RNA Isolation and DNA microarray analysis

Total RNA was prepared from PMEF cells grown in a monolayer in 150 mm dishes using the Trizol reagent (Invitrogen) according to the manufacturer's directions. For DNA microarray analysis, mRNA was isolated from total RNA using the Oligotex mRNA purification system (Qiagen). Incyte Genomics reverse transcribed AG and PG polyA⁺ RNA with either Cy3 or Cy5 label. The labeled cDNA was then hybridized to the Incyte mouse GEM1 cDNA microarray according to their established protocol.

Reverse transcription and polymerase chain reaction (RT-PCR)

Total RNA from PMEFs or mouse tissues was treated with RQ1 RNase-free DNase (Promega) to remove residual genomic DNA, followed by heat inactivation of the endonuclease. 5 µg RNA was reverse transcribed by Superscript II RNase H- RT (Invitrogen) in 60 µl reaction volumes according to the manufacturer's procedure, except that the reaction contained both 25 ng/µl Oligo dT and 5 ng/µl random hexamers. PCR was performed using 1 µl of the RT reaction as a starting material according to standard procedures. PCR cycling parameters were typically 4 minutes at 94°C, followed by thirty cycles of 30 sec at 94°C, 30 sec at 58°C, 90 sec at 72°C, and a final seven minute extension at 72°C. Supplemental Table S1 has the sequences of oligonucleotide primers used in this study.

Imprinting analysis

RT-PCR products were generated from PMEF or brain RNA that was obtained from appropriate embryos or adult mice, respectively, derived from 129S1 and CzechII/Ei matings. Subsequently, RT-PCR products were cleaned using the MinElute PCR purification kit (Qiagen). RT-PCR samples were then treated with and without the indicated restriction enzymes obtained from New England Biolabs. Digests were analyzed by agarose gel electrophoresis with ethidium bromide staining. Supplemental Table S2 has 129S1 and CzechII/Ei restriction site polymorphisms discovered during the course of this study.

cDNA Library screening and RNA Ligase Mediated-RACE (RLM-RACE)

An arrayed mouse day 19 embryo cDNA library (Origene MEA-1001) was screened by PCR to isolate Irm, Meg8, Meg9, Peg11, and anti-Peg11 clones, while an adult mouse brain cDNA library was used for Dlk1/“DAT” cloning (Origene MAB-1001). The gene-specific primers for cDNA library screening are denoted by “#” in Supplemental Table S1. RLM-Race (Ambion) was performed for Irm according to the manufacturer's directions, with the initial PCR utilizing the 5′ RACE Outer and Irm 197Dn primers. The subsequent amplification used the same upstream primer and the Irm 120Dn primer.

Identification of Irm as a candidate maternally expressed gene in mice

For each of the forty ESTs that are listed in Table 1, RT-PCR was performed on total RNA from wild-type (WT), AG, and PG PMEFs to determine if expression was consistent with being imprinted. Figure 1 shows a typical RT-PCR result and demonstrates that appropriate imprinted gene expression is maintained in these uniparental cell lines. Grb10, Meg3/Gtl2, Peg3, and Dlk1 were selected as imprinted gene controls. As expected, the biallelic gene histone acetyltransferase 1 (Hat1) and imprinted genes (Grb10, Meg3/Gtl2, Peg3 and Dlk1) showed appropriate expression by RT-PCR in the three cell lines (Figure 1). Ultimately, nine of the forty ESTs (denoted by stars in Table 1) were highly differentially expressed between AG and PG PMEFs by RT-PCR (data not shown). Of these nine, five ESTs corresponded to imprinted genes that were known at the time (Grb10, Igf2r, Meg3/Gtl2, Peg3, and U2af35-rs1). This result suggested the possibility that a subset of the four remaining ESTs (corresponding to Postn, Mpeg1, Racgap1, and Irm) may in fact be imprinted. To demonstrate conclusively the imprinting status of a gene, an assessment of allele specific transcription was performed using an informative polymorphism for the four ESTs whose imprinting status was unknown. We found that Postn, Mpeg1, and Racgap1 were biallelically expressed (data not shown), suggesting that the genes corresponding to these ESTs may be regulated by passage number or by an imprinted gene product. Intriguingly, the final EST W89392 (Irm) with its high RNA levels in PG versus AG PMEFs connected to other ESTs that mapped to the same BAC (AC040981.2) as the known mouse imprinted gene Meg3/Gtl2. Meg3/Gtl2 is the founding member of a much larger imprinted gene region that has been implicated as having parent of origin effects in three mammalian species. Hence, an exhaustive screen was undertaken for a 1.96 Mb region around Irm to investigate the imprinting status of all contained mRNAs. For the sake of clarity, results will be presented from centromere to telomere. Figure 2 shows the genomic organization of this region in mice.

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Figure 1

Androgenetic and Parthenogenetic PMEFs maintain appropriate imprinted gene expression.

RT-PCR was performed on indicated PMEF cell lines: W (Wild-type), A (Androgenetic), and P (Parthenogenetic). Histone acetyltransferase 1 (Hat1) is a biallelically expressed control. Dlk1 and Peg3 are controls for paternal expression while Grb10 and Meg3/Gtl2 are controls for maternal expression. With Irm primers 1307Up and 1581Dn, Irm RNA expression was undetectable in AG PMEF, while its expression was abundant in WT and PG PMEFs. Moreover, Meg8 and Meg9, two other genes in the large imprinted gene cluster that contains Irm, have expression patterns that are consistent with being imprinted with maternal expression. The PCR primers were Hat1 510Up/758Dn, Grb10 270Up/770Dn, Meg3 267Up/578Dn, Peg3 4430Up/4956Dn, Dlk1 830Up/1274Dn, Irm 988Up/1140Dn, Meg8 1Up/294Dn (AF498299) and Meg9 2614-3009.

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Figure 2

Genomic Organization of Dlk1-Dio3 Imprinted Region.

This figure summarizes our results about the imprinting and structure of genes in this mouse region. Arrows denote transcriptional orientations. Genes written in black were shown to be biallelically expressed. Genes drawn in blue are paternally expressed, while those in red are maternally expressed. Genes written in yellow were undetectable by RT-PCR. For Begain transcribed from promoter 2, this transcript was coded blue as it is paternally expressed in sheep in a tissue specific manner, although in this study, RT-PCR failed to amplify a transcript derived from this promoter. Of these, accession numbers, AK141557, AK163826, AK048151, and AK044800, correspond to unspliced “transcripts” whose cDNA sequence ends in a polyA stretch of genomic DNA, suggesting that these “cDNAs” may be genomic DNA contamination in the Riken mouse cDNA libraries. For the bottom panel, exons are dark colored while introns are light. Purple lines represent the position of microRNA precursors. In all, fifty-two mouse microRNA precursors as listed in Table 2 map to this imprinted region.

Defining the centromeric and telomeric ends of the imprinted gene cluster in PMEFs

Six mRNAs (Yy1, Slc25a29, Hdcmp, Wars, AK147993, and Begain) as depicted in Figure 2 that are on the centromeric side of Dlk1 were tested by RT-PCR in WT, AG, and PG PMEFs to determine if they were differentially expressed between these samples. Similarly, two mRNAs, Ppp2r5c and Dync1h1, on the telomeric side of Dio3 were also tested. For seven of the eight genes, RT-PCR products were obtained from the three cell lines at similar levels, indicating that these genes are biallelically expressed in PMEFs (data not shown). Mouse Hdcmp (accession number AI132487) was undetectable by RT-PCR. Since the neighboring genes, Yy1, Slc25a29, Wars, and AK147993, are not imprinted in PMEFs, it is unlikely that Hdcmp is imprinted (see Figure 2).

For Begain RT-PCR, our original primer set fell within the region that is common to Begain mRNA irrespective of which of the two mouse Begain promoters is used. Our results were consistent with biallelic expression. In sheep, Begain has been recently shown to be biallelically expressed in all tissues when transcription is initiated at one of its two promoters [48]. Transcripts derived from the other promoter in sheep are characterized by imprinted expression in a tissue specific manner. Since our RT-PCR product would detect transcripts derived from both promoters, multiple primer sets were designed to investigate expression from each mouse promoter. Unfortunately, the alternative first exons of Begain are quite GC rich and specific amplifications products were not observed. Hence, we can only conclude that at least one of the two Begain promoters is biallelic.

Imprinting, Expression, and cDNA Cloning of Dlk1 and Its Alternative Polyadenylated Transcript “DAT”

Dlk1 is paternally expressed in mice [25], [27] and in other mammalian species with the exception of marsupials [26], [49]. A DraIII restriction site polymorphism between CzechII/Ei and 129S1 mice in the Dlk1 cDNA was identified and used to confirm the imprinted status of Dlk1 (Figure 3A). Northern analysis showed that Dlk1 is expressed in many adult mouse tissues, including brain, kidney, testis, and thymus. Mouse brain expressed the most Dlk1 mRNA and is characterized by an additional and abundant 4.5 kb transcript that was not detected in the other investigated tissues (Figure 3B).

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Figure 3

Dlk1 is paternally expressed and has an abundant alternatively polyadenylated transcript in the mouse brain.

(A) A DraIII restriction site polymorphism between 129S1 and CzechII/Ei mice was utilized to determine that Dlk1 is paternally expressed. PCR primers were Dlk1 2Up/317Dn. (B) Northern analysis using the same Dlk1 PCR fragment as probe revealed that Dlk1 is widely expressed. Of the tissues investigated, only the adult brain is characterized by having an additional and abundant transcript that is roughly 4.5 kb in length. (C) Several ESTs cluster to the syntenic region of “DAT” (Dlk1 associated transcript). RT-PCR was done with “DAT” primers 1533Up/2044Dn. Using a EcoO109I restriction site polymorphism, “DAT” was shown to be like Dlk1 in its paternal expression. (D) Northern analysis on adult tissue samples of “DAT” detected a 4.5 kb transcript only in the brain. This band is identical in size to the long transcript of Dlk1 found in the brain. Moreover, the length of this transcript is greater than the distance (~2.7 kb) between mouse Dlk1 polyA and “DAT” polyA. (E) RT-PCR was performed with various primer sets. Left most panel showed that Dlk1 is expressed in brain, heart and muscle. Middle panel shows that “DAT” is only detectable in brain and is consistent with Northern findings. For the right panel, PCR was performed with a primer set in which the upstream primer (Dlk1 1237Up) was before the canonical Dlk1 polyA site while the downstream primer was after this site (Dlk1 1805Dn). A band was detected only in the brain, indicating that “DAT” is an alternatively polyadenylated transcript of Dlk1. This result was confirmed by cDNA cloning (Accession numbers: EU434914-EU434917).

In sheep and humans, a neighboring gene “DAT” (DLK1 Associated Transcript) is also paternally expressed [23]. In addition to their common paternal expression, DLK1 and “DAT” lie in the same transcriptional orientation with the polyA of DLK1 roughly 3.1 kb upstream of the polyA of “DAT” in humans. In mice, several ESTs from Unigene cluster Mm.458824 map to the syntenic region of “DAT”. An EcoO109I restriction site polymorphism demonstrated that “DAT” is paternally expressed in mice (Figure 3C). Northern analysis detected “DAT” only in the brain and its 4.5 kb length was identical to the brain-specific long form of mouse Dlk1 (Figure 3D). This finding in conjunction with the fact that the genomic distance between the polyAs of Dlk1 and “DAT” in mice is ~2.7 kb indicated that “DAT” was most likely an alternatively polyadenylated transcript derived from the Dlk1 gene. To test this hypothesis, RT-PCR was performed with primers before and after the canonical Dlk1 polyA site and yielded a product only in mouse brain (Figure 3E). Moreover, four independent cDNA clones from the Origene mouse brain cDNA library (MAB-1001) were isolated and sequenced, demonstrating unequivocally that the “DAT” sequences result from an alternative polyadenylated product from the Dlk1 gene (Accession numbers: EU434914-EU434917). The lengths of our two longest clones that contain the entire Dlk1 ORF are consistent with the size of the 4.5 kb form of alternatively polyadenylated Dlk1. The significance of this alternative pre-mRNA processing event is unknown, but EST analysis indicates that is conserved across mammals (data not shown). In addition to alternative polyadenylation, our isolated cDNA clones are characterized by alternative splicing within the Dlk1 ORF. The existence of these alternatively spliced products was independently confirmed by several mouse Dlk1 mRNAs and ESTs in GenBank.

Meg3/Gtl2: Imprinting, Expression, and miR-770

Figure 4A shows that Meg3/Gtl2 is maternally expressed using an identified Alw26I restriction site polymorphism between CzechII/Ei and 129S1 mice. This result confirms several earlier studies [24], [50]. By Northern analysis, Meg3/Gtl2 is highly expressed in the brain and testis (see Figure 4B). In adult mouse brain, the predominant form is 2.4 kb, while in testis, a 6.5 kb transcript is most abundant. In addition, Meg3/Gtl2 contains in its last intron (Relative to Accession number Y13832) the evolutionarily conserved microRNA miR-770 (see Figure 2 and Table 2). This observation is intriguing since H19, a maternally expressed noncoding RNA near Igf2, also is characterized by having an evolutionarily conserved microRNA miR-675 [51]. Table 2 shows several mRNAs that include Bmp1, Bmp15, Fosb, Lmna, Sspn, Tmod1, and Trp53 that are predicted to be regulated by miR-770. If these genes are bona fide targets, loss of miR-770 that occurs in the associated pUPD syndromes could contribute to the observed phenotypes by increasing the levels of its targeted genes, many of which are implicated in placental, bone, and muscle biology.

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Figure 4

Meg3 is maternally expressed and highly expressed in the brain and testis.

(A) Alw26I restriction digests on RT-PCR products confirm that Meg3 is maternally expressed. For PCR, Meg3 267Up/578Dn primers were used. (B) Northern blots reveal that Meg3 is characterized by alternative splicing. In the adult mouse brain, the predominant band is roughly 2.4 kb in length while in testis is 6.5 kb.

Peg11 and Anti-Peg11: Imprinting, Expression, cDNA Cloning, and MicroRNAs

Unlike other genes in this region where oligo(dT) and random primed reverse transcription reactions were used as a substrate for PCR, we also performed strand-specific RT reactions to ascertain if the observed product corresponded to Peg11 or its antisense transcript. As Figure 5A–C demonstrates, Anti-Peg11 was expressed from the maternal allele in both PMEFs and adult brain. In contrast, Peg11 was undetectable in PMEFs and was expressed at lower levels in the brain in comparison to its antisense transcript. Our results showed that Peg11 was paternally expressed in brain. Northern analyses were performed with double stranded DNA probes and then sequentially with two single-stranded probes. Figure 5D shows that Peg11 is broadly expressed but at low levels in many tissues. Anti-Peg11 on the other hand is far more restricted in its expression with a prominent signal only detected in the brain.

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Figure 5

Imprinting and expression analysis of mouse Peg11 and anti-Peg11.

(A) RT-PCR was performed on RNA derived from wild-type {W}, Androgenetic {A} and Parthenogenetic {P} PMEF cell lines. RT reactions were performed with either random hexamers or the strand-specific primers for Peg11 “477” or Anti-Peg11 “1052”. For PCR, Anti-Peg11 522Up/847Dn primers were used. These results demonstrate that in PMEFs Anti-Peg11 is expressed but not Peg11. (B) An NlaIII restriction site polymorphism between 129S1 and CzechII/Ei was utilized to demonstrate that Anti-Peg11 is maternally expressed in PMEFs. (C) As in panel A, three RT reactions were performed with the same PCR primers but on adult brain RNA derived from a mouse with a CzechII/Ei mother and 129S1 father. These results demonstrate that Anti-Peg11 and Peg11 are both expressed, although Anti-Peg11 is more abundant in mouse brain. Peg11 was paternally expressed, while Anti-Peg11 was maternally expressed. PCR on genomic DNA (Gen) revealed that the primers did not preferentially amplify either the 129S1 or CzechII/Ei alleles. (D) Northern for Peg11 and Anti-Peg11 using a double stranded DNA probe corresponding to Anti-Peg11 477-1052. Blue arrows indicate the position of Peg11 transcripts, while red arrows correspond to Anti-Peg11. This annotation was possible as the Northern was repeated with single stranded probes for both Peg11 and Anti-Peg11 (data not shown).

To date, there are no reports of a cloned cDNA corresponding to either Peg11 or Anti-Peg11 in any species. In mice, a computationally predicted open reading frame (ORF) termed Retrotransposon-like 1 (Rtl1) has been deposited in GenBank (NM_184109) that maps to Peg11. Hence, an arrayed day 19 embryonic cDNA library (Origene MEA-1001) was screened by PCR with Anti-Peg11 477Up and 1052Dn primer pair. This primer set does not distinguish between strands and would yield a product for both Peg11 and Anti-Peg11. Ultimately, two Peg11 cDNAs were isolated and sequenced. Both clones (Accession numbers: EU434914 and EU434918) exceeded 6 kb in length, revealing that Peg11 has two exons (see Figure 2). Given the length of the Peg11 as determined by Northern, our cDNA clones are nearly full length. Likewise, a 2.3 kb Anti-Peg11 cDNA was isolated and sequenced (Accession number EU434921). As Figure 2 shows, a large number of microRNAs map to the Anti-Peg11/Peg11 region and all are in the same transcriptional orientation as Anti-Peg11. Our Anti-Peg11 cDNA clone most likely represent a cleavage product of a longer Anti-Peg11 message that resulted as the primary miRNA transcript is processed into pre-miRNAs. This conclusion is based on the observation that our cDNA begins at the end of miR-431 precursor and ends at the beginning of miR-127 precursor. Multiple experiments were unsuccessful in isolating cDNA clones that were upstream or downstream of our cloned sequence. Since Anti-Peg11 serves as a primary microRNA transcript, it would be nearly impossible to detect a full length Anti-Peg11 under conditions where the Microprocessor complex is functioning.

A large number of microRNAs map to the Anti-Peg11/Peg11 region (see Figure 2 and Table 2). Three microRNAs in this region, miR-431, miR-127, and miR-136, were shown previously to regulate Peg11 through a siRNA-like mechanism [52]. Also, miR-127 was shown to regulate Bcl6 [53].Since individual microRNAs regulate large sets of genes often numbering in the hundreds [40], [41], we investigated the predicted targets for all microRNAs in this region. As Table 2 shows, several predicted targets for microRNAs in this region have been implicated in processes that when perturbed could result in phenotypes associated with the human and mouse UPD syndromes. These genes include Bmp2, Casq2, E2f1, E2f3, Grb10, Igf2, Jak2, Junb, Ldb3, Peg10, Plagl2, Magel2, Nnat, Pax3, Ryr1, Sgce, Sntb2, Sspn, Tcf21, Tln1, Tpm2, Trp53, Ttn, and Xpo5.

Meg8: Imprinting, Expression, cDNA Cloning and MicroRNAs

As Figure 6A demonstrates, Meg8 is a maternally expressed gene in mice as deduced from digests of RT-PCR products using an informative HgaI restriction site polymorphism between 129S1 and Czech II/Ei mice. To date, no reports have documented the cloning of Meg8 in any mammalian species. As such, the Origene arrayed day 19 cDNA library (MEA-1001) was screened for a full length Meg8 cDNA. The sequence of the isolated Meg8 cDNA has been deposited in GenBank (Accession number: EU434919). Comparison of Meg8 cDNA with the mouse genome revealed that this transcript has 26 exons (Figure 2). Conceptual translation of Meg8 revealed that it is a noncoding RNA that lacks significant ORFs (data not shown). Importantly, we found that Meg8 and Irm share ten common exons. Northern analysis for the region using a probe that is unique to Meg8 cDNA showed that it is expressed highly in the adult mouse brain and skin (Figure 6B). Significantly lower levels of Meg8 were detected in the heart. In contrast, Irm is barely detectable in the skin (Figure 7B), demonstrating that Meg8 and Irm differ in their expression patterns. Moreover, only Meg8 transcripts have the intron-encoded microRNAs miR-341, miR-1188, and miR-370 that lie upstream of Irm (see Figure 2 and Table 2). Of note, miR-370 is also conserved in humans. Several genes that are predicted to be regulated by these microRNAs are shown in Table 2 and include Grb10, Lmna, Peg10, and Trp53.

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Figure 6

Meg8 is expressed from the maternal allele and highly in the brain and skin.

(A) The Meg8 alleles contain an informative restriction site polymorphism in that a HgaI site is present in the 129S1 allele in comparison to CzechII/Ei. RT-PCR products with AF498299 1Up/294Dn primers for Meg8 from the four intraspecific crosses were digested with and without HgaI, verifying the imprinted expression of this gene. (B) Northern blots were performed on adult tissues (left) and embryos harvested at different time points (right). Meg8 levels were highest in brain and testis. In addition, Meg8 RNA increases as a functional of embryonic development.

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Figure 7

Irm is maternally expressed, alternatively spliced, and abundant in the adult brain.

(A). A single nucleotide polymorphism in Irm cDNA was identified at 1094A→T between 129S1 and CzechII/Ei mouse strains that creates a NlaIII restriction site in the CzechII/Ei Irm cDNA. RT-PCR was performed with Irm primers 988Up and 1140Dn. Restriction digests of RT-PCR products from intraspecific crosses between these two subspecies by NlaIII demonstrated that Irm is expressed in a monoallelic manner from the maternal allele. (B) An adult tissue (left) and total embryo from different gestational days (right) polyA⁺ RNA blot (left) was hybridized with a Irm/Rian exon 1 cDNA probe. A 2.5 kb transcript was predominantly detected in the brain and to a significantly lesser extent in testis, stomach, and muscle. Higher molecular weight bands of lower relative abundance in comparison to the 2.5 kb message were also seen that reflect the heterogeneity in spliced variants. Since Rian RNA is approximately 5.4 kb, the predominant RNA product from the Irm/Rian promoter is Irm RNA, while Rian accounts for at most 5% of total transcripts. This result is consistent with the relative abundance of ESTs that are specific for each of the alternatively spliced products.

Irm: Imprinting, Expression, RLM-RACE and cDNA cloning

From the original large scale transcription profiling of AG and PG PMEFs, EST W89392 was an attractive candidate for a novel imprinted transcript. Since no polymorphisms were detected within this EST, a cDNA library derived from mouse day 19 embryos (Origene) was screened to obtain a full-length clone. The sequence of the longest of the three isolated cDNA clones (2361 nt) has been deposited in Genbank (Accession number AF498294). This cDNA was named Irm, Imprinted RNA near Meg3/Gtl2. Initial blast searches with Irm cDNA of the nonredundant Genbank database yielded only a single cDNA match to AK017440, an uncharacterized RNA that differs from Irm by alternate 5′ splice site choice in exon 8. Subsequent searches yielded partial matches to two additional RNAs, the noncoding RNA Rian [29] and the long form of MBII-343 snoRNA [30]. In addition, four snoRNAs are present in introns of Irm [54]. Conceptual translation of Irm revealed that it is a noncoding RNA as it only has several short ORFs that lack significant homology to any known protein as assayed by blast searches (data not shown).

Corresponding regions in Irm cDNA from 129S1 and CzechII/Ei mice were analyzed, leading to the identification of a single nucleotide polymorphism at position 1094A→T creating a NlaIII restriction site in the CzechII/Ei cDNA (Accession numbers: AF498295 and AF498296). RT-PCR was performed on RNA derived from individual PMEF lines with the following genotypes, 129S1, CzechII/Ei, 129S1×CzechII/Ei, or CzechII/Eix129S1. RT-PCR products were purified and digested with NlaIII restriction enzyme. The resulting restriction digestion pattern shown in Figure 7A demonstrates that this cDNA is indeed maternally expressed.

Since Meg8 and Irm cDNAs overlap substantially and are both maternally expressed, one possibility is that Irm represents a 5′ truncated transcript derived from the Meg8 promoter. An alternate hypothesis is that Meg8 and Irm have distinct promoters. Even though our Northern results support the latter hypothesis, RLM-RACE was performed to identify the transcriptional start site of Irm [55]. In this procedure, RNA is incubated with calf intestinal phosphatase (CIP), leading to the 5′ dephosphorylation of tRNA, rRNA, and fragmented transcripts. After CIP inactivation, samples are treated with tobacco acid pyrophosphatase that catalyzes the removal the ⁷mG cap with the liberation of a free 5′ monophosphate. An RNA adapter is subsequently ligated and RT-PCR performed with adapter- and gene-specific primers. Sequencing of the resulting RT-PCR products revealed that the transcriptional start site of Irm is located twelve nucleotides upstream of the original Irm cDNA sequence (5′gccaatgatgac with Irm 1-120, data not shown). This site is identical to the Irm promoter and corresponding transcriptional start site predicted by a neural network based algorithm (http://www.fruitfly.org/seq-tools/promoter.html). Moreover, the 620 bp length of the first Irm exon supports that the fact that it is the first transcribed exon, as less than 1% of internal exons are longer than 400 bp [56].

Over 60 mouse ESTs corresponding to Irm RNA are present in the Genbank database, providing considerable insight into the Irm expression pattern. In adult mice, Irm ESTs are found in the pancreas, eye, testis, and different areas of the brain, including the hippocampus, hypothalamus, amygdala, and striatum. In mouse embryos, Irm ESTs are found in the developing pancreas, brain, heart, testis, optic vesicle, and the neural retina. Given the profile of Irm ESTs, Irm expression was evaluated using Northern and whole mount in situ hybridization techniques using a probe common to exon 1 of both Irm and Rian RNAs. Northern analysis on adult tissues (Figure 7B) revealed high levels and multiple transcripts for Irm RNA in the brain. Significantly lower Irm expression was detected after longer exposure times in adult mouse testis, stomach, skin, heart, and muscle. Northern analysis of embryos at different developmental stages revealed that Irm expression is temporally regulated during development (Figure 7B).

As mentioned previously, Irm RNA partially overlaps a cloned noncoding RNA termed Rian for RNA Imprinted and Accumulated in the Nucleus [29]; however, Rian Northerns have not been reported to verify its existence and size. Rian is characterized by several peculiar features. Most notably, it contains three retained introns, lacks major polyadenylation signals at its 3′ end, and terminates in a region of genomic DNA that has 15 adenosine residues. Therefore, the identified Rian cDNA most likely represents an internally primed Irm RNA that is reversed transcribed from the polyA stretch and does not exist in the cell as a mature species. Since our probe for the Irm Northern would detect Rian equally well, our results demonstrate that Irm is the major transcript derived from the Irm promoter (~2.5 kb) and raises doubts about the existence of Rian.

The spatio-temporal distribution of Irm transcripts within the embryo was determined by whole mount in situ hybridization at different stages of development. Prominent and persistent expression of Irm RNA was detected in the somites from embryonic day 9.5 (E9.5) onwards (Figures 8A–C). Other tissues expressing Irm include the first and second branchial arches and the limb cartilage (Figures 8A–C). In extraembryonic tissues, Irm expression exhibited a weak, but scattered expression in the yolk sac and to the maternal side of placenta (Figures 8A and 8C). Irm transcripts are also found in the brain, most prominently in the forebrain (Figure 8A) and later at E10.5 in the layers enriched with neural precursors in the midbrain (Figure 8B). Other regions within the nervous system that were positive for Irm expression were the cranial ganglia (predominantly the trigeminal ganglion), telencephalon, and midline of the caudal neural tube (Figures 8D and 8E). In summary, Northern, whole mount embryonic in situ hybridization, and EST analyses demonstrate that Irm RNAs are predominantly expressed in the brain but is not strictly brain-specific. RT-PCR experiments on seven adult mouse tissues for the long form of MBII-343 yielded a similar conclusion [30] and is noteworthy since this RNA overlaps Irm exon 6.

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Figure 8

Tissue distribution of the Irm transcripts in the mouse embryo revealed by in situ hybridization.

(A). On embryonic day 9.5 (E9.5), Irm expression is detected in the forebrain area (FB), first and second branchial arches (BA), in developing somites (Sm), and in the caudal neural tube (NT). A dispersed staining is observed in the extraembryonic tissues, particularly in the yolk sac (YS). (B). By E10.5 the brain expression domain shifts more in the midbrain area (MB) where the neuroepithelial lining shows the Irm staining. Similar to E9.5, expression persists both in the branchial arches and in the somites. (C). In E12 mouse conceptus, Irm transcripts could be also visualized in the forming limb cartilage (LmC), still clearly detectable in the telencephalon (TE) and caudal somites. The yolk sac (YS) and the maternal side of placenta (MP) are positive for Irm by in situ staining as well. The expression along the midline in the caudal neural tube (NT) is most likely associated with the floor plate area (D). In the head, prominent Irm transcript accumulation is observed in the trigeminal area (Tg), ear pinna (EP), and the developing whiskers hair follicle (WF, see panel E). (F). Hybridization with the sense Irm probe under the same conditions resulted in no specific signal.

Imprinting of AK050713 and AK053394

Multiple cDNA clones derived from Riken libraries map to the region between Irm and Meg9. Their accession numbers are from centromere to telomere as follows: AK050713, AK141557, AK163826, AK053394 and AK046809. For AK141557 and AK163826, RT-PCR with four sets of primer pairs did not detect a transcript in PMEFs or four adult tissues: brain, heart, muscle, and white adipose. These two putative cDNAs are unspliced, lack major polyadenylation signals, and end in a stretch of polyA mouse genomic sequence, suggesting that they may represent genomic DNA contaminants. For AK050713, we found that this transcript is maternally expressed in PMEFs (Figure 9A). Likewise, AK053394 is expressed exclusively from the maternal chromosome in PMEFs (Figure 9B). AK053394 is a spliced transcript with two exons and contains the entire sequence of AK046809.

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Figure 9

AK050713 and AK053394 are maternally expressed.

(A) The AK050713 alleles contain an informative restriction site polymorphism in that a MscI site is present in the CzechII/Ei allele in comparison to 129S1. RT-PCR products with primers 784Up/1213Dn from the four intraspecific crosses were digested with and without MscI, verifying the imprinted expression of this gene. (B) Similarly, a BslI informative restriction site was utilized to demonstrate that AK053394 is maternally expressed using PCR primers 1647Up/2100Dn.

Meg9/Mirg: Imprinting, Expression, cDNA Cloning and MicroRNAs

Several spliced mRNAs and ESTs such as AK013406, CB193969, and BQ175773 are located approximately 73–88 kb downstream of the 3′ end of Irm. Sequences corresponding to AK013406 were determined for 129S1 and CzechII/Ei mice (Accession numbers: AF498297 and AF498298). The two alleles differ by a 14 bp insertion that abolishes an MboII restriction site in CzechII/Ei mice. Utilization of this informative restriction site on RT-PCR products revealed that this gene is maternally expressed. We thus named it Meg9 for Maternally expressed gene 9 (Figure 10A). A day 19 mouse embryo cDNA library was screened, resulting in the identification of three transcripts that are all longer than 3 kb (Accession numbers: EU434920, EU616812, and EU616813). These transcripts overlap the recently identified Mirg (MicroRNA containing gene) [34]. In comparison to Mirg, the longest Meg9 clone has nine additional upstream exons and contains four more microRNAs: miR-382, miR-134, miR-668, and miR-485. The location of the 5′ end of Mirg indicated that this truncated transcript results as result of processing of the exonic miR-485 precursor. As Figure 10 shows, Northerns using a probe that would detect both Meg9 and Mirg demonstrate that Meg9/Mirg is widely expressed in many adult mouse tissues with the highest levels being in the brain, skin, and testis. Meg9/Mirg expression is also regulated during embryogenesis.

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Figure 10

Meg9 transcripts are derived from the maternal allele and are widely expressed in adult tissues.

(A) Meg9 alleles between 129S1 and CzechII/Ei differ by a polymorphism that abolishes a MboII restriction site in the CzechII/Ei allele. Moreover, the CzechII/Ei allele harbors a fourteen nucleotide long insertion. RT-PCR (bottom) and MboII restriction digestion (top) of the Meg9 RT-PCR products demonstrated that this gene is maternally expressed. (B) Northern demonstrate that Meg9, like Dlk1 (see Figure 3B), is widely expressed in adult tissues and that the abundance of its transcripts increase as a function of embryonic age.

In total, Meg9 has thirteen microRNAs as shown in Figure 2 and Table 2. Since several hundred genes are now known that contain at least one microRNA (see http://microrna.sanger.ac.uk/sequences/), we propose that this transcript for the sake of clarity be referred to as Meg9/Mirg to avoid unnecessary confusion with the vast number of other microRNA containing genes. In this imprinted region alone, Meg3/Gtl2, Anti-Peg11, and Meg8 also contain microRNAs as already presented. The name Meg9/Mirg has already been used in four previous publications [57], [58], [59], [60]. For the thirteen microRNAs that are contained in the Meg9/Mirg transcription unit, examples of genes that are predicted to be targeted by individual microRNAs as determined by miRanda are shown in Table 2. Some noteworthy mRNAs are Atp2a2, Bmp1, Calm2, Camk2g, Ctcf, Dicer1, Dlk1, Fgf10, H19, Kras, Mycn, Myf5, Myog, Myot, Mypn, Obscn, Peg10, Plagl1, Pdlim5, Sgce, Trp53, and Vim. Moreover, miR-134 was recently shown to regulate negatively Limk1 [61], Nanog, and LRH1 [62].

We thank Drs. Dedra Cutler and Jr-Gang Cheng, Lidia Hernandez, and Lori Sewell for excellent technical assistance and fruitful discussions.