation preinitiation complexes (Kedersha and Anderson, 2002). Thus this observation was largely unexpected because Bsr RNA does not contain any obvious protein-coding potential. The intracellular behavior of two other nuclear-retained RNAs is also altered upon a cellular stress: CTN-RNA is cleaved and released to the cytoplasm (Prasanth et al., 2005), whereas heat shock causes Hsr-omega RNA-containing nuclear speckles to coalesce into larger clusters (Prasanth et al., 2000). In addition, several noncoding RNAs are specifically induced and/or play a role in response to oxidative stress (Crawford et al., 1996a,b; Wang et al., 1996) or heat shock (Jolly et al., 2004; Shamovsky et al., 2006). Therefore, Bsr RNAs might sequester nuclear RNA-binding proteins and serve as storage sites that modulate their intracellular availability, depending on environmental and/or internal stimuli. Alternatively, because SGs contain endoribonuclease activities (Tourriere et al., 2003), the possibility that Bsr RNAs might undergo slow decay in these bodies cannot be formally excluded. More sophisticated experiments are now required to fully appreciate the potential interplay between ncRNAs and the function and/or the organization of the SGs.
Nuclear poly(A)+ RNA species have been reported (Perry et al., 1974; Carter et al., 1991; Visa et al., 1993; Huang et al., 1994), and taking into account that noncoding RNAs represent a major outcome of mammalian transcripts (Carninci et al., 2005; Cheng et al., 2005), many other nuclear RNAs with roles in various aspects of nuclear functions and/or cell organization are expected to be described in the near future.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Present address: Département de Biologie, Faculté des Sciences Université de Sherbrooke, 2500 Boulevard de l'Université Sherbrooke, Québec, J1K 2R1, Canada.
Address correspondence to: Jérôme Cavaillé (cavaille{at}ibcg.biotoul.fr).
Abbreviations used: ncRNA, noncoding RNA; miRNA, microRNA; REF, rat embryonic fibroblast; PWS, Prader-Willi syndrome; SG, stress granule; PBs, P-bodies.
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), the intron-containing RNA signals at the transcription site (
), and the spliced RNA signals at the transcription site was assayed (
). A time-course analysis is shown. A minimum of 100 nuclei was analyzed for each actinomycin D treatment. Right, analysis of the number of nuclear foci per cell in control cells (n = 112) and actinomycin D–treated cells (n = 109). The number of dot-like signals in control cells was set to 1.