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First published online December 16, 2009
doi: 10.1242/jcs.054957

Journal of Cell Science 123, 70-83 (2010)
Published by The Company of Biologists 2010
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Research Article

Long nuclear-retained non-coding RNAs and allele-specific higher-order chromatin organization at imprinted snoRNA gene arrays

Patrice Vitali1,2,*,{ddagger}, Hélène Royo1,2,*,§, Virginie Marty1,2, Marie-Line Bortolin-Cavaillé1,2 and Jérôme Cavaillé1,2,¶

1 Université de Toulouse, UPS; Laboratoire de Biologie Moléculaire Eucaryote, F-31000 Toulouse, France
2 CNRS; LBME, F-31000 Toulouse, France

Author for correspondence (cavaille{at}ibcg.biotoul.fr)

Accepted 12 October 2009


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The imprinted Snurf-Snrpn domain, also referred to as the Prader-Willi syndrome region, contains two ~100-200 kb arrays of repeated small nucleolar (sno)RNAs processed from introns of long, paternally expressed non-protein-coding RNAs whose biogenesis and functions are poorly understood. We provide evidence that C/D snoRNAs do not derive from a single transcript as previously envisaged, but rather from (at least) two independent transcription units. We show that spliced snoRNA host-gene transcripts accumulate near their transcription sites as structurally constrained RNA species that are prevented from diffusing, as well as multiple stable nucleoplasmic RNA foci dispersed in the entire nucleus but not in the nucleolus. Chromatin structure at these repeated arrays displays an outstanding parent-of-origin-specific higher-order organization: the transcriptionally active allele is revealed as extended DNA FISH signals whereas the genetically identical, silent allele is visualized as singlet DNA FISH signals. A similar allele-specific chromatin organization is documented for snoRNA gene arrays at the imprinted Dlk1-Dio3 domain. Our findings have repercussions for understanding the spatial organization of gene expression and the intra-nuclear fate of non-coding RNAs in the context of nuclear architecture.

Key words: Chromatin, Epigenetic, Genomic imprinting, Intranuclear RNA trafficking, Non-coding RNA, Nuclear architecture


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Recent studies have indicated that a previously unappreciated, huge proportion of the mammalian genome gives rise to long, non-protein-coding transcripts [also referred to as non-coding RNAs (ncRNAs) or mRNA-like transcripts]. These ncRNA gene loci are rather complex and still poorly defined, with some ncRNAs overlapping with, or embedded within, protein-coding genes or even other ncRNA gene loci. Although most of their functions remain to be deciphered, ncRNA transcripts are now recognized as key regulatory players in a broad range of cellular and molecular pathways (reviewed by Mercer et al., 2009Go; Ponting et al., 2009Go; Prasanth and Spector, 2007Go). Remarkably, among the best-studied non-coding RNAs, several locate to the nucleus and act either as cis- or trans-acting epigenetic regulators of chromatin. This is the case for Xist that triggers X-chromosome inactivation (Chaumeil et al., 2006Go; Clemson et al., 1996Go), Kcnq1ot1 and Air that control genomic imprinting (Mancini-Dinardo et al., 2006Go; Nagano et al., 2008Go; Pandey et al., 2008Go; Redrup et al., 2009Go; Sleutels et al., 2002Go; Terranova et al., 2008Go) and HOTAIR that regulates homeobox (HOX) genes (Rinn et al., 2007Go). Some other nuclear-retained transcripts concentrate in nuclear bodies: CTN-RNA and NEAT1/Men {epsilon}/β accumulate in paraspeckle domains (Clemson et al., 2009Go; Prasanth et al., 2005Go; Sasaki et al., 2009Go; Sunwoo et al., 2009Go) and MALAT1 associates with speckle domains (Hutchinson et al., 2007Go; Wilusz et al., 2008Go). Remarkably, NEAT1/Men {epsilon}/β acts as `architectural RNA' and controls paraspeckle formation (Clemson et al., 2009Go; Sasaki et al., 2009Go; Sunwoo et al., 2009Go). The nuclear stress-induced RNAs Sat III or hsr{omega}-n (in human and Drosophila, respectively) are retained at their transcription sites and, by their association with RNA-processing factors, form nuclear stress granules and omega speckles, respectively (Jolly et al., 2004Go; Prasanth et al., 2000Go). Finally, several other nuclear transcripts such as DMPK, Bsr or Gomafu RNAs display punctuated nuclear staining patterns without any obvious association with known bodies (Davis et al., 1997Go; Royo et al., 2007Go; Sone et al., 2007Go). Therefore, understanding the intra-nuclear fate of long ncRNAs becomes a crucial issue in comprehending their functions.

Imprinted gene clusters are chromosomal domains (of several 100 kb in length) wherein genes are mono-allelically expressed in a parent-of-origin-specific manner, e.g. for a given gene the paternal allele is turned on and the maternal allele is turned off (Fig. 1). Imprinted gene expression is regulated by allele-specific epigenetic marks introduced during gametogenesis on complex cis-acting regulatory elements, the so-called imprinting centre regions (ICRs), which coordinate monoallelic expression in cis and in a domain-wide manner (Lewis and Reik, 2006Go; Reik and Walter, 2001Go). Most of the imprinted gene clusters examined so far contain one (or several) ncRNA gene(s) whose expression is regulated by ICRs. Interestingly, they also exhibit reciprocal imprinted expression relative to their flanking protein-coding genes (Koerner et al., 2009Go) and two of them (Airn and Kcnq1ot1) play a role in regulating genomic imprinting. Indeed, these two moderately stable, nuclear transcripts are only synthesized from the paternal allele and, through still poorly known mechanisms, silence in cis the flanking, maternally expressed protein-coding genes (Kanduri, 2008Go; Mancini-Dinardo et al., 2006Go; Pauler et al., 2007Go; Sleutels et al., 2002Go). Similarly to Xist that remains associated with its own locus (Clemson et al., 1996Go) and induces the formation of a nuclear repressive compartment (Chaumeil et al., 2006Go), a leitmotif is now emerging whereby long ncRNAs recruit chromatin-modifying complexes in a lineage-specific manner (in placenta and extra-embryonic cells) and trigger parental-specific higher-order chromatin organization (Nagano et al., 2008Go; Pandey et al., 2008Go; Redrup et al., 2009Go; Terranova et al., 2008Go) (reviewed by Koerner et al., 2009Go; Mohammad et al., 2009Go).


Figure 1
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  		Fig. 1. Complex arrays of repeated C/D snoRNA genes at the imprinted Dlk1-Dio3 and Snurf-Snrpn domains. Rat Dlk1-Dio3 domain (top) is characterized by a large array of maternally expressed non-coding RNA genes (Gtl2, anti-Rtl1, Bsr and microRNA-HG), whereas Snurf-Snrpn domain (bottom) contains a large array of paternally expressed non-coding RNA genes including alternative U-exons (IC transcript), C/D snoRNA host-gene transcripts (snoRD116-HG and snoRD115-HG), IPW and antisense transcript to Ube3a. C/D snoRNAs are embedded within ~1-2 kb repeated units spanning an intron containing the C/D snoRNA and one (or two) flanking non-protein-coding exons (grey rectangles). MicroRNAs and C/D snoRNAs are indicated as triangles and ovals, respectively. Other imprinted protein-coding genes are rectangles. Paternally or maternally expressed alleles are blue and pink, respectively, whereas epigenetically silenced alleles are black. ICR, imprinting centre region. Differentially methylated regions are symbolized as lollipops with black and white circles indicating methylated and unmethylated alleles, respectively. The scheme is not to scale.

 

The imprinted Snurf-Snrpn domain (also referred to as PWS/AS region in human) contains two large arrays of repeated C/D small nucleolar (sno)RNA genes (here referred to as snoRD115 and snoRD116) with most, if not all of them, embedded within, and post-transcriptionally processed from, introns of larger, paternally expressed ncRNAs (Cavaille et al., 2000Go; Royo and Cavaille, 2008Go; Runte et al., 2001Go). In mouse, C/D small RNAs have been proposed to derive from Lncat, a huge ~1 Mbase transcript overlapping ICR–snoRD116-Ipw-snoRD115-anti-Ube3a antisense regions (Landers et al., 2004Go). In human, a large ncRNA (470 kb) encompassing both C/D small RNA gene clusters has also been reported (Runte et al., 2001Go). Our knowledge of long ncRNAs at the Snurf-Snrpn domain, however, is still limited due to the lack of relevant transgenic mouse models together with the complexity of their repeated gene organization, i.e. small RNA that are embedded and processed form longer ncRNAs. Most studies have concentrated on the potential roles of the embedded small RNA species (Royo and Cavaille, 2008Go), and long RNA species have been considered as small RNA vehicles. Although the role of these long ncRNAs, if any, is far from being demonstrated, it is worth noting that the 3'-part of Lncat overlaps, in the antisense orientation, the Ube3a gene expressed only from the maternal allele. As a consequence, it was proposed that Lncat might function in cis to silence the paternal allele of the Ube3a gene in neurons (Chamberlain and Brannan, 2001Go; Johnstone et al., 2006Go; Kishino, 2006Go; Rougeulle et al., 1998Go; Runte et al., 2001Go).

We address important issues regarding the biogenesis and the intra-nuclear fate of long, imprinted non-coding RNAs from the Snurf-Snrpn domain, in order to better understand how these long RNAs are generated and how they interact with nuclear structure. We show that these snoRNA host-gene mRNA-like transcripts are basically not exported to the cytoplasm but rather accumulate in the vicinity of their transcription sites as large RNA species that are unable to diffuse freely. Interestingly, chromatin structure at the paternal allele displays a more decondensed state than at the maternal silent allele. Furthermore, we report that chromatin at another tandemly repeated snoRNA gene array that maps to rat Dlk1-Dio3 domain displays the same allele-specific chromatin organization. The peculiar intra-nuclear fate of these imprinted mRNA-like transcripts and the parental-specific higher-order chromatin organization of their genes are discussed.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
snoRD115-HG and snoRD116-HG transcripts are nuclear-retained RNAs
The rodent Snurf-Snrpn imprinted domain is characterized by two large arrays of tandemly repeated, intron-encoded C/D small RNA genes – snoRD115 and snoRD116 (formerly named MBII-52 and MBII-85, respectively) that are embedded within the ~1-2 kb repeat units synthesized as long non-protein coding precursors that are mostly, if not exclusively, expressed in neurons in rodents (Cavaille et al., 2000Go). The Snurf-Snrpn domain therefore shares common genomic and epigenetic features with the Dlk1-Dio3 domain (Cavaille et al., 2002Go; Cavaille et al., 2001Go; Royo et al., 2007Go; Royo and Cavaille, 2008Go) (Fig. 1). After RNA splicing and post-transcriptional processing, snoRD115 and snoRD116 gene loci give rise to mature C/D snoRNAs as well as spliced mRNA-like species that consist of the same repeated, non-protein-coding exons (A, G1 and G2 exons, Fig. 1). For clarity, these poorly characterized spliced C/D small RNA host-gene transcripts at the Snurf-Snrpn domain will be referred to as snoRD115-HG and snoRD116-HG (HG for host gene). Due to gaps in the genome sequence, the exact size of these snoRNA gene arrays in rodents is unknown. However, based on quantitative PCR and sequence analysis, they are predicted to extend over ~100-200 kb (supplementary material Table S1).

To resolve issues regarding the intra-nuclear fate of spliced C/D RNA host transcripts, we took advantage of their highly repeated structure to develop high-resolution fluorescence in situ hybridization (FISH) methodologies using short DNA oligonucleotides designed to specifically recognize either exon-exon junctions or intronic regions different from embedded snoRNA sequences. We used primary cultures of hypothalamic or hippocampal neurons prepared from either rat or mouse embryos. Consistent with neuronal-specific expression, the vast majority of, if not all, GFAP-negative and MAP2-positive cells (not shown) highly expressed snoRD-HG genes, as exemplified in Fig. 2A for snoRD-116HG. As shown in Fig. 2B,C spliced snoRD115-HG and snoRD116-HG transcripts are visualized as characteristic elongated, strong nuclear RNA signals (several µm in length) in 70-80% of examined nuclei. They often occupy a significant part of the DAPI-stained nucleus and partially overlap with the signal of unspliced RNA species detected by intronic probes, i.e. spliced RNA signals tend to be larger and more punctuated than unspliced RNA signals. Therefore, spliced snoRD115-HG and snoRD116-HG transcripts accumulate in close proximity to their genes and, consistent with their monoallelic expression, a single RNA signal per nucleus is revealed (see later). In some nuclei, intron-containing transcripts also adopt a linear configuration along which RNA splicing appears to be spatially oriented, as judged by detection of two gradients of decreasing and increasing intensities for the unspliced and spliced RNA signals, respectively (Fig. 2B, lower panel). In addition to strong RNA signals at the transcription sites, spliced-specific probes enable the visualization of many weaker, punctuated RNA signals not detected by intronic-probes. These were observed throughout the entire nucleus (10 to >100 per nucleus) but only very rarely in the cytoplasm and never in the nucleolus (Fig. 2B,C). The use of the snoRNA-specific probe also reveals a single nucleoplasmic signal that merges perfectly with that detected by the intronic-probe. However, in contrast to the intronic-probe, the snoRNA-specific probe also stains the nucleolus, here detected by pre-ribosomal RNA-specific probes matching the rRNA 18S-ITS1 junction (Fig. 2D).


Figure 2
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  		Fig. 2. SnoRD116 and snoRD115 HG transcripts are nuclear-retained transcripts. (A) Primary rat hypothalamus cells were hybridized with Cy3-labeled spliced snoRD116-HG probes (red) and then stained with GFAP (green) antibodies that revealed glial cells. White arrows indicate the spliced-specific RNA signals (only a single focal plane is shown); * shows a GFAP-positive cell. (B,C) The intra-nuclear fate of spliced and unspliced snoRD116-HG and snoRD115-HG is visualized by RNA FISH with fluorescent oligonucleotide probes as indicated on the panels (red and green signals correspond to Cy3- and Alexa-Fluor-488-labeled probes, respectively). (D) Fully processed snoRD116 (top row) and snoRD115 (bottom row) accumulate within the nucleolus. Top row: primary rat hypothalamus cells were hybridized with Cy3-labeled intron probes (red) and Alexa-Fluor-488-labeled snoRNA-probe (green). Bottom row: primary rat hypothalamus cells were hybridized with Cy3 labeled snoRNA (red) and Alexa-Fluor-488-labeled intron (green) probes. Nucleoli were revealed with Cy5-labeled pre-rRNA probes (blue). Cb, Cajal bodies (as demonstrated by colocalization with coilin, not shown); No, nucleolus. White arrows indicate the sites of transcription. The relative location of oligo-probes is indicated above the panels by small black rectangles. Scale bars: 5 µm.

 
From these data, we conclude that fully processed snoRD115 and snoRD116 accumulate within the nucleolus and, more importantly, that spliced snoRD115-HG and snoRD116-HG transcripts represent nuclear-retained RNA species. Large accumulations of spliced RNA species near their transcription sites will be collectively referred to as `RNA grapes', whereas spliced RNA species dispersed in the nucleus will be referred to as `RNA dots'. Importantly, these two terms do not imply any structural and/or functional meaning.

snoRD115-HG and snoRD116-HG are structurally constrained around their transcription sites
We observed that most of the snoRD115-HG RNA grape signals do not merge with those of snoRD116-HG in rat neurons, with at best a few overlapping signals, thus leaving the impression that they cannot intermingle (Fig. 3Ac,f,i and Fig. 3E, left histogram). This non-overlapping pattern is also seen in mouse neurons (Fig. 3B, left panel). Such observations were largely unanticipated given the proximity of their transcribed DNA regions, e.g. snoRD115 and snoRD116 genes are located approximately 15 kb and 54 kb apart in rat and mouse, respectively. In addition, RNA grapes at snoRD115 genes are not affected in neurons prepared from a knockout mouse deleted for the paternal snoRD116 cluster (Skryabin et al., 2007Go), indicating that these two RNA grapes behave as separate structural entities (Fig. 3B, right panel).


Figure 3
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  		Fig. 3. SnoRD116 and snoRD115 HG transcripts are prevented from diffusing near their transcription sites. (A) Spliced snoRD116-HG and snoRD115-HG are simultaneously visualized by RNA FISH in rat hypothalamic neurons with Alexa-488-conjugated spliced probes (green) and Cy3-conjugated spliced probes (red), respectively. Three representative nuclei with non-overlapping RNA signals at their transcription sites are shown with compact, strong RNA signals (a-c) or more dispersed, elongated RNA signals (d-f and g-i). Note that mouse snoRD115 and snoRD116 genes are located 54 kb apart; in rat, the exact intergenic sequence length is not known due to two small gaps. (B) Spliced snoRD116-HG and snoRD115-HG are simultaneously visualized by RNA FISH in mouse hippocampal neurons with Alexa-Fluor-488-conjugated spliced probes (green) and Cy3-conjugated spliced probes (red), respectively. Neurons were prepared from wild-type mouse (left panel) or mouse deleted for the paternal snoRD116 alleles (right panel). (C) Lncat is visualized by RNA FISH in rat hypothalamic neurons with two Cy3-conjugated U-exon probes (red), Alexa-Fluor-488-conjugated snoRD116-HG probes (green) and Cy5-conjugated snoRD115-HG probes (blue). The limit of nuclear DAPI staining is shown by a white line. A few additional dot-like signals (1-3 per nucleus, small white arrows) were routinely detected with U-specific probes but their significance was not pursued. (D) Intron-containing snoRD116 and snoRD115 transcripts are simultaneously detected by RNA FISH in rat hypothalamic neurons with Alexa-Fluor-488-conjugated intronic (green) and Cy3-conjugated intronic probes (red), respectively. Enlarged intronic RNA signals are shown in the inserts. Scale bars: 5 µm. (E) The percentages of nuclei with RNA FISH signals as symbolized below the histograms were scored. The values represent the mean, and error bars the standard deviation.

 
We visualized U-exon-containing transcripts (also referred to as IC transcripts) initiating within a ~500 kb region upstream of the snoRD116 gene locus. RNA FISH experiments were carried out in rat neurons, where many repeated U-exons have been identified (Landers et al., 2004Go), hence facilitating their detection by our DNA oligo-probe methodologies. As shown in Fig. 3C, the use of U-exon-specific probes reveals a single, major nuclear RNA signal in 40-50% of examined nuclei. These signals are juxtaposed to spliced snoRD115-HG and snoRD116-HG transcripts but again did not exhibit any significant overlap. Remarkably, in many nuclei the apparent order of RNA FISH signals at the interphasic nucleus level mirrors that of the linear organization at the genome level (Fig. 3C, bottom panel and Fig. 3E, center histogram). Finally, compartmentalized RNA FISH signals were observed for unspliced snoRD116-HG and snoRD115-HG when simultaneously tested with intronic-probes (Fig. 3D and Fig. 3E, right histogram).

The compartmentalization of spliced transcripts seems paradoxical, because snoRD115-HG and snoRD116-HG are thought to be part of a single, huge transcript (Landers et al., 2004Go; Runte et al., 2001Go). The model of a unique transcript is based on the ability to detect overlapping transcripts by RT-PCR throughout snoRD gene domains. At best, such experiments indicate that the entire domain is potentially transcribed, but they do not demonstrate formally the existence of this putative single transcript.

Several lines of evidence argue against a single transcript. First, many human ESTs overlapping snoRD116 genes end within evolutionarily conserved sequences annotated as highly conserved elements (HCE) and frequently found associated with 3'-UTRs (Siepel et al., 2005Go), strongly indicating the presence of a transcriptional stop signal between snoRD115-HG and snoRD116-HG genes (supplementary material Fig. S1). Second, that human snoRD115 is brain-specific whereas snoRD116 is detected in most tissues implies that these two snoRNAs derive from different transcripts (supplementary material Fig. S2A). Third, even though snoRD115 and snoRD116 are both brain-specific in rodents (Cavaille et al., 2000Go), their temporal expression patterns do not overlap during rat brain development (supplementary material Fig. S2B) or during in vitro differentiation of mouse P19 cells (Landers et al., 2004Go): snoRD116 is expressed earlier than snoRD115. We therefore propose a model whereby snoRD115-HG and snoRD116-HG transcripts are generated by (at least) two different transcription units. Our model does not exclude occasional read-through transcription that might explain the ability to experimentally link snoRD115 and snoRD116 HG transcripts by RT-PCR. However, two transcription units do not fully explain why spliced RNA species generated from two genes so close to one another look structurally constrained, because intra-nuclear RNA trafficking occurs largely by energy independent, diffusion-like mechanisms (Politz et al., 1999Go; Shav-Tal et al., 2004Go; Singh et al., 1999Go; Vargas et al., 2005Go).

snoRD115-HG and snoRD116-HG do not display marked accumulation within poly(A)-rich domains
Speckle domains are subnuclear regions enriched in RNA metabolic factors and polyadenylated RNAs, including some specific mRNAs (Hall et al., 2006Go). Recent studies suggest that speckle domains, besides serving as storage sites for splicing factors, might also act as dynamic hubs that spatially organize early steps of gene expression by linking post-splicing steps to subsequent intra-nuclear trafficking and export mRNA pathways (Hall et al., 2006Go; Smith et al., 2007Go). Transcripts entering speckles are unable to freely diffuse out of them (Shopland et al., 2002Go), so speckles might have the potential to impose structural constraints on newly made, released transcripts. We therefore analyzed the intra-nuclear distribution of RNA grapes relative to speckle domains, delineated here by detection of poly(A) RNAs. As shown in Fig. 4Aa,b, neither RNA grapes nor RNA dots are preferentially found within poly(A)-rich domains, even though they are frequently adjacent to them. We thus conclude that stable accumulation of RNA grapes within speckles is unlikely to contribute significantly to non-overlapping RNA track patterns. It is worth noting that compartmentalized RNA grapes are not affected significantly in neurons treated with latrunculin A (an actin polymerization inhibitor), nocodazol (a microtubule inhibitor), sodium azide and D-glucose (energy deprivation), puromycin or cycloheximide (translation inhibitors) or by various temperature treatments (not shown).


Figure 4
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  		Fig. 4. Intra-nuclear distribution and relative stability of spliced snoRD115 and snoRD116 transcripts. (A) Rat hypothalamic neurons treated with ethanol (control, a-c) or with actinomycin D (AMD, 5 µg/ml for 5 hours, a'-c'). Spliced snoRD116-HG, snoRD115-HG and RBII-36 HG transcripts are visualized with Cy3-conjugated (red) spliced probes and poly(A)-rich domains are revealed with Alexa-Fluor-488-conjugated (green) oligo-dT probes. Note that transcription inhibition dramatically affects the appearance of most poly(A)-rich domains that adopt characteristic donut-like shapes. White arrowheads indicate remnants of snoRD115-HG and snoRD116-HG RNA grapes still detected in AMD-treated cells. (B) Spliced RBII-36-HG (blue), snoRD116-HG (green) and snoRD115-HG (red) are detected by RNA FISH in rat hypothalamic neurons hybridized with Cy5-conjugated probes, Alexa-Fluor-488-conjugated probes and Cy3-conjugated probes, respectively. White arrowheads indicate some overlapping snoRD116-HG and snoRD115-HG RNA dots in the region of the nucleus delineated by the white rectangle. (C) More examples of multiple colocalizing snoRD115-HG (red) and snoRD-116-HG (green) dots. Scale bars: 5 µm. (D) The percentages of nuclei with spliced RNA FISH signals within, without or partially associated with poly(A)-rich domains (as symbolized below the histograms) were scored. The values represent the mean, and error bars the standard deviation.

 
snoRD115-HG and snoRD116-HG exhibit an intra-nuclear trafficking distinct from that of RBII-36 HG transcripts
We have previously shown that another structurally related, long ncRNA mapping to the imprinted Dlk1-Dio3 domain (here referred to as RBII-36-HG) displays a punctuated nuclear staining pattern highly reminiscent of snoRD115-HG and snoRD116-HG (Royo et al., 2007Go). We subsequently asked whether these three repeated exon-containing mRNA-like transcripts occupy the same nuclear region. As shown in Fig. 4B, RNA dots made by RBII-36-HG do not show any significant association with those made by snoRD115-HG or snoRD116-HG. By contrast, a significant fraction of RNA dots made by spliced snoRD116-HG and snoRD115-HG, i.e. ~49% (n=1122) and 38% (n=1458), respectively, are tightly associated to each other, or even overlap completely (Fig. 4B,C). These data indicate that a subset of spliced snoRD115-HG and snoRD116-HG transcripts concentrate independently as separate entities in the same nuclear region. Alternatively, such overlapping RNA dots might represent chimeric transcripts (aggregates or single RNA molecules) owing to read-through transcription as discussed previously.

We next evaluated the relative stability of snoRD115-HG and snoRD116-HG by treating neurons with actinomycin D (AMD) at high concentration (5 µg/ml) in order to rapidly inhibit on-going transcription. As shown in Fig. 4Aa',b', the detection of RNA grapes at transcription sites is severely impaired after 5 hours of treatment, even though RNA remnants are still detected in a substantial proportion of nuclei. Importantly, snoRD115-HG and snoRD116-HG dots are only slightly affected in transcriptionally arrested cells after 5 hours of treatment, indicating that they represent metabolically stable RNA species resistant to rapid nuclear RNA decay.

The overall nuclear staining of RBII-36 HG, snoRD115-HG and snoRD116-HG appear highly similar. However, there are significant differences notably in response to drug treatments. First, RBII-36-HG RNA grapes associate more tightly with poly(A)-rich domains in untreated cells (Fig. 4Ac, Fig. 4D) and then completely disappear in AMD-treated cells without any detectable RNA remnants (Fig. 4Ac'). Second, more than 90% of RBII-36-HG RNA dots, but not those made by snoRD115-HG or snoRD116-HG, concentrate within poly(A)-rich domains in AMD-treated neurons (Fig. 4Ac'), and also in cells incubated with okadaic acid, an inhibitor of phosphatases known to affect speckle morphology (supplementary material Fig. S3) (Hall et al., 2006Go). These data provide additional evidence showing that transcripts generated at Snurf-Snrpn and Dlk1-Dio3 domains have complex and distinct intra-nuclear paths.

Spatial organization of snoRD115-HG and snoRD116-HG relative to their genes: evidence for RNA tracks
The spatial configuration of RNA grapes relative to their genes was investigated to determine whether they represent accumulation of processed transcripts detached from their genes, the so-called `RNA tracks' previously reported at several gene loci (Lawrence et al., 1989Go; Melcak et al., 2000Go; Shopland et al., 2002Go; Smith et al., 1999Go; Xing et al., 1993Go). This is an important issue because if RNA grapes correspond to nascent transcripts still attached to their DNA, the so-called trees, then their association with their genes might provide the structural basis that prevents them from diffusing. Inspired by the work of others (Smith et al., 1999Go), we reasoned that RNA accumulation extending largely beyond one side of the gene should indicate RNA tracks, whereas trees should be visualized as RNA accumulation that mostly coincides with their genes.

PFA-fixed primary neurons were heat-denatured and DNA was first detected with a mixture of four oligo-probes antisense to the template strand of either snoRD115 (not shown) or snoRD116 genes, followed by detection of RNA grapes. As shown in Fig. 5A, in most nuclei elongated RNA signals tend to localize to one extremity of the DNA signals, suggesting that RNA grapes represent RNA tracks. However, due to the large size of RNA signals and the inherent limit of such analysis, some RNA signals can occasionally superimpose significantly with DNA signals, especially when snoRD gene loci are detected as multiple DNA FISH signals (Fig. 5A, middle and bottom panels; see below for further explanation).


Figure 5
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  		Fig. 5. Extended DNA FISH signals are preferentially associated with the transcriptionally active allele. (A) RNA/DNA-FISH performed on rat hypothalamic neurons with a mixture of four Cy3-conjugated DNA probes designed to detect the template strand of snoRD116 gene arrays (red) and Alexa-Fluor-488-conjugated spliced probes to reveal spliced snoRD116-HG transcripts (green). Representative nuclei with S-S (top row), S-D* (middle row) or D*-D* (bottom row). Scale bars: 5 µm. (B) Representative DNA FISH patterns at snoRD116 and snoRD115 gene arrays detected by a mixture of four Cy3-labeled DNA probes (red) and four Cy5-labeled DNA probes (green), respectively. Scale bar: 2.5 µm. (C) Histograms show length (µm) of beaded DNA structures with 3, 4, 5 or 6-7 pinpoint DNA FISH signals (n=24, 29, 21 and 20, respectively). The values represent the mean, and error bars the standard deviation. (D) Histograms show percentage of rat hypothalamic neurons with S-S, S-D* and D*-D* FISH patterns observed at snoR115 and snoRD116 genes (the green zigzag line representing nascent RNAs). Note that D* alleles mean two or more pinpoint DNA signals. The mean averages 300-350 nuclei scored independently by two of us, and error bars represent standard deviation. (E) RNA/DNA-FISH performed on rat hypothalamic neurons with a mixture of four Cy3-conjugated DNA probes designed to detect the template strand of RBII-36 genes (red) and Alexa-Fluor-488-conjugated spliced probes to reveal RBII-36 HG transcripts (green). Scale bar: 5 µm. Other examples of extended DNA FISH signals at the active RBII-36 genes are shown below. Scale bar: 2.5 µm. (F) Histograms show percentages of rat hypothalamic neurons with S-S, S-D* and D*-D* FISH patterns (as indicated) observed at RBII-36 genes. The mean averages 377 nuclei scored independently by two of us, and error bars represent standard deviation.

 
The transcriptionally active allele is seen as decondensed DNA FISH signals
We discovered that snoRD gene loci are not only observed as a single, punctuated FISH signal (S signal) as illustrated in Fig. 5Ba, but also as doublet, triplet or even more complex FISH signals (up to 6-7 pinpoint signals for a given allele), especially in the case of snoRD116 genes. These punctuated FISH signals, collectively termed D*, are organized either as `linear string-like' (Fig. 5Bd,e) or as more interlaced structures (Fig. 5Bc). Even though DNA FISH signals are regularly and closely spaced along beaded structures, in some nuclei the apparent distance between two pinpoint signals can vary substantially (Fig. 5Bb). Of particular interest are linear extended DNA FISH signals with 6-7 pinpoint signals that can be as long as 2-3 µm (Fig. 5C).

Careful examination reveals that in 50-60% of the examined nuclei, one allele is seen as S signal whereas the other is detected as D* signal (pattern S-D* illustrated in Fig. 5A, middle panel). Only 10-30% of nuclei possess two parental alleles as two singlet DNA FISH signals (pattern S-S illustrated in Fig. 5A, top panel) whereas in remaining nuclei each parental allele is revealed as more than two FISH signals (pattern D*-D* illustrated in Fig. 5A, bottom row). The exact percentages of the three DNA FISH signal patterns observed at snoRD115-HG and snoRD116-HG gene clusters are given in Fig. 5D. Interestingly, in nuclei with S-D* pattern, the active allele (i.e. DNA FISH signals associated with RNA signals) is preferentially visualized as D* signals in ~80% of examined nuclei (Fig. 5D). Remarkably, extended D* FISH signals characterized by beaded structures are also visualized at the 100 kb RBII-36 HG locus (Fig. 5E). Furthermore, similarly to snoRD116 and snoRD115 genes, RNA signals are found at D* signals in the vast majority of examined nuclei with S-D* patterns (Fig. 5F).

Asynchronous DNA replication has been described at some imprinted gene clusters with singlet and doublet-like FISH signals corresponding to late- and early-replicating alleles, respectively (Gribnau et al., 2003Go). However, given that we are using primary non-dividing neurons, DNA replication cannot explain doublet FISH signals. Doublet FISH signals might arise if there were an underlying difference in chromatin structure between the two parental alleles, i.e. a differential cohesion of sister chromatids. In this regard, singlet-doublet DNA FISH signals independent of asynchronous DNA replication, the so-called SIAR, have been described at the X chromosome center (Mlynarczyk-Evans et al., 2006Go). Again, this possibility is extremely unlikely because we failed to obtain any evidence for primary neurons arrested at the G2-M transition, as judged by the lack of duplicated centrosomes (not shown). Finally, and more importantly, none of these DNA-replication-based explanations can account for more than two DNA FISH signals for a given allele.

One can argue, however, that the active paternal allele, perhaps because of its highly repeated structure, might be prone to DNA amplification. To exclude this possibility, we compared the fluorescence intensity of D* signals to that of S signals. We reasoned that if the two alleles were genetically identical, then in a nucleus with S-D* pattern the total fluorescence intensity of the FISH signals at the D* allele should be in the same range of magnitude as that of the S allele, i.e. the ratio of fluorescence intensity of S/D* should be ~1. As shown in supplementary material Fig. S4, these experimentally determined ratios are centered to 1 in nuclei with S-S pattern and, even more importantly, they do not change significantly in nuclei with various S-D* patterns, thus strongly arguing against allele-specific gene amplification. We therefore conclude that D* and S FISH signals reflect two different chromatin states with the active paternally inherited allele (D*) tending to be more decondensed than the genetically identical, silent maternally inherited allele (S).

Decondensed DNA FISH signals are visualized in highly condensed chromatin
To establish a link between higher-order chromatin structure and gene activity, we performed DNA FISH experiments in primary mouse embryonic fibroblasts (MEFs) that do not express snoRD115-HG or snoRD-116-HG (not shown). Intuitively, one could anticipate that D* signals will be underrepresented, or even absent in these non-expressing cells. However, D* FISH were still detected in MEFs (Fig. 6A, left panel) and the relative proportions of S-S, S-D* and D*-D* FISH patterns at snoRD116-HG or snoRD115-HG were not significantly different from those observed in rat neurons (Fig. 6B), even though extended DNA FISH signals with more than 3-4 pinpoint signals were less frequent (~10-15%). Perhaps more surprisingly, D* FISH signals (mostly doublet-triplet) were also seen in 50-60% of condensed anaphase chromosomes observed in asynchronously proliferating MEFs (as illustrated in Fig. 6A, right panel) with 40-50% and ~10% of daughter cells displaying S-D* and D*-D* FISH patterns, respectively (Fig. 6B). These observations are rather unexpected because it is assumed that only DNA regions separated by >500-1000 kb produce readily resolvable FISH signals on metaphasic chromosomes (Lawrence et al., 1990Go), although in some cases DNA sequences less than 100 kb apart have been resolved (Lemieux et al., 1994Go).


Figure 6
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  		Fig. 6. Extended DNA FISH signals in highly condensed chromatin structures. (A) Examples of decondensed DNA FISH signals observed in non-expressing, primary MEFs: interphase nuclei (left panel) or in anaphase (right panel). A single focal plane is shown. snoRD116 (red) and snoRD115 (green) genes are visualized by DNA FISH with Alexa-Fluor-568- and ATTO-488-labeled BAC clones, respectively. Scale bar: 5 µm. (B) Histograms show the percentages of DNA FISH patterns at snoRD115 and snoRD116 genes that have been scored in interphase or in anaphase as indicated. (C) Examples of extended DNA FISH signals observed in male germ cells. SnoRD115 and snoRD116 genes (a-c; the Snurf-Snrpn domain) and a region of the Dlk1-Gtl2 cluster overlapping snoRNA genes (d-f) are visualized with digoxygenin-labeled BAC clones (green), or biotin-labeled BAC clones (red). Scale bars: 5 µm. (D) Histograms show percentages of S and D* FISH signals in late spermatids, round spermatids and primary spermatocytes (two independent experiments). Note that in primary spermatocytes (diploid cells that are undergoing chromosome pairing), homologous chromosomal regions are not easily resolved (c), although in some nuclei the two chromosomal regions can be discriminated (f). As a consequence, we only scored primary spermatocytes in which the two parental alleles were reasonably separated. The `late spermatid' fraction comprises late elongating spermatids, condensing spermatids and spermatozoa. Meiotic cells and spermatids were staged based on their appearance with DAPI.

 
To further appreciate the level of chromatin decondensation at those repeated snoRNA gene loci, we investigated the appearance of their FISH signals in hypercondensed chromatin of haploid spermatozoa and late spermatids. As illustrated in Fig. 6C,D, a substantial number of late spermatids harbor D* FISH signals (mostly doublet-like signals) with the two closely linked snoRD115 and snoRD116 gene loci relatively well resolved in some nuclei (Fig. 6Ca). Similar patterns of DNA FISH were observed in late spermatids at the Dlk1-Gtl2 domain (Fig. 6Cd), even though doublet-like FISH signals might appear less resolved. Consistent with our interpretation that DNA FISH signal appearance reflects the opening of chromatin, the proportion of doublet and triplet FISH signals increases in early, round spermatids known to have less condensed DNA (Fig. 6Cb,e), whereas in primary spermatocytes with highly decondensed chromatin (diploid cells undergoing recombination and pairing), the three repeated loci are always detected as D* patterns, with large string-like DNA FISH signals (up to 25 pinpoint signals per allele, Fig. 6Cc,f). Altogether, our ability to visualize D* signals in highly packaged chromatin, either on mitotic chromosomes or in late spermatids and/or spermatozoa, supports the notion that those imprinted gene loci might escape, to some extent, normal chromatin compaction during mitosis or spermiogenesis.


   Discussion
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The aim of this study was to better understand the metabolism of two long non-coding RNAs from the imprinted Snurf-Snrpn domain. We found that their intranuclear fate is reminiscent of RBII-36-HG/Bsr, another related non-coding transcript mapping at the imprinted rat Dlk1-Dio3 domain (Royo et al., 2007Go): these three spliced, repeated-exon containing mRNA-like transcripts accumulate near their transcription sites as large RNA grapes and as numerous stable punctuated foci dispersed in the nucleoplasm (Figs 2, 4). Consequently, this work identifies imprinted, C/D small RNA host-gene transcripts as members of the growing list of mammalian nuclear-retained RNAs.

Although the function and the cellular pathway underlying their nuclear retention have not yet been identified, we hypothesize that RNA dots are multimerized, or even single RNA molecules that are unable to enter the nucleocytoplasmic transport pathway. In this context, they might represent previously unidentified, subnuclear compartments wherein imprinted long non-coding RNA function and/or are stored. By analogy to two other repeated nuclear non-coding RNAs (hsr{omega} and sat III transcripts in Drosophila and in human cells, respectively) thought to be involved in the dynamics of RNA processing factors in heat-shocked cells (Jolly and Lakhotia, 2006Go), spliced snoRNA host-gene transcripts might associate specifically with proteins and serve as storage sites, regulating their intracellular availability depending on environmental cues and/or stress. One should note that RBII-36-HG has complex intracellular paths because it associates with nuclear poly(A)-rich domains and cytoplasmic stress granules in transcriptionally arrested neurons (Fig. 4) and in arsenite-treated fibroblasts, respectively (Royo et al., 2007Go). We are aware that drug treatments might have indirect effects on nuclear structures that are difficult to interpret. Thus, we cannot rule out the possibility that RNA dots are entrapped within structurally altered speckle domains.

To our best knowledge, this is the first time that the early intra-nuclear trafficking of two such closely linked transcripts has been reported. That RNA grapes do not intermingle deserves further comment as such an observation could be counterintuitive because it gives the impression that these two spliced mRNA-like species are delivered to distinct subnuclear environments and/or are tethered to different underlying nuclear structures that restrict their movement (Fig. 3). These interpretations might also appear at odds with the notion that RNP complexes travel through the nucleus by random, diffusion-like motion (Politz et al., 1999Go; Shav-Tal et al., 2004Go; Singh et al., 1999Go; Vargas et al., 2005Go), even though energy-dependent processes increase the nuclear mobility of macromolecular entities (Calapez et al., 2002Go; Carmo-Fonseca et al., 2002Go). Thus, our data show that freshly made spliced snoRNA host transcripts are compartmentalized and retained in the vicinity of their transcription sites. We wish to emphasize, however, that RNA dots away from their transcription sites are found randomly in the nucleoplasm, and thus only RNA species around their transcription sites seem to be prevented from diffusing. Highly localized accumulation of transcripts detached from their genes but in close proximity to their transcription sites, the so-called `RNA tracks', have been well documented. These observations have been incorporated into a paradigm whereby transcription, pre-mRNA metabolism and the early steps of intra-nuclear trafficking are spatially organized within the nucleus (Lawrence et al., 1989Go; Melcak et al., 2000Go; Shopland et al., 2002Go; Smith et al., 1999Go; Xing et al., 1993Go). Of notable interest, MyHC and dystrophin RNAs synthesized from two different chromosomes display a marked difference in their nuclear compartmentalization relative to speckle domains (inside and outside, respectively) and they do not intermingle even when they are extremely close (Smith et al., 1999Go). Given that snoRD115 and snoRD116 RNA grapes do not significantly enter speckle domains (Fig. 4), the putative underlying nuclear structure that might represent a physical barrier remains highly elusive.

An appealing hypothesis stipulates that RNA grapes, in conjunction with bound proteins and also perhaps through their highly repeated structure, might have the inherent ability to organize into cytological defined entities. Airn is able to form an RNA cloud that frequently envelops the paternally silenced Slc22a3 in placenta (Nagano et al., 2008Go) as well as poorly characterized RNA clusters in differentiated ES cells (Braidotti et al., 2004Go). By analogy to Airn and Kcnq1ot1 transcripts that recruit chromatin-modifying factors and create repressive nuclear compartments (Nagano et al., 2008Go; Pandey et al., 2008Go; Redrup et al., 2009Go; Terranova et al., 2008Go), it is tempting to speculate that RNA grapes made at Snurf-Snrpn and Dlk1-Dio3 imprinted domains play a role in transcriptional gene silencing, in particular silencing of the flanking, protein-coding genes at the 3' distal part of the domains, Ube3A and Dio3, respectively. These display reciprocal imprinted expression, and no allele-specific epigenetic marks have been described so far (Chamberlain and Brannan, 2001Go; da Rocha et al., 2008Go; Johnstone et al., 2006Go; Kishino, 2006Go; Rougeulle et al., 1998Go; Runte et al., 2001Go). More sophisticated experiments are now required to fully appreciate the potential function of this complex population of large imprinted ncRNAs.

Although allele-specific epigenetic marks (e.g. DNA methylation and histone modifications) have been extensively studied, our knowledge of higher-order chromatin organization at imprinted gene clusters is still limited. The paternal allele at the mouse Kcnq1 cluster displays a more contracted state than the maternal counterpart (Terranova et al., 2008Go), whereas no significant topological difference between parental alleles was detected at the human PWS domain (Mahy et al., 2002Go; Nogami et al., 2000Go; Rauch et al., 2008Go). Based on DNA FISH signals, we provide compelling evidence that the active paternal allele at the rodent snoRNA gene clusters exhibits a more decondensed state than the silent maternal allele (Fig. 5). To our best knowledge, such extended DNA FISH signals have never been reported at any other imprinted gene loci. Importantly, chromatin decondensation at those snoRNA gene arrays must be considered as an indication of potential transcription and not of transcription per se (Fig. 6A). That D* signals are still detected in highly packaged chromatin throughout mitosis and also in haploid late spermatids was rather unexpected. Extended DNA signals correlate with the methylation status of ICRs at the Dlk1-Gtl2 region and the snoRD115-116 region. It is, however, very unlikely that D* signals are faithfully inherited. First, in the male germ line, the two snoRNA regions with reciprocal imprinted expression harbor similar DNA FISH patterns (Fig. 6C,D). Second, the two daughter cells display different DNA FISH patterns in ~50% of cells in anaphases we examined (not shown). Altogether, these observations support the notion that genetic material is not homogenously packaged along mitotic chromosomes or within late spermatids, even though one cannot formally exclude that, under some circumstances, hybridization procedures might yield such pinpoint signals.

Visualization of different levels of chromatin compaction in interphase nuclei in relation to gene activity has been extensively reported for large artificial, repeated transgene arrays (Dietzel et al., 2004Go; Hu et al., 2009Go; Janicki et al., 2004Go; Muller et al., 2001Go; Tsukamoto et al., 2000Go; Tumbar et al., 1999Go). Interestingly, string-like DNA FISH signals described here (Fig. 5) are reminiscent of the beaded structures revealed at some reporter genes (Hu et al., 2009Go; Muller et al., 2001Go; Tsukamoto et al., 2000Go), suggesting that conclusions drawn from these artificial systems might be relevant for endogenously expressed, repeated arrays in their native chromatin context. We noticed with interest that beaded structures were visualized at two different non-repeated human, ~400 kb regions (MHCII and 22q) in their natural chromatin context (Muller et al., 2004Go), again strengthening the notion that beaded DNA FISH signals are common features of decondensed chromatin rather than simply due to the repeated structure or the imprinted status of snoRD genes. While this manuscript was under revision, LaSalle's group independently reported outstanding, neuronal allele-specific decondensation (highly reminiscent of our results) at the same two loci investigated by us (Leung et al., 2009Go). With 200 kb as an upper limit, we estimate a packing ratio at the snoRNA gene loci ranging from ~1/70 to ~1/30 for bead-like DNA FISH signals with for three and 6-7 pinpoint DNA signals, respectively. These values are consistent with snoRD gene loci being organized into a 30-nm chromatin fiber (30- to 40-fold compaction ratio) and thus differ from recent studies showing that active transcription occurs at a compaction ratio greater than expected for the 30-nm fiber (Hu et al., 2009Go). It is important to stress that decondensation at snoRD genes is not absolutely required, as exemplified by high expression level at S signals in neurons.

It has been shown that actively transcribed DNA sequences at the ~2 Mbase mammary tumor virus (MMTV) array are extruded from transcription sites as highly decondensed domains not detected by conventional DNA FISH protocols (Muller et al., 2007Go). Although we failed to obtain any experimental evidence for extended loops, whether snoRNA gene arrays reflect the same chromatin configuration as MMTV array remains an open question. This is an important issue because highly decondensed loops might provide, at least partially, some explanation regarding the structural organization of RNA grapes. In other words, newly spliced RNA species might not overlap because their transcribed DNA sequences are looped out from their transcription sites, and consequently they are more distant from each other than we anticipated on the basis of visible DNA FISH signals.

In addition to furthering our understanding of the spatial organization of gene expression, this study also provides insights into the relationship between the lack of expression of snoRD116 gene loci and a complex neurologic human disorder, the Prader-Willi syndrome (PWS) (Nicholls and Knepper, 2001Go). Indeed, most recent studies focused on snoRD116 genes as potential candidate genes (Ding et al., 2008Go; Gallagher et al., 2002Go; Sahoo et al., 2008Go; Schule et al., 2005Go; Skryabin et al., 2007Go; Wirth et al., 2001Go). However, it has never been demonstrated formally that PWS results from the lack of expression of snoRD166, or snoRD116-HG, or both. Our data show that snoRD116 and snoRD115 gene clusters most probably represent two independent transcription units generating metabolically stable nuclear RNA species in neurons, which opens new avenues for future studies to test the physiological roles of long, imprinted non-protein-coding RNA in PWS.

In conclusion, our study reveals that two evolutionarily distinct imprinted snoRNA gene clusters at the Dlk1-Dio3 and Snurf-Snrpn domains share common features: firts, they are sources of highly expressed, long nuclear-retained mRNA-like transcripts; and second, they exhibit an outstanding parental-specific chromatin organization that can be directly visualized at the light microscopy level. Future studies will unravel the hypothetical interplay between long non-coding RNA, control of genomic imprinting and higher-ordered chromatin organization in neurons and also perhaps in neurological diseases.


   Materials and Methods
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Cell cultures
Rat primary hypothalamic neurons were prepared from fetal Sprague Dawley rat hypothalami (E17) as previously described (Royo et al., 2007Go). The same method was used to prepare mouse primary hippocampal neurons from fetal mouse hippocampi (E18) except that they were cultured in neurobasal medium (Gibco) supplemented with B27, 5 mM glutamine and antibiotics (penicillin or streptomycin).

Fluorescence In situ hybridization (FISH), probe preparation and microscopes
For RNA FISH, neurons or fibroblasts were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 minutes at room temperature and permeabilized overnight at 4°C in 70% ethanol. RNA FISH was carried out overnight at 37°C in 15% formamide, 2x SSPE, 10% dextran sulfate, 150 µg/ml yeast tRNA and 10 ng oligo-probe (30 µl). Cells were washed at room temperature in 15% formamide, 2x SSPE (20 minutes, twice) and in 1x SSPE (10 minutes). For DNA-RNA FISH, coverslips were first heat-denaturated at 85°C for 12 minutes 30 seconds in 70% formamide, 2x SSPE, kept on ice (20 seconds), dehydrated in 70% ethanol (30 seconds), air dried (2 minutes) and hybridized overnight at 37°C. Cells were then washed in 15% formamide, 2x SSPE (20 minutes, room temperature), 50% formamide, 2x SSPE (20 minutes, room temperature) and in 0.1x SSPE (10 minutes, 50°C), prior to RNA FISH as described above. Coverslips were mounted in Moviol DAPI (0.1 µg/ml). Note that for DNA FISH a mixture of four oligonucleotides antisense to the template strand of DNA was used. Amino-allyl-modified oligonucleotides were labeled with fluorolink Cy3 (Amersham Biosciences, Piscataway, NJ), Cy5 (Amersham Biosciences), or Alexa Fluor 488 (Invitrogen) according to the manufacturer's instructions (their sequences are given in supplementary material Table S2). DNA FISH on MEFs was performed using BAC clones RP24-177J9 (snoRD-116 gene cluster) and RP24-571B24 (snoRD-115 gene cluster), labeled with ChromaTide Alexa-Fluor-568–5-dUTP (Molecular Probes) or aminoallyl-dUTP-ATTO-488 (Jena Bioscience) by random priming (Bioprime DNA Labeling System; Invitrogen) and hybridized using the same conditions described for oligo-probes. Images were captured with a CoolSnap ES camera (Roper Scientific, Tucson, AZ) mounted on a microscope (model DMRA, Leica, Deerfield, IL) with Leica 100x plan Apo 1.4. DNA FISH on germ cells were carried out as described (Mahadevaiah et al., 2009Go) (see below for further details) with biotin- or digoxigenin-labeled probes, prepared with a Biotin or a Digoxigenin Nick Translation Kit (Roche) on BACs RP24-177J9 (snoRD116 gene cluster), RP24-571B24 (snoRD115 gene cluster) and RP24-378G4 (12q cluster). Slides were mounted in Vectashield DAPI. Image acquisition was carried out on an inverted microscope (Ix70; Olympus) using a 100x1.35 U-PLAN-APO oil immersion objective (Olympus) and a computer-assisted (DeltaVision) CCD camera (CH350L; Photometrics).

DNA FISH on germ cells
Mouse testes were macerated using scalpel blades in RPMI medium and the resulting cell suspension was applied to boiled slides. Slides were covered in CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES, 0.5% Triton X-100, 1 mM EGTA and 2 mM vanadyl ribonucleoside, pH 6.8) for 10 minutes, and then in 4% paraformaldehyde (pH 7-7.4) for 10 minutes. We rinsed slides in PBS before dehydration through an ethanol series (2x 70%, 85%, 90%, 95%, 100%) and freezing at –20°C. Slides were rinsed in PBS and in 75°C 2x sodium citrate/sodium acetate buffer (SSC) before denaturation in 70% formamide, 2x SSC at 75°C for 3 minutes We transferred slides to ice-cold 70% ethanol and submitted them to a second ethanol series. For hybridization, we used biotin- or digoxigenin-labeled probes, prepared with a Biotin or a Digoxigenin Nick Translation Kit (Roche) on BACs RP24-177J9 (snoRD116 gene cluster), RP24-571B24 (snoRD115 gene cluster) and RP24-378G4 (12q cluster). Hybridization reactions consisted of 0.3 µg of labeled probe, 3 µg of labeled Cot-1 and 20 µg of salmon sperm DNA in 2x SSC, 10% dextran sulfate, 1 mg/ml BSA and 2 mM vanadyl ribonucleoside, and were carried out at 37°C overnight. We then subjected slides to stringency washes at 45°C in 2x SSC (4x 3 minutes) and at 60°C in 0.1x SSC (4x 3 minutes) and to blocking in (4x SSC, 5% casein) for 30 minutes at 37°C. Detection of biotin-coupled probes was performed by using streptavidin-Cy3 (1:100, 30 minutes at 37°C), followed by amplification using biotinylated antibody to streptavidin (1:100, 30 minutes at 37°C) and then one further round of streptavidin-Cy3 (1:100, 30 minutes at 37°C). Detection of digoxigenin-coupled probes was carried out with anti-DIG-FITC (1:10, 1 hour at 37°C). Slides were washed 3x 2 minutes in (4x SSC, 0.1% Tween) between each step.

RNA isolation, northern blot analysis, RT-PCR
To measure the spatiotemporal expression pattern of snoRD115 and snoRD116 (supplementary material Fig. S2), the following methods were used. Total RNA was prepared using Trizol (Invitrogen) according to the manufacturer's instructions and treated with RQ1 DNase (Promega) and proteinase K (Sigma). Human total RNAs were purchased from Ambion (The FirstChoice human total RNAsurvey panel, #AM6000). For RT-PCR analysis, 2 µg of total RNA was reverse-transcribed with random hexamerprimers, using Superscript II RTase (Invitrogen) at 42°C for 75 minutes, 1/10 of the RT reaction was amplified by PCR (cycles n=25 using GoTaq polymerase, Promega) using the oligonucleotides exon59, exon61, Par1for and Par1rev. For northern blot analysis, total RNA was fractionated by electrophoresis on a 6% acrylamide, 7 M urea denaturing gel. Electrotransfer was performed onto nylon membranes, followed by UV light irradiation. Northern blot hybridization was carried out with 5'-[32P]-labeled-DNA oligonucleotide probes, with an overnight incubation at 50°C in 5x SSPE, 0.1% SDS, 5x Denhardt's, 150 mg/ml yeast tRNA. Membranes were washed twice with 0.1% SSPE, 0.1% SDS at room temperature before autoradiography. The snoRD-115 and snoRD-116 were detected using the oligonucleotides snoRD-115rev and snoRD-116rev, respectively (supplementary material Table S2).


   Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/123/1/70/DC1

* These authors contributed equally to this work Back

{ddagger} Present address: Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK Back

§ Present address: Division of Stem Cell and Developmental Genetics, MRC NIMR, Mill Hill, London, NW7 1AA, UK Back

We thank Caroline Monod, Emmanuel Käs, Kerstin Bystriky and Stéphane Labialle for careful reading of the manuscript and for their continuous and helpful discussions. We thank James Turner and Dee Scadden for providing us with material and reagents during the revision of the manuscript. We are grateful to Guillaume Canal for help with the R software and Alexander Ludwig for advice in the preparation of mouse primary hippocampal neurons. We are very indebted to Juergen Brosius and Boris Skryabin for providing us with neurons prepared from a knockout mouse deleted for the paternal snoRD116 cluster. The modified oligonucleotide probes for RNA FISH were synthesized by J. Marc Escudier (`Plateforme de synthèse de l'Interface Chimie Biologie de l'ITAV'). This work was supported by grants from the European Union (STREP Prader Willi Syndrome) and from l'Agence Nationale de la Recherche (ANR blanche snosca).


   References
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 Summary
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 Results
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 References
 

Braidotti, G., Baubec, T., Pauler, F., Seidl, C., Smrzka, O., Stricker, S., Yotova, I. and Barlow, D. P. (2004). The Air noncoding RNA: an imprinted cis-silencing transcript. Cold Spring Harb. Symp. Quant. Biol. 69, 55-66.[Abstract/Free Full Text]

Calapez, A., Pereira, H. M., Calado, A., Braga, J., Rino, J., Carvalho, C., Tavanez, J. P., Wahle, E., Rosa, A. C. and Carmo-Fonseca, M. (2002). The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive. J. Cell Biol. 159, 795-805.[Abstract/Free Full Text]

Carmo-Fonseca, M., Platani, M. and Swedlow, J. R. (2002). Macromolecular mobility inside the cell nucleus. Trends Cell Biol. 12, 491-495.[CrossRef][Medline]

Cavaille, J., Buiting, K., Kiefmann, M., Lalande, M., Brannan, C. I., Horsthemke, B., Bachellerie, J. P., Brosius, J. and Huttenhofer, A. (2000). Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization. Proc. Natl. Acad. Sci. USA 97, 14311-14316.[Abstract/Free Full Text]

Cavaille, J., Vitali, P., Basyuk, E., Huttenhofer, A. and Bachellerie, J. P. (2001). A novel brain-specific box C/D small nucleolar RNA processed from tandemly repeated introns of a noncoding RNA gene in rats. J. Biol. Chem. 276, 26374-26383.[Abstract/Free Full Text]

Cavaille, J., Seitz, H., Paulsen, M., Ferguson-Smith, A. C. and Bachellerie, J. P. (2002). Identification of tandemly-repeated C/D snoRNA genes at the imprinted human 14q32 domain reminiscent of those at the Prader-Willi/Angelman syndrome region. Hum. Mol. Genet. 11, 1527-1538.[Abstract/Free Full Text]

Chamberlain, S. J. and Brannan, C. I. (2001). The Prader-Willi syndrome imprinting center activates the paternally expressed murine Ube3a antisense transcript but represses paternal Ube3a. Genomics 73, 316-322.[CrossRef][Medline]

Chaumeil, J., Le Baccon, P., Wutz, A. and Heard, E. (2006). A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced. Genes Dev. 20, 2223-2237.[Abstract/Free Full Text]

Clemson, C. M., McNeil, J. A., Willard, H. F. and Lawrence, J. B. (1996). XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132, 259-275.[Abstract/Free Full Text]

Clemson, C. M., Hutchinson, J. N., Sara, S. A., Ensminger, A. W., Fox, A. H., Chess, A. and Lawrence, J. B. (2009). An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol. Cell. 33, 717-726.[CrossRef][Medline]

da Rocha, S. T., Edwards, C. A., Ito, M., Ogata, T. and Ferguson-Smith, A. C. (2008). Genomic imprinting at the mammalian Dlk1-Dio3 domain. Trends Genet. 24, 306-316.[CrossRef][Medline]

Davis, B. M., McCurrach, M. E., Taneja, K. L., Singer, R. H. and Housman, D. E. (1997). Expansion of a CUG trinucleotide repeat in the 3' untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc. Natl. Acad. Sci. USA 94, 7388-7393.[Abstract/Free Full Text]

Dietzel, S., Zolghadr, K., Hepperger, C. and Belmont, A. S. (2004). Differential large-scale chromatin compaction and intranuclear positioning of transcribed versus non-transcribed transgene arrays containing beta-globin regulatory sequences. J. Cell Sci. 117, 4603-4614.[Abstract/Free Full Text]

Ding, F., Li, H. H., Zhang, S., Solomon, N. M., Camper, S. A., Cohen, P. and Francke, U. (2008). SnoRNA Snord116 (Pwcr1/MBII-85) deletion causes growth deficiency and hyperphagia in mice. PLoS ONE 3, e1709.[CrossRef][Medline]

Gallagher, R. C., Pils, B., Albalwi, M. and Francke, U. (2002). Evidence for the role of PWCR1/HBII-85 C/D box small nucleolar RNAs in Prader-Willi syndrome. Am. J. Hum. Genet. 71, 669-678.[CrossRef][Medline]

Gribnau, J., Hochedlinger, K., Hata, K., Li, E. and Jaenisch, R. (2003). Asynchronous replication timing of imprinted loci is independent of DNA methylation, but consistent with differential subnuclear localization. Genes Dev. 17, 759-773.[Abstract/Free Full Text]

Hall, L. L., Smith, K. P., Byron, M. and Lawrence, J. B. (2006). Molecular anatomy of a speckle. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 288, 664-675.[Medline]

Hu, Y., Kireev, I., Plutz, M., Ashourian, N. and Belmont, A. S. (2009). Large-scale chromatin structure of inducible genes: transcription on a condensed, linear template. J. Cell Biol. 185, 87-100.[Abstract/Free Full Text]

Hutchinson, J. N., Ensminger, A. W., Clemson, C. M., Lynch, C. R., Lawrence, J. B. and Chess, A. (2007). A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics 8, 39.[CrossRef][Medline]

Janicki, S. M., Tsukamoto, T., Salghetti, S. E., Tansey, W. P., Sachidanandam, R., Prasanth, K. V., Ried, T., Shav-Tal, Y., Bertrand, E., Singer, R. H. et al. (2004). From silencing to gene expression: real-time analysis in single cells. Cell 116, 683-698.[CrossRef][Medline]

Johnstone, K. A., DuBose, A. J., Futtner, C. R., Elmore, M. D., Brannan, C. I. and Resnick, J. L. (2006). A human imprinting centre demonstrates conserved acquisition but diverged maintenance of imprinting in a mouse model for Angelman syndrome imprinting defects. Hum. Mol. Genet. 15, 393-404.[Abstract/Free Full Text]

Jolly, C. and Lakhotia, S. C. (2006). Human sat III and Drosophila hsr omega transcripts: a common paradigm for regulation of nuclear RNA processing in stressed cells. Nucleic Acids Res. 34, 5508-5514.[Abstract/Free Full Text]

Jolly, C., Metz, A., Govin, J., Vigneron, M., Turner, B. M., Khochbin, S. and Vourc'h, C. (2004). Stress-induced transcription of satellite III repeats. J. Cell Biol. 164, 25-33.[Abstract/Free Full Text]

Kanduri, C. (2008). Functional insights into long antisense noncoding RNA Kcnq1ot1 mediated bidirectional silencing. RNA Biol. 5, 208-211.[Medline]

Kishino, T. (2006). Imprinting in neurons. Cytogenet. Genome Res. 113, 209-214.[CrossRef][Medline]

Koerner, M. V., Pauler, F. M., Huang, R. and Barlow, D. P. (2009). The function of non-coding RNAs in genomic imprinting. Development 136, 1771-1783.[Abstract/Free Full Text]

Komine, Y., Tanaka, N. K., Yano, R., Takai, S., Yuasa, S., Shiroishi, T., Tsuchiya, K., and Yamamori, T. (1999). A novel type of non-coding RNA expressed in the rat brain. Brain Res. Mol. Brain Res. 66, 1-13.[Medline]

Landers, M., Bancescu, D. L., Le Meur, E., Rougeulle, C., Glatt-Deeley, H., Brannan, C., Muscatelli, F. and Lalande, M. (2004). Regulation of the large (approximately 1000 kb) imprinted murine Ube3a antisense transcript by alternative exons upstream of Snurf/Snrpn. Nucleic Acids Res. 32, 3480-3492.[Abstract/Free Full Text]

Lawrence, J. B., Singer, R. H. and Marselle, L. M. (1989). Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57, 493-502.[CrossRef][Medline]

Lawrence, J. B., Singer, R. H. and McNeil, J. A. (1990). Interphase and metaphase resolution of different distances within the human dystrophin gene. Science 249, 928-932.[Abstract/Free Full Text]

Lemieux, N., Malfoy, B., Fetni, R., Muleris, M., Vogt, N., Richer, C. L. and Dutrillaux, B. (1994). In situ hybridization approach at infragenic level on metaphase chromosomes. Cytogenet. Cell Genet. 66, 107-112.[Medline]

Leung, K. N., Vallero, R. O., DuBose A. J., Resnick, J. L. and LaSalle, J. M. (2009). Imprinting regulates mammalian snoRNA-encoding chromatin decondensation and neuronal nucleolar size. Hum. Mol. Genet. 18, 4227-4238.[Abstract/Free Full Text]

Lewis, A. and Reik, W. (2006). How imprinting centres work. Cytogenet. Genome Res. 113, 81-89.[CrossRef][Medline]

Mahadevaiah, S. K., Costa, Y. and Turner, J. M. (2009). Using RNA FISH to study gene expression during mammalian meiosis. Methods Mol. Biol. 558, 433-444.[CrossRef][Medline]

Mahy, N. L., Perry, P. E. and Bickmore, W. A. (2002). Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH. J. Cell Biol. 159, 753-763.[Abstract/Free Full Text]

Mancini-Dinardo, D., Steele, S. J., Levorse, J. M., Ingram, R. S. and Tilghman, S. M. (2006). Elongation of the Kcnq1ot1 transcript is required for genomic imprinting of neighboring genes. Genes Dev. 20, 1268-1282.[Abstract/Free Full Text]

Melcak, I., Cermanova, S., Jirsova, K., Koberna, K., Malinsky, J. and Raska, I. (2000). Nuclear pre-mRNA compartmentalization: trafficking of released transcripts to splicing factor reservoirs. Mol. Biol. Cell 11, 497-510.[Abstract/Free Full Text]

Mercer, T. R., Dinger, M. E. and Mattick, J. S. (2009). Long non-coding RNAs: insights into functions. Nat. Rev Genet 10, 155-159.[CrossRef][Medline]

Mlynarczyk-Evans, S., Royce-Tolland, M., Alexander, M. K., Andersen, A. A., Kalantry, S., Gribnau, J. and Panning, B. (2006). X chromosomes alternate between two states prior to random X-inactivation. PLoS Biol. 4, e159.[CrossRef][Medline]

Mohammad, F., Mondal, T. and Kanduri, C. (2009). Epigenetics of imprinted long noncoding RNAs. Epigenetics 4, 277-286.[Medline]

Muller, W. G., Walker, D., Hager, G. L. and McNally, J. G. (2001). Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter. J. Cell Biol. 154, 33-48.[Abstract/Free Full Text]

Muller, W. G., Rieder, D., Kreth, G., Cremer, C., Trajanoski, Z. and McNally, J. G. (2004). Generic features of tertiary chromatin structure as detected in natural chromosomes. Mol. Cell. Biol. 24, 9359-9370.[Abstract/Free Full Text]

Muller, W. G., Rieder, D., Karpova, T. S., John, S., Trajanoski, Z. and McNally, J. G. (2007). Organization of chromatin and histone modifications at a transcription site. J. Cell Biol. 177, 957-967.[Abstract/Free Full Text]

Nagano, T., Mitchell, J. A., Sanz, L. A., Pauler, F. M., Ferguson-Smith, A. C., Feil, R. and Fraser, P. (2008). The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322, 1717-1720.[Abstract/Free Full Text]

Nicholls, R. D. and Knepper, J. L. (2001). Genome organization, function, and imprinting in Prader-Willi and Angelman syndromes. Annu. Rev. Genomics Hum. Genet. 2, 153-175.[CrossRef][Medline]

Nogami, M., Kohda, A., Taguchi, H., Nakao, M., Ikemura, T. and Okumura, K. (2000). Relative locations of the centromere and imprinted SNRPN gene within chromosome 15 territories during the cell cycle in HL60 cells. J. Cell Sci. 113 (Pt 12), 2157-2165.[Abstract]

Pandey, R. R., Mondal, T., Mohammad, F., Enroth, S., Redrup, L., Komorowski, J., Nagano, T., Mancini-Dinardo, D. and Kanduri, C. (2008). Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol. Cell 32, 232-246.[CrossRef][Medline]

Pauler, F. M., Koerner, M. V. and Barlow, D. P. (2007). Silencing by imprinted noncoding RNAs: is transcription the answer? Trends Genet. 23, 284-292.[CrossRef][Medline]

Politz, J. C., Tuft, R. A., Pederson, T. and Singer, R. H. (1999). Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells. Curr. Biol. 9, 285-291.[CrossRef][Medline]

Ponting, C. P., Oliver, P. L. and Reik, W. (2009). Evolution and functions of long noncoding RNAs. Cell 136, 629-641.[CrossRef][Medline]

Prasanth, K. V. and Spector, D. L. (2007). Eukaryotic regulatory RNAs: an answer to the `genome complexity' conundrum. Genes Dev. 21, 11-42.[Abstract/Free Full Text]

Prasanth, K. V., Rajendra, T. K., Lal, A. K. and Lakhotia, S. C. (2000). Omega speckles-a novel class of nuclear speckles containing hnRNPs associated with noncoding hsr-omega RNA in Drosophila. J. Cell Sci. 113 Pt 19, 3485-3497.[Abstract]

Prasanth, K. V., Prasanth, S. G., Xuan, Z., Hearn, S., Freier, S. M., Bennett, C. F., Zhang, M. Q. and Spector, D. L. (2005). Regulating gene expression through RNA nuclear retention. Cell 123, 249-263.[CrossRef][Medline]

Rauch, J., Knoch, T. A., Solovei, I., Teller, K., Stein, S., Buiting, K., Horsthemke, B., Langowski, J., Cremer, T., Hausmann, M. et al. (2008). Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei. Differentiation 76, 66-82.[Medline]

Redrup, L., Branco, M. R., Perdeaux, E. R., Krueger, C., Lewis, A., Santos, F., Nagano, T., Cobb, B. S., Fraser, P. and Reik, W. (2009). The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing. Development 136, 525-530.[Abstract/Free Full Text]

Reik, W. and Walter, J. (2001). Genomic imprinting: parental influence on the genome. Nat. Rev. Genet. 2, 21-32.[Medline]

Rinn, J. L., Kertesz, M., Wang, J. K., Squazzo, S. L., Xu, X., Brugmann, S. A., Goodnough, L. H., Helms, J. A., Farnham, P. J., Segal, E. et al. (2007). Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311-1323.[CrossRef][Medline]

Rougeulle, C., Cardoso, C., Fontes, M., Colleaux, L. and Lalande, M. (1998). An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nat. Genet. 19, 15-16.[Medline]

Royo, H. and Cavaille, J. (2008). Non-coding RNAs in imprinted gene clusters. Biol. Cell 100, 149-166.[CrossRef][Medline]

Royo, H., Basyuk, E., Marty, V., Marques, M., Bertrand, E. and Cavaille, J. (2007). Bsr, a nuclear-retained RNA with monoallelic expression. Mol. Biol. Cell 18, 2817-2827.[Abstract/Free Full Text]

Runte, M., Huttenhofer, A., Gross, S., Kiefmann, M., Horsthemke, B. and Buiting, K. (2001). The IC-SNURF-SNRPN transcript serves as a host for multiple small nucleolar RNA species and as an antisense RNA for UBE3A. Hum. Mol. Genet. 10, 2687-2700.[Abstract/Free Full Text]

Sahoo, T., del Gaudio, D., German, J. R., Shinawi, M., Peters, S. U., Person, R. E., Garnica, A., Cheung, S. W. and Beaudet, A. L. (2008). Prader-Willi phenotype caused by paternal deficiency for the HBII-85 C/D box small nucleolar RNA cluster. Nat. Genet. 40, 719-721.[CrossRef][Medline]

Sasaki, Y. T., Ideue, T., Sano, M., Mituyama, T. and Hirose, T. (2009). MEN{epsilon}/β noncoding RNAs are essential for structural integrity of nuclear paraspeckles. Proc. Natl. Acad. Sci. USA 8, 2525-2530.

Schule, B., Albalwi, M., Northrop, E., Francis, D. I., Rowell, M., Slater, H. R., Gardner, R. J. and Francke, U. (2005). Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome. BMC Med. Genet. 6, 18.[CrossRef][Medline]

Shav-Tal, Y., Darzacq, X., Shenoy, S. M., Fusco, D., Janicki, S. M., Spector, D. L. and Singer, R. H. (2004). Dynamics of single mRNPs in nuclei of living cells. Science 304, 1797-1800.[Abstract/Free Full Text]

Shopland, L. S., Johnson, C. V. and Lawrence, J. B. (2002). Evidence that all SC-35 domains contain mRNAs and that transcripts can be structurally constrained within these domains. J. Struct. Biol. 140, 131-139.[CrossRef][Medline]

Siepel, A., Bejerano, G., Pedersen, J. S., Hinrichs, A. S., Hou, M., Rosenbloom, K., Clawson, H., Spieth, J., Hillier, L. W., Richards, S. et al. (2005). Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034-1050.[Abstract/Free Full Text]

Singh, O. P., Bjorkroth, B., Masich, S., Wieslander, L. and Daneholt, B. (1999). The intranuclear movement of Balbiani ring premessenger ribonucleoprotein particles. Exp. Cell Res. 251, 135-146.[CrossRef][Medline]

Skryabin, B. V., Gubar, L. V., Seeger, B., Pfeiffer, J., Handel, S., Robeck, T., Karpova, E., Rozhdestvensky, T. S. and Brosius, J. (2007). Deletion of the MBII-85 snoRNA gene cluster in mice results in postnatal growth retardation. PLoS Genet. 3, e235.[CrossRef][Medline]

Sleutels, F., Zwart, R. and Barlow, D. P. (2002). The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 415, 810-813.[Medline]

Smith, K. P., Moen, P. T., Wydner, K. L., Coleman, J. R. and Lawrence, J. B. (1999). Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific. J. Cell Biol. 144, 617-629.[Abstract/Free Full Text]

Smith, K. P., Byron, M., Johnson, C., Xing, Y. and Lawrence, J. B. (2007). Defining early steps in mRNA transport: mutant mRNA in myotonic dystrophy type I is blocked at entry into SC-35 domains. J. Cell Biol. 178, 951-964.[Abstract/Free Full Text]

Sone, M., Hayashi, T., Tarui, H., Agata, K., Takeichi, M. and Nakagawa, S. (2007). The mRNA-like noncoding RNA Gomafu constitutes a novel nuclear domain in a subset of neurons. J. Cell Sci. 120, 2498-2506.[Abstract/Free Full Text]

Sunwoo, H., Dinger, M. E., Wilusz, J. E., Amaral, P. P., Mattick, J. S. and Spector, D. L. (2009). MEN {epsilon}/β nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and are essential components of paraspeckles. Genome Res. 5, 668-679.

Terranova, R., Yokobayashi, S., Stadler, M. B., Otte, A. P., van Lohuizen, M., Orkin, S. H. and Peters, A. H. (2008). Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos. Dev. Cell 15, 668-679.[CrossRef][Medline]

Tsukamoto, T., Hashiguchi, N., Janicki, S. M., Tumbar, T., Belmont, A. S. and Spector, D. L. (2000). Visualization of gene activity in living cells. Nat. Cell Biol. 2, 871-878.[CrossRef][Medline]

Tumbar, T., Sudlow, G. and Belmont, A. S. (1999). Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain. J. Cell Biol. 145, 1341-1354.[Abstract/Free Full Text]

Vargas, D. Y., Raj, A., Marras, S. A., Kramer, F. R. and Tyagi, S. (2005). Mechanism of mRNA transport in the nucleus. Proc. Natl. Acad. Sci. USA 102, 17008-17013.[Abstract/Free Full Text]

Wilusz, J. E., Freier, S. M. and Spector, D. L. (2008). 3' end processing of a long nuclear-retained noncoding RNA yields a tRNA-like cytoplasmic RNA. Cell 135, 919-932.[CrossRef][Medline]

Wirth, J., Back, E., Huttenhofer, A., Nothwang, H. G., Lich, C., Gross, S., Menzel, C., Schinzel, A., Kioschis, P., Tommerup, N. et al. (2001). A translocation breakpoint cluster disrupts the newly defined 3' end of the SNURF-SNRPN transcription unit on chromosome 15. Hum. Mol. Genet. 10, 201-210.[Abstract/Free Full Text]

Xing, Y., Johnson, C. V., Dobner, P. R. and Lawrence, J. B. (1993). Higher level organization of individual gene transcription and RNA splicing. Science 259, 1326-1330.[Abstract/Free Full Text]


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