Article

• The EMBO Journal (2006) 25, 3565 - 3575
• doi:10.1038/sj.emboj.7601245

Published online: 27 July 2006

• Subject Categories:

  • Chromatin and Transcription | RNA

The imprinted Air ncRNA is an atypical RNAPII transcript that evades splicing and escapes nuclear export

Christine IM Seidl^a, Stefan H Stricker^a and Denise P Barlow

1. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, c/o Institute of Genetics, Max F Perutz Laboratories, Vienna Biocenter, Dr Bohr-Gasse 9/4, A-1030 Vienna, Austria

Correspondence to:

Denise P Barlow, CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, c/o Institute of Genetics, Max F Perutz Laboratories, Vienna Biocenter, Dr Bohr-Gasse 9/4, 1030 Vienna, Austria. Tel.: +43 1 4277 54 610; Fax: +43 1 4277 9546; E-mail: denise.barlow@univie.ac.at

^aThese authors contributed equally to this work

Received 4 April 2006; Accepted 28 June 2006

• Keywords:

  • genomic imprinting,
  • ncRNA,
  • nuclear export,
  • RNA stability,
  • transcription interference

Introduction

A surprisingly large number of noncoding RNAs (ncRNAs) that have been suggested to constitute a new epigenetic regulatory system have recently been identified by diverse transcriptome analyses in mammalian cells (Mendes Soares and Valcarcel, 2006). Functional classification of these ncRNAs has not yet started but categories with known gene regulatory functions include short or micro-ncRNAs that participate in RNA interference and post-transcriptional regulatory pathways, intergenic transcripts that regulate local chromatin activity, cis-acting long ncRNAs such as Xist involved in chromosome inactivation and the imprinted Air and Kcnq1ot1 ncRNAs involved in domain silencing (Heard, 2004; Pauler and Barlow, 2006).

The Air ncRNA regulates genomic imprinting of a cluster of autosomal genes on mouse chromosome 17 (Braidotti et al, 2004). Many details of the epigenetic mechanism regulating genomic imprinting in mammals have been described, and it is now appreciated that most imprinted genes are found in clusters that contain at least one long ncRNA (Verona et al, 2003). In a typical mammalian imprinted gene cluster of which there are six well-characterized examples, the expression of multiple imprinted protein-coding genes is restricted to one parental chromosome, whereas the other parental chromosome silences these mRNA genes and instead expresses an ncRNA. Thus, imprinted ncRNAs show discordant expression in cis with imprinted protein-coding mRNA genes, indicating that they may act as silencers. Of the three imprinted ncRNAs so far tested, a silencing function has been shown for the Air and Kcnq1ot1 ncRNAs (Sleutels et al, 2002; Mancini-Dinardo et al, 2006). The H19 ncRNA, however, does not act as a silencer, despite showing a similar discordant expression pattern (Schmidt et al, 1999). Although a direct role for other imprinted ncRNAs has not yet been tested, deletion of the ncRNA promoters in three other imprinted clusters is associated with loss of silencing of cis-linked imprinted mRNA genes, indicating that these ncRNAs may also possess silencing functions (Chamberlain and Brannan, 2001; Lin et al, 2003; Williamson et al, 2006).

Targeted manipulations in mice have shown that expression of the 108 kb long Air ncRNA exerts a silencing effect in cis on three protein-coding genes Igf2r, Slc22a2 and Slc22a3, spread over 300 kb (Sleutels et al, 2002). Thus, Air exerts a domain silencing effect (Figure 1A). It is not yet known if Air is needed to induce silencing, or acts at a later stage to maintain the silenced state. It is also unknown if silencing requires the Air RNA itself or, merely, the act of Air transcription through its 108 kb locus. The Air promoter is located in the second intron of Igf2r and transcription occurs only on the paternal chromosome in antisense orientation through the Igf2r promoter, terminating 108 kb downstream in the last intron of the next flanking gene, Mas1 (Figure 1A and B).

Figure 1.

The Air ncRNA gene and promoter. (A) Map showing the position and orientation of Air ncRNA relative to flanking genes. The Air promoter lies in Igf2r intron 2 and the 3'-end in the last Mas1 intron. Arrows: expressed genes; P: paternal allele; M: maternal allele; ^*: Air promoter. (B) Promoter organization of Air. Stripes indicate the Air CpG island (http://www.ebi.ac.uk/emboss/cpgplo
t/), T1–3: transcription starts; SD: splice donor site described in Figure 5. Consensus sites are indicated for AP1, AP2, SP1, Myc, GC-box (GCB) and a TATA sequence. (C) Steady-state levels of Igf2r and Air determined by RPA in cells and adult mouse using probe F3B (Air), EX46/47 (Igf2r): 1: probe+yeast RNA-RNase; 2: size marker; 3: probe+yeast RNA+RNase; 4: adult brain; 5: adult liver; 6: adult kidney; 7: adult lung; 8: adult heart; 9: NIH3T3. CypA (loading control). ^*Undigested probe. (D) QPCR (with q-assay Air middle) showing different Air transcript levels in adult mouse kidney (Ki), adult mouse heart (He), mouse placenta (Pl) 11.5 dpc and 16.5 dpc. Air transcript levels from four biological replicates were normalized to 18S rRNA and levels in kidney were set to 1; the numbers below the bars indicate transcript levels relative to kidney. IE: imprinted expression; BE: biallelic expression; NE: not expressed.

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The mature Air transcript is polyadenylated but, in contrast to most mature transcripts, is rich in interspersed repeats and appears to have few or no introns (Lyle et al, 2000). The other two genes silenced by Air (Slc22a2 and Slc22a3) do not overlap nor share sequence homology of single-copy DNA with Air. Silencing by Air is also regulated in a tissue- and developmental-specific manner in the brain and placenta, the basis of which is unknown (Sleutels et al, 2002; Yamasaki et al, 2005). The Air promoter is silent on the maternal chromosome that expresses the three protein-coding genes and carries a DNA methylation imprint that is established during oocyte development (Stoger et al, 1993). In agreement with the finding that expression of Air silences genes on the paternal chromosome, we can reason that DNA methylation silences the Air promoter on the maternal chromosome to allow expression of the protein-coding genes. In support of this, it has been shown that mouse embryos lacking DNA methylation repress Igf2r on both parental chromosomes (Li et al, 1993). However, the effect of DNA methylation on Air expression has not yet been directly tested.

Although it is not yet known how Air expression leads to gene silencing, one model that is based on similarities between genomic imprinting and X-chromosome inactivation (XCI) in mammals is receiving much attention. These similarities arise from the observation that both imprinting and XCI use an epigenetic cis-acting silencing mechanism dependent on an ncRNA (named Xist in XCI), that XCI is also an imprinted phenomenon in marsupials and in extra-embryonic mouse tissues, and that both imprinting and XCI share a similar evolutionary distribution (Reik and Lewis, 2005). If the Air ncRNA performed a function similar to that of the Xist transcript in XCI, it would be expected to coat the 300 kb region containing the silenced genes and to recruit repressive chromatin as has been described for the action of Xist (Heard, 2004). The small size of the genomic region silenced by the Air ncRNA precludes much of the immunofluorescent analyses that have been performed for the Xist RNA to show recruitment of repressive chromatin to the whole 180 Mbp X chromosome. Conversely, the large size of the Air RNA makes it laborious to define functional regions along its 108 kb length as has been performed for the 17.9 kb Xist RNA (Wutz et al, 2002).

A more detailed understanding of the transcriptional and post-transcriptional features of the Air ncRNA may, however, help to distinguish which model of Air-mediated gene silencing is operating. Here, we report that the Air promoter is transcribed by RNA polymerase II (RNAPII) in a DNA methylation-sensitive manner and, similar to other RNAPII transcripts, has a 7mGcap. However, in contrast to most RNAPII transcripts, we find that Air is inefficiently spliced such that most nascent transcripts constitute the unspliced mature 108 kb form of this ncRNA. The unspliced 108 kb Air ncRNA is highly unstable relative to the Igf2r mRNA and is not exported to the cytoplasm, whereas the spliced Air variants have a similar stability to the Igf2r mRNA and are exported. The data presented here show that Air is a new member of the nuclear regulatory RNA family, whose properties indicate that its ability to silence genes in cis may not depend on the RNA itself but, instead, may be related to transcription through its 108 kb locus.

Expression and abundance of the Air ncRNA

The organization of the imprinted Igf2r cluster and details of the Air promoter are shown in Figure 1A and B. RNase protection assays (RPA) were used to determine the steady-state levels of Igf2r and Air in adult mouse tissues and cell lines expressing different levels of these two genes. A multiprobe RPA was performed that simultaneously detected Air, Igf2r and cyclophilin A (CypA) transcripts. Air could be detected in all tissues examined, but was consistently less abundant than Igf2r in all tested tissues (Figure 1C). The levels of Air relative to Igf2r varied between different tissues and cell types from 17% (adult kidney) to 67% (adult lung) (Figure 1C, lanes 6 and 7). Similar ratios were found using two different Air and Igf2r probes (data not shown). The reason for the differing ratios of Igf2r/Air in different cell types is not clear but may reflect single cell variation between cells that lack Air and express Igf2r biallelically, and cells that show imprinted expression of Air and Igf2r, as recently reported for mouse brain (Yamasaki et al, 2005). Figure 1D shows that the abundance of Air does not correlate with its silencing effects on genes within the Igf2r cluster. Air is equally abundant in the heart and 11.5 dpc placenta. However, only Igf2r shows imprinted expression in the heart, whereas Igf2r, Slc22a2 and Slc22a3 show imprinted expression in 11.5 dpc placenta (Sleutels et al, 2002). In 16.5 dpc placenta, only Igf2r and Slc22a2 show imprinted expression but Air expression is three-fold higher compared to 11.5 dpc placenta.

The multiple transcriptional start sites (T1–3; Figure 1B) of the Air ncRNA are located at the 5'-border of a CpG island. The CpG island is unmethylated on the paternal allele that expresses Air and methylated on the maternal chromosome that lacks Air expression. To test if DNA methylation represses Air on the maternal chromosome, we analysed embryos, which were wild-type, heterozygous or homozygous for a null mutation of Dnmt1 (Li et al, 1993). Figure 2 shows the expression levels of four imprinted genes. The two protein-coding genes Igf2r and Igf2 show reduced expression in Dnmt1 null embryos compared to heterozygotes as previously described (Li et al, 1993). Igf2 mRNA levels are reduced to background in this assay, whereas Igf2r mRNA levels are reduced to 26% of controls. In contrast, the two ncRNAs Air and H19 show more than two-fold increased expression in Dnmt1 null embryos compared to heterozygotes. The increase in H19 ncRNA level has previously been shown to occur from re-expression of the normally silent paternal H19 promoter (Li et al, 1993). These results show that the Air ncRNA promoter is repressed by DNA methylation in mouse embryos.

Figure 2.

The Air ncRNA promoter is repressed by DNA methylation. Expression of Igf2r (q-assay ex48), Air (q-assay Air middle), Igf2 (q-assay Igf2) and H19 (q-assay H19) was assayed by qPCR in E9.5 embryo cDNAs from one litter containing one wild-type (WT), six heterozygotes and three homozygotes for a Dnmt1 null mutation (Li et al, 1993). RNA from individual embryos was assayed separately and used to calculate the standard deviation. Values are normalized to Gapdh. Results from WT and heterozygous embryos were similar and were pooled and set to 1. Igf2r and Igf2 mRNAs show a reduction in Dnmt1 null embryos of 0.26 and 0.02 (arrow), respectively. Air and H19 ncRNAs show enrichments of 2.89 and 2.54, respectively.

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Air is a non-coding RNAPII transcript

To determine which RNAP transcribes Air, we made use of -amanitin, which inhibits RNAPII at low (5 g/ml) concentrations. RNAPIII can also be inhibited by high concentrations, whereas RNAPI remains unaffected by -amanitin. Figure 3A shows that at 5 g/ml -amanitin, the RNAPII-transcribed Myc gene is fully repressed at 24 h, whereas the RNAPIII 5S RNA promoter is not affected. The same RNA preparation analysed in Figure 3A was then used to quantify Air by qPCR, which shows that Air levels decrease to 13–30% by 24 h treatment (Figure 3B). This indicates that the Air ncRNA is transcribed by RNAPII.

Figure 3.

The Air ncRNA is an RNAPII transcript. (A) RNA blot of NIH3T3 cells exposed to -amanitin (-am). Total cell RNA from control (-) and poisoned (+) cells was hybridized with the RNAPII transcript Myc and the RNAPIII transcript 5S RNA (probes are listed in Supplementary data). One of three biological replicates is shown demonstrating inhibition of RNAPII but not RNAPIII transcription at 24 h. Methylene blue staining of the 28S and 18S rRNA bands is shown underneath as loading control, as RNAPI is not affected by -am. (B) Air expression analysed by qPCR (q-assay Air middle) normalized to 18S rRNA shows reduced Air expression in all three biological replicates (BR). The untreated control was set to 100%.

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We next investigated if the Air ncRNA is modified with a methyl-7-guanosine (7mGcap) characteristic of RNAPII transcripts. We used a monoclonal antibody to immunoprecipitate capped RNAs using the capped Igf2r mRNA as a positive control, and the uncapped ribosomal 18S rRNA as a negative control to monitor unspecific binding of RNA to the antibody. Figure 4A shows that Air and Igf2r are found in the immunoprecipitated fraction, whereas the 18S rRNA is not. The efficiency of immunoprecipitation (IP) of Air and Igf2r relative to 18S rRNA shows a large variation for the three biological replicates (Figure 4B). However, control reactions lacking antibody or with an unrelated antibody of the same IgG isotype (Figure 4C and D) consistently yielded only a low level of unspecific bound RNA. Therefore, these results demonstrate that the Air ncRNA carries a 7mGcap.

Figure 4.

The Air ncRNA bears a 7mGcap. (A) qPCR of RNA immunoprecipitated (IP) by the cap-specific antibody H20. RNA from four fractions from biological replicate 2 (BR2), supernatant (SN), first wash (1W), fifth wash (5W) and antibody-bound RNA (IP), was analysed by qPCR for Air (white bars, q-assay Air middle), Igf2r (light grey bars, q-assay ex48) and 18S (dark grey bars, q-assay 18S). The SN fraction was set to 1 and the other fractions are displayed as relative enrichment/reduction. Arrows indicate a negligible value. (B) Ratio of IP RNA to RNA in the supernatant (IP/SN) is shown for Air and Igf2r for three biological replicates relative to the same ratios for 18S rRNA. (C) Control reactions for beads only without antibody (Mock-IP) used to monitor unspecific binding to Sepharose G-beads. (D) IP with an unrelated antibody of the same isotype (IgG-IP) used to determine unspecific binding of RNA to the IgG epitope.

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Air has reduced splicing potential

Air was initially reported as an unspliced RNA (Lyle et al, 2000). To investigate whether any Air transcripts are spliced, EST databases were scanned for discontinuous alignments to the Air sequence. A total of 204 EST sequences were found and of these, 13% showed a discontinuous alignment indicating that they spanned an intron. A total of 13 independent clones were identified that showed the same orientation as the Air ncRNA and were classified into five groups (splice variant (SV)1 (DQ275617), SV1a (DQ275618), SV2 (DQ275619), SV3 (DQ275620), SV4) whose organization and general features are shown in Figure 5A and B. All splice variants except SV4, whose 5'-end is missing, have their 5'-end close to the Air main transcription start (T1) and all use the same splice donor 53 bp downstream of T1 (SD; Figure 1B). Therefore, all splice variants share the first exon.

Figure 5.

Air has reduced splicing potential. (A) Map showing relative positions Air, Igf2r and Mas1. Indicated below are full-length unspliced Air and five groups of spliced variants (SV1, SV1a, SV2, SV3, SV4) that share the same 5'-splice donor 53 bp downstream of the transcriptional start. Black bars: exons; grey lines: introns; locations of primers (one forward primer (FP) combined with different reverse primers (RP)) and Taqman probe (TP) used for qPCR are shown underneath. (B) Summary of spliced Air EST clones found in databases (www.ensembl.org and http://www.ncbi.nlm.nih.gov/BLAST). n: number of clones; bp: length of SV; Rep: % repeats determined by http://www.repeatmasker.org/; ORF: length of longest open reading frame. (C–G) qPCR assays showing relative expression levels (normalized to cyclophilin q-assay CypA ex3/4) in adult mouse tissues of unspliced (C, q-assay Air 5') and spliced (D-G q-assays SV1, SV1a, SV2, SV3) Air ncRNA. Te: testis; Br: brain; Ki: kidney; Lu: lung; He: heart. (H) RNA prepared from cell lines and tissues was analysed by RPA with probe MlMs1 spanning the multiple Air transcription start sites showing spliced and unspliced Air transcripts. 1: size marker; 2: probe+yeast RNA-RNase; 3: probe +yeast RNA+RNase; 4: Thp/DB104 MEFs; 5: DB104/Thp MEFs; 6: MEFF Thp/+ cells; 7: NIH3T3 cells; 8: adult heart from wild-type mice. The maternal allele is written on the left side. See Materials and methods for details of cells. Transcription starts for unspliced Air: T1, T2 and T3; for spliced Air: SVT1, SVT2 and SVT3. Protected bands for SVT2 and SVT3 are visible on original exposures. Using bands marked by an asterisk in lane 4, the abundance of spliced Air is 23–44% that of unspliced Air in lanes 4, 6, 7 and 8.

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Splice variants SV1 and SV1a share a common downstream exon, whereas the remaining variants have different downstream exons. None of the splice variants end at the same polyadenylation site identified for full-length unspliced Air. The 3'-ends of SV1 and SV3 are A-rich and these clones may have been obtained by oligo-d(T) priming of internal stretches of multiple A's. However, SV2 and SV4 both end in a region without obvious enrichment of A's. It is notable that one splice variant (SV3) has its transcription end more than 17 kb downstream of the mapped 3'-end of the full-length unspliced Air (at 929 bp in sequence AJ249895). The existence of the previously described polyadenylation site (18 464 bp in AJ249895) was confirmed in mouse tissues and cells used here (data not shown) (Lyle et al, 2000). The lengths of the spliced variants range from 500 to 1392 bp and the longest open reading frames range from 105 to 339 bp; however, all contain interspersed repeats (12–77%) (Figure 5B). This indicates that these spliced variants are likely to be ncRNAs.

The relative expression levels of unspliced and spliced Air transcripts were determined in organs that show imprinted Igf2r expression (kidney, lung, heart) and in organs that partly lack imprinted expression (testis, brain). Figure 5C–G shows that similar relative amounts of unspliced and spliced Air are found in all tested tissues, indicating a lack of correlation between splicing of Air and loss of Igf2r imprinted expression. We used RPA to quantify the proportion of Air that is spliced (Figure 5H), using a probe that overlaps the three known transcriptional start sites and the common 53 bp splice donor. Both unspliced and spliced Air are only expressed from the paternal (Figure 5H, lanes 4 and 6) and not from the maternal chromosome (lane 5). As all splice variants use the same exon 1 splice donor, the RPA probe detects the sum of splice variants (bands labelled SV in Figure 5H). All three transcriptional start sites could be detected for unspliced (T1, T2, T3) and spliced Air ncRNA (SV^T1, SV^T2, SV^T3). However, the main transcriptional start site for the spliced variants (SV^T1) is seen as three separate bands and the second start site SV^T2 is also seen as two faint bands on the original image, which indicates that these start sites are used with loose stringency. The abundance of steady-state spliced relative to unspliced Air was determined as between 23 and 44% (Figure 5H).

The Air ncRNA is not exported to the cytoplasm

Nuclear, cytoplasmic and total cellular RNAs were analysed to test cellular localization of Air. Figure 6 shows the result of two experiments using a qPCR assay and RNA blots. In the qPCR assays, all values are normalized to CypA and expressed as enrichment relative to this mRNA and for each histogram the value in the total cellular RNA is set to 1. Thus, an mRNA localized to the cytoplasm such as Gapdh shows no enrichment relative to CypA, and cytoplasmic and nuclear levels approximately equal 1 (Figure 6A). In contrast, the nuclear-localized 45S rRNA shows a mean nuclear/cytoplasmic ratio of 621:1 (Figure 6A). The imprinted H19 ncRNA shows a mean nuclear/cytoplasmic ratio of 1.0:1, confirming a previous report that it is exported to the cytoplasm (Brannan et al, 1990) (Figure 6A). Analysis of the unspliced Air ncRNA in these same samples shows a mean nuclear/cytoplasmic ratio of 55:1, indicating that unspliced Air is nuclear localized (Figure 6B). The nuclear localization of Air can also be visualized by RNA blots, although this type of analysis is not efficient owing to the degradation of the 108 kb RNA (Figure 6C).

Figure 6.

The unspliced Air ncRNA is not exported to the cytoplasm. qPCR assay of nuclear (N), cytoplasmic (C) and total cell (T) RNAs. Bars show values for two biological replicates normalized to CypA. The value in total RNA (T) is set to 1 (asterisk). Cyclophilin (CypA) is a known cytoplasmic mRNA. Other cytoplasmic mRNAs show the same distribution as CypA and show no enrichment in N and C fractions. (A) Distribution of control RNAs. Gapdh (q-assay Gapdh ex5) shows no enrichment in N and C relative to CypA (q-assay CypA ex3/4) and is located in the cytoplasm; 45S pre-rRNA (q-assay 45S pre-RNA) shows N/C ratios of 342/1 and 901/1 and is located in the nucleus; H19 ncRNA (q-assay H19) shows N/C ratios of 0.9/1 and 1.1/1 and is located in the cytoplasm. (B) Distribution of unspliced Air (q-assay Air middle) shows N/C ratios of 30/1 and 80/1 and is located in the nucleus. Igf2r (q-assay ex48) shows N/C ratios of 2.4/1 and 2.1/1 and is exported to the cytoplasm but also present in the nucleus. (C) RNA blot of samples analysed in panel B showing the distribution of Gapdh, CypA, Myc, Igf2r, Air (probes listed in Supplementary data) and rRNA (methylene blue staining). (D) Air splice variants are exported to the cytoplasm and show similar N/C ratios as Igf2r (q-assay SV1: 6.8/1 and 6.4/1; q-assay SV1a: 3.0/1 and 2.4/1; q-assay SV2: 3.0/1 and 2.8/1; q-assay SV3: 2.8/1 and 2.2/1).

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Igf2r mRNA assayed in the same RNA samples shows a mean nuclear/cytoplasmic ratio of 2.2:1 (Figure 6B, note the relatively strong nuclear signal for Igf2r and Myc compared to Gapdh and CypA). A similar result was obtained with two independent qPCR assays, RNA blots and the use of two different methods to prepare the nuclear and cytoplasmic RNA fractions (data not shown, see Materials and methods). In contrast to Air, however, Igf2r mRNA is clearly exported to the cytoplasm (Figure 6B and C). All Air splice variants are also exported with a similar efficiency as Igf2r (Figure 6D). This indicates that spliced Air in contrast to the majority of unspliced Air can be exported to the cytoplasm.

Unspliced Air is an unstable transcript

To determine the stability of the Air ncRNA, inhibition of total cellular transcription by Actinomycin D was used. Myc and Gapdh were chosen as controls for short and long half-life mRNAs. Figure 7A–C shows an RNA blot analysis of mouse embryo fibroblast (MEF) cells treated for up to 8 h with Actinomycin D demonstrating that Myc mRNA is largely depleted after 1 h, whereas Gapdh and Igf2r show no significant changes. Figure 7D shows that the half-life of the unspliced Air ncRNA is 2.1 h, whereas that of Igf2r is 14.3 h. In contrast, the Air splice variants SV1 and SV3 show increased stability with half-lives of 15.4 and 16.7 h.

Figure 7.

Full-length Air is an unstable transcript. (A–C) RNA blots of NIH3T3 exposed to 10 g/ml of Actinomycin D (ActD). A representative set of blots from 1 of 3 biological replicates is shown. Total RNA was prepared from control (-) and poisoned (+) cells and hybridized to Myc (A), Igf2r (B) and Gapdh (C). Probes are listed in Supplementary data. Myc mRNA is depleted after 2 h, whereas Igf2r and Gapdh mRNA levels were unchanged after 8 h of ActD treatment. (D) qPCR assays for stability of the Air (q-assay Air middle) and Igf2r (q-assay ex48) transcripts using the RNA samples analysed in panels A–C. Each value was normalized to Gapdh (q-assay Gapdh ex5) (up to 8 h) or 18S rRNA (q-assay 18S) (up to 48 h). Control untreated samples were set to 100% and ActD samples are shown as a % of controls. Values average three biological replicates each performed in technical duplicate. A one-phase exponential decay curve was calculated for these results and the half-life values are calculated by Prism4 (span=100, plateau=0, k0) as unspliced Air 2.1 h (black triangle), Igf2r 14.3 h (black square), SV1 15.4 h (light grey triangle) and SV3 16.7 h (dark grey triangle). (E) qPCR assays for stability of Air, Igf2r and Xist (q-assay Xist) with RNA of primary E13.5 dpc cells. Analysis was performed as in panel D; half-lives: Air 1.6 h (black triangle), Igf2r 14.3 h (black square), Xist 4.6 h (light grey square).

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The above experiments were performed in established MEF cells between passages 12 and 20 that show full repression of the paternal Igf2r allele as determined by RNA FISH and acquisition of DNA methylation (Braidotti et al, 2004; data not shown). In order to investigate if Air shows a different stability at an earlier developmental time point when Igf2r silencing is incomplete in terms of repression and methylation (Stoger et al, 1993; Lerchner and Barlow, 1997), we investigated Air stability in cells obtained directly from 13.5 dpc embryos (Figure 7E). The half-lives for Air and Igf2r in these cells were similar (1.6 and 14.3 h, respectively) to those determined in established MEF cells. In order to compare the stability of the unspliced Air ncRNA with that of a spliced ncRNA, we also determined the stability of the Xist ncRNA in these primary cells as 4.6 h (in agreement with a recent report; Sun et al, 2006), which is three-fold more stable than Air (Figure 7E).

The Air ncRNA is an atypical RNAPII transcript

We show here that the Air ncRNA is transcribed by RNAPII and bears a 7mGcap and poly(A) tail, typical for such transcripts. However, it is atypical because although it shows some characteristics of RNAPII transcripts, it lacks others. Air transcripts mostly evade the splicing machinery, are unstable and are not exported to the cytoplasm. While there are some known unspliced mRNAs that are exported to the cytoplasm (Sakharkar et al, 2005), and two known spliced ncRNAs that are nuclear-localized (namely Xist and the CTN-RNA; Heard, 2004; Prasanth et al, 2005), the Air ncRNA is different as nuclear localization is only shown by unspliced variants, whereas the spliced variants are exported to the cytoplasm.

In addition to the three well-known mammalian RNAPs (RNAPI that transcribes ribosomal genes, RNAPII that transcribes mRNAs and ncRNAs, and RNAPIII that transcribes short tRNAs and small RNAs), a new nuclear-localized RNAP RNAPIV, which is an alternatively spliced product from the mitochondrial encoded RNAP gene, was recently found to be involved in the transcription of several mouse nuclear-encoded genes (Kravchenko et al, 2005). However, we can exclude that Air is transcribed by this polymerase, as RNAPIV was shown to be insensitive to -amanitin. The finding that Air is transcribed by RNAPII is in agreement with bioinformatics analysis of the Air promoter that identified binding sites for typical RNAPII transcription factors such as SP1, commonly found at CpG islands (Figure 1B). It thus seems likely that long imprinted ncRNAs are generally transcribed by RNAPII, as the Xist, Tsix and H19 ncRNAs are also products of RNAPII (Brannan et al, 1990; Navarro et al, 2005). The H19 ncRNA was also shown to be modified with both 7mGcap and poly(A) tail (Pachnis et al, 1988). Both these 5'- and 3'-modifications are typical features of RNAPII transcripts (Vasudevan and Peltz, 2003).

Repression of an RNAPII promoter by DNA methylation

Silencing of a CpG island promoter by DNA methylation is an unusual mechanism in normal embryonic development and has only been found at imprinted and X-chromosome inactive genes (Antequera, 2003). In this study, we show that DNA methylation is necessary to silence Air expression in the mouse embryo. Although the Igf2r promoter becomes methylated in later embryonic stages, it has been previously shown that lack of DNA methylation reduces Igf2r transcription to very low levels (Li et al, 1993). This indicated, contrary to expectation, that DNA methylation was required for Igf2r expression. The observation in this study of a doubling of Air expression upon genomic demethylation, in combination with the previous demonstration that Air expression silences Igf2r in cis (Sleutels et al, 2002), explains this paradox.

Lack of nuclear export of an RNAPII unspliced transcript

The 108 kb unspliced Air is localized to the nucleus and not exported to the cytoplasm, in contrast to spliced Air variants and mRNAs such as Igf2r and Myc. It is generally considered that mRNAs are bound by multiple RNA-binding proteins during transcription, which are recognized by nuclear export factors and rapidly transported to the cytoplasm (Vasudevan and Peltz, 2003). However, mRNAs can show differing efficiencies in terms of nuclear export. As a result, mature transcripts for inefficiently exported mRNAs such as C-jun or -actin can be found in almost equal amounts in both nuclear and cytoplasmic compartments, whereas efficiently exported mRNAs such as the CypA or H4-histone mRNAs are mostly found in the cytoplasm (Gondran et al, 1999). Our result shows that Igf2r mRNA and Air splice variants are exported, albeit inefficiently compared to CypA. However, unspliced Air completely escapes nuclear export.

The reason for nuclear retention of some mature mRNA transcripts is unclear. The lack of nuclear export of the full-length unspliced Air ncRNA can be explained neither by the fact that it is an ncRNA nor by its 108 kb size, as both the 2.4 kb H19 and the 1000 kb Ube3aAS ncRNAs are efficiently exported to the cytoplasm (Brannan et al, 1990; Le Meur et al, 2005). Additionally, low stability cannot explain why unspliced Air is not detected in the cytoplasm, as mRNAs like Myc with five-fold lower stability are exported and detected in the cytoplasm (Gondran et al, 1999). It is important to note that single exon genes can also be efficiently exported to the cytoplasm (Sakharkar et al, 2005). Lack of splicing may, however, be a significant factor in the nuclear retention of the 108 kb full-length Air, as all four spliced variants are exported to the cytoplasm with efficiency similar to that of Igf2r and Myc mRNAs. This identifies splicing as a key regulator of Air ncRNA metabolism.

Inefficient splicing of an RNAPII transcript

It was previously shown that the 108 kb Air transcript was either completely unspliced or contained few and short introns (Lyle et al, 2000). Here we show that between 23 and 44% of Air steady-state level transcripts are spliced. The splice variants are, however, eight-fold more stable than full-length Air, which indicates that the amount of nascent transcripts that become spliced is a small minority (less than 5%) of total Air transcription. The majority of Air transcripts thus appear unspliced (with the caveat that small introns inside repetitive elements would not be detected in the EST screen).

The percentage of spliced Air in steady-state populations is similar to that found for the Tsix ncRNA that overlaps the Xist ncRNA in antisense orientation, where 30–60% of the transcripts are spliced and the function of the spliced product is unknown (Shibata and Lee, 2003). Many factors argue against a silencing role for the Air spliced variants. First, the sequence apart from the first 53 bp is different for each of the four spliced Air variants. Second, nuclear export of the spliced Air variants indicates that they would be unable to specifically target the paternal chromosome. Third, there is no correlation between expression of spliced variants and tissues that lack imprinted Igf2r expression. Last, it is unlikely that the splice variants are post-transcriptional regulators of cytoplasmic Igf2r mRNA, as they share no sequence homology with the mature transcript and a complete absence of Air expression does not increase maternal Igf2r expression (Sleutels et al, 2002). All of these features argue against a functional role for the Air spliced variants.

It is unknown what prevents Air transcripts from being spliced—especially as multiple splice sites would be predicted to occur within 108 kb. We have previously shown that short Air mouse transgene constructs are spliced efficiently and that a rabbit -globin splice acceptor inserted 3 kb downstream of the Air transcription start is used for 80% of transcripts (Sleutels and Barlow, 2001; Sleutels et al, 2002). We speculate that some sequence elements within the Air transcript, but outside the region contained in the above transgenes, could regulate the splicing ability of Air. The possibility that the atypical splicing behaviour of Air results from transcription in a nuclear compartment deficient in splicing factors also cannot be excluded, but appears less likely as in adult tissues the flanking non-imprinted Slc22a1 gene is expressed and spliced from the paternal chromosome that also expresses the unspliced Air ncRNA.

The Air ncRNA is an unstable transcript

We show here that the unspliced Air ncRNA half-life is between 1.6 and 2.1 h and that it is approximately 10-fold less stable than the Igf2r mRNA that it silences. Air is also three-fold less stable in MEFs than the nuclear-localized Xist ncRNA. The ratio of stability between Igf2r and Air is reminiscent of what was recently described for the Xist/Tsix sense/antisense ncRNAs. The half-life of Xist was determined to be 3–4 h in fibroblast cells, whereas the half-life of unspliced Tsix was less than 1 h (the half-life of spliced Tsix has not been determined) (Sun et al, 2006). We show here that splicing of Air restores stability to a level comparable with that of the Igf2r mRNA. This may indicate that the 108 kb Air ncRNA is rapidly degraded because it is not spliced.

The co- and post-transcriptional features of the Air ncRNA described here show that Air is an inefficiently processed and unstable, nuclear-localized ncRNA. The possibility exists that these three features depend on each other. Absence of splicing could result in nuclear retention, as it has been shown that exon splice junctions are bound by exon junction complexes that enhance nuclear export (Vasudevan and Peltz, 2003). Absence of splicing may be a mechanism to trap the Air ncRNA close to the site of transcription to keep the silencing effect on the paternal chromosome and prevent it acting in trans on the maternal chromosome in the same nucleus. In support of this, it has been shown that -globin mutants defective in splicing stay close to the site of transcription and are not exported to the cytoplasm (Custodio et al, 1999). The results presented here show that instability of the Air ncRNA is associated with a lack of splicing. Spliced Air variants are 10 times more stable but they are exported to the cytoplasm, away from the site of repression. This may indicate that Air transcription is more important than the ncRNA for its silencing function.

Models of Air-mediated gene silencing

Although it is not yet known how Air expression leads to gene silencing, three models have been considered (Sleutels et al, 2003). The first model proposes a form of 'expression competition' between Air and the three silenced protein-coding genes, which is based on common transacting factors required for promoter or enhancer activation (Barlow, 1997). This model is not supported by the demonstration that Air can be expressed in cis with the three protein-coding genes when truncated from 108 to 3 kb (Sleutels et al, 2002). The second model is based on the antisense orientation of Air with respect to Igf2r that results in a 28 kb transcription overlap between the two genes. In this 'sense–antisense' model, double-stranded RNA from Igf2r/Air transcription overlap could lead to RNAi–mediated silencing of the Igf2r promoter. The silencing of the Igf2r promoter would then lead in a second step to recruitment of silencing factors to the distant Slc22a2 and Slc22a3 genes. Two findings argue against this model; the Air promoter is included in the transcription overlap but not silenced, and imprinted expression of Air, Slc22a2 and Slc22a3 is maintained in the absence of the Igf2r/Air transcription overlap (Sleutels et al, 2003).

The third model of 'RNA-directed targeting' is based on the strong similarities between genomic imprinting and X inactivation (Reik and Lewis, 2005). This model proposes that the Air ncRNA is localized to the silenced 300 kb region and attracts repressive chromatin proteins to the three silenced genes. The most prominent example of 'RNA-directed targeting' is mammalian XCI in which the Xist ncRNA coats one entire X chromosome and induces gene silencing by recruiting repressive histone modifications and DNA methylation (Heard, 2004). This model has not yet been directly tested for the Air ncRNA but analysis of DNase I hypersensitive sites in a 220 kb region that includes the complete Igf2r/Air genes does not indicate that expression of Air recruits silent chromatin to the paternal chromosome (Pauler et al, 2005). In addition, the results presented here, on the lack of a correlation between Air abundance and the number of genes silenced, combined with the finding that Air is unstable, do not indicate a role for the RNA itself.

An earlier suggestion that transcription rather than the RNA induces silencing could not readily be transferred to the whole Igf2r cluster, as Air does not overlap all silenced genes in the cluster (Rougeulle and Heard, 2002). However, in view of the fact that transcription-based silencing could incorporate instability of the mature unspliced Air transcript, we present here a model that is based on 'transcription interference' (TI) (Figure 8). In this model, the Air ncRNA is suggested to silence Igf2r and the downstream Slc22a2 and Slc22a3 genes on the paternal chromosome by the act of transcription through a domain regulatory element. TI has recently received new attention as a regulatory feature in eukaryotic genomes (Shearwin et al, 2005) and would depend only on the process of transcription to ensure that RNAPII from the Air promoter continually traverses the promoter of Igf2r, and a key long-range cis-acting domain regulator needed for placental expression of the Slc22a2 and Slc22a3 genes (Figure 8). This putative cis-acting domain regulator has not yet been identified and is predicted to lie anywhere within the 108 kb Air transcription unit. As the nature of this element may not correspond to a known cis-regulator such as an enhancer and it likely acts only in the placenta, we are currently pursuing genetic approaches in mice to determine functional regions within Air to test for the existence of an independent cis-regulatory element. Although the instability of the Air ncRNA indicates some support for a TI model, the nuclear retention of the transcript does not. However, as argued above, nuclear retention of Air may simply be a consequence of lack of splicing.

Figure 8.

TI model of Air ncRNA-mediated gene silencing. The expression pattern of 11.5 dpc placenta is shown (note this is the only embryonic tissue to express Slc22a2 and Slc22a3). On the paternal chromosome, transcription of the Air ncRNA (RNAPII with dotted lines) is predicted to interfere with RNAPII binding to the Igf2r promoter, and to interfere with activation of a placental-specific domain regulator (DR) needed to express the Slc22a2 and Slc22a3 genes. On the maternal chromosome, the Air promoter is pre-emptively silenced by a DNA methylation imprint acquired in the oocyte. RNAPII can access the Igf2r promoter and domain regulator binding proteins (DR-BP) can access the placental-specific domain regulator and activate the Slc22a2 and Slc22a3 genes (striped arrow). A domain regulator is envisaged to be a cis-acting element such as an enhancer, or a less well-defined element such as a matrix attachment region or may even be an unknown cis-acting element. The low stability of the Air ncRNA is indicated by dotted lines of the nascent transcript.

View full figure (85 KB)

Although Air is often compared to the Xist ncRNA, the results presented here indicate greater similarities to the Tsix ncRNA. These similarities include an antisense overlap with a silenced sense gene, inefficient splicing, a short half-life and nuclear retention. Tsix has been shown to function at the level of the RNA by recruiting a DNA methylating enzyme and has also been suggested to induce transient chromatin changes by transcription through the Xist promoter (Sun et al, 2006). The silencing function of Tsix, however, is limited to the overlapped Xist gene, whereas Air can also silence non-overlapped genes in a methylation-independent manner. Thus, the details of the two systems are likely to differ. In summary, this characterization of the Air ncRNA favours a role for Air transcription in the silencing mechanism but does not exclude a role for the RNA itself, and it remains possible that Air could act at different levels to mediate cis-acting silencing of the Igf2r cluster. The data presented here provide a base for further studies that will allow features of the TI model to be compared with other models of ncRNA-mediated gene silencing at this and other imprinted gene clusters.

Cells, RNA and transcription inhibitors

MEFs used: NIH3T3 (+/+), MEFF (+/Thp), Thp/DB104 and DB104/Thp. (+) wild-type chromosome 17; the Thp chromosome contains a 6 Mbp deletion that contains the complete Igf2r/Air imprinted cluster and DB104 contains a two-copy transgene each carrying a fully imprinted Igf2r cluster (Wutz et al, 1997). Primary MEFs used for Figure 6 were prepared from seven 13.5 dpc FVB embryos and used at p2. Cytoplasmic and nuclear RNA was prepared using either the Paris™ kit (Ambion) or standard protocols. For Actinomycin D and -amanitin treatment, 5 10⁵ cells seeded per 10 cm dish were cultured for 42 h. At time point 0, the media were removed, cells washed with PBS and incubated with media supplemented with 10 g/ml Actinomycin D or 5 g/ml -amanitin (both dissolved in ethanol). Control dishes were incubated with media plus ethanol. At each time point, cells from a control dish and a treated dish were harvested for RNA using standard techniques. RNA blots and RPA were performed using standard techniques (probes are described in Supplementary data).

RNA analysis

RNA blot refers to 'Northern' blot that were prepared from formaldehyde gels in standard conditions. RPA refers to 'RNase protection assay' that was performed using the RPA III™-kit (Ambion) according to instructions. RPA probe name, accession number, position, and size of the unprotected (upr) and protected (pr) bands are given in Supplementary data. RNA blot and RPA signals were obtained from Phosphoimager screens scanned by a Typhoon8600 scanner (Molecular Dynamics, Amersham) and quantified by ImageJ software (http://rsb.info.nih.gov/ij/index.ht
ml).

Real-time qPCR

Taqman probes and primers were designed by PrimerExpress and qPCR performed with the ABI PRISM 7000 with the following primers (900 nM) and Taqman probe (200 nM) under the following cycling conditions: 2 min 50°C, 10 min 95°C, 40 cycles of 15 s 95°C and 1 min 60°C (62°C for Air 5' and splice variants). RNA quantification was made by the standard curve method using serial dilutions of plasmids (Air 5' and splice variants) or cDNA (adult mouse heart or total NIH3T3 RNA for Air middle, Gapdh ex5, Igf2r ex48, CypA ex3/4, 45S pre-rRNA, 18S rRNA). Assay specificity for full-length unspliced and spliced Air variants was tested by serial dilutions of plasmids. Relative quantification and statistics were performed as described in the manufacturer's protocol (Applied Biosystems). Primers and Taqman probes used for qPCR assays are described in Supplementary data.

Immunoprecipitation

IP was performed with mouse monoclonal antibody (clone H20, cat. no. 201001, Synaptic Systems) to 3mG and 7mGcap structures and 10 g DNase I-treated total RNA from NIH3T3 cells. Equal volumes of the supernatant, first and fifth wash and bound RNA were analysed after reverse transcription by qPCR after precipitation and resuspension in equal volumes. Three biological replicates were performed each including a mock IP control (beads without antibody) and an IP with an unrelated antibody of the same isotype (IgG1, MeCP2, Upstate) processed simultaneously.

EST search and promoter analysis

Transcription factor binding sites at the Air ncRNA promoter were determined by MatInspector (www.genomatix.de). EST sequences that align to the sequence between Mas1 and exon 3 of Igf2r found by the Ensembl 30 database (http://www.ensemble.org) were tested for discontinuous alignment to Air (AJ249895) by PipMaker (http://pipmaker.bx.psu.edu/pipmake
r/). Spliced ESTs were additionally aligned against the Blast database (http://www.ncbi.nlm.nih.gov/BLAST/) to find similar sequences. Repeat content was analysed by Repeat masker (http://www.repeatmasker.org/).

We thank R Kakkad, M Koerner, F Pauler, L Spahn and A Wutz, for reading the manuscript, C Unger and K Warczok for technical assistance and F Pauler for bioinformatics analysis. S Stricker is supported by Boehringer Ingelheim Fonds, C Seidl by FWF Austrian Science Fund and SFB Modulators of RNA fate and function and the laboratory is supported by the Austrian Academy of Sciences and is a member of the EU Framework 6 Network of Excellence 'The Epigenome'.

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