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

Abstract

Expression of the Air ncRNA is necessary to silence multiple genes in cis in the imprinted Igf2r cluster. However, its mode of action is unknown. Here, we characterize co- and post-transcriptional features of Air that identify it as a new member of the class of nuclear regulatory RNAs. We show that Air is transcribed from a DNA methylation-sensitive promoter by RNA polymerase II (RNAPII). However, although it is capped and polyadenylated similar to other RNAPII transcripts, the majority of Air transcripts evade cotranscriptional splicing resulting in a mature 108 kb ncRNA. As a consequence, the mature unspliced Air is nuclear localized and highly unstable. These features show that Air is an atypical RNAPII transcript whose properties indicate that its mode of action in gene silencing may not depend on the RNA per se but instead is related to its actual transcription.

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/cpgplot/), 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.

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.

Figure 3

The Air ncRNA is an RNAPII transcript. (A) RNA blot of NIH3T3 cells exposed to α-amanitin (α-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 α-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%.

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.

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.

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).

Figure 7

Full-length Air is an unstable transcript. (A–C) RNA blots of NIH3T3 exposed to 10 μg/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, k⩾0) 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).

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.