Expression analysis of AK003491, an imprinted noncoding RNA, during mouse development

ABSTRACT

The Dlk1-Dio3 imprinted domain on mouse chromosome 12qF1 contains three paternally expressed protein-coding genes and multiple maternally expressed long or short noncoding RNA genes. All these imprinted genes are regulated by IG-DMR located between Dlk1 and Meg3/Gtl2. Recently, several novel imprinted noncoding RNAs were identified in the intergenic region of this domain, although the exact number of imprinted genes within the region is unclear. Here, we report that a novel noncoding RNA, AK003491, located between Meg3/Gtl2 and Meg8, is maternally expressed in E15.5 brain, tongue, heart, lung, liver and kidney tissues. In situ hybridization analysis at E15.5 shows AK003491 is predominantly expressed in forebrain, tongue, thymus, somites, lung and liver. Quantitative real-time RT-PCR analysis confirms this expression pattern and detects highest expression in tongue. While the AK003491 expression pattern at postnatal day 1 is similar to E15.5, AK003491 expression at postnatal day 30 is mainly restricted to the brain. These results expand the number of known imprinted long noncoding RNAs in this domain, and contribute to further investigation of their regulatory mechanism and function.

INTRODUCTION

Genomic imprinting is an epigenetic phenomenon enabling genes to be monoallelically expressed in a parent-of-origin-dependent manner. To date, approximately 100 imprinted genes have been identified in mouse, with most located in clusters harboring differentially methylated regions (Bartolomei and Ferguson-Smith, 2011). Genes within large imprinted domains can be regulated by long-range cis-acting elements carrying parental-specific epigenetic modifications (Edwards and Ferguson-Smith, 2007).

Recently, the extent of activation of the Dlk1-Dio3 imprinted domain on mouse chromosome 12qF1 was shown to be positively correlated with pluripotency levels of mouse induced pluripotent stem (iPS) cells. Long noncoding RNAs, such as Meg3/Gtl2 and Rian, and numerous miRNAs clustered within this domain, are activated in fully pluripotent embryonic stem cells but aberrantly silenced in mouse iPS cells showing low pluripotency and reprogramming efficiency (Liu et al., 2010; Stadtfeld et al., 2010). Within the Dlk1-Dio3 imprinted domain (Fig. 1A), there are three paternally expressed protein-coding genes (Dlk1, Rtl1/Peg11 and Dio3), multiple maternally expressed long noncoding RNA genes (including Meg3/Gtl2, anti-Rtl1, Meg8, Irm and Meg9/Mirg), and numerous microRNAs and small nucleolar RNAs (snoRNAs) (Miyoshi et al., 2000; Schmidt et al., 2000; Takada et al., 2000; Tierling et al., 2006; da Rocha et al., 2008; Hagan et al., 2009). An intergenic germline-derived differentially methylated region (IG-DMR), located ~13 kb upstream of the Meg3/Gtl2 promoter, functions as the imprinting control region for this domain (Takada et al., 2002; Lin et al., 2003). To date, the total number of noncoding RNAs located within this region is unclear, and the precise genomic organization and functional relationship between these noncoding RNAs is not known. Multiple Riken cDNA clones and ESTs map to intergenic regions in the Dlk1-Dio3 domain, therefore the purpose of this study was to identify novel imprinted genes within this domain and determine their expression patterns during mouse development.

Fig. 1.

Schematic representation of the mouse Dlk1-Dio3 imprinted domain and genomic location of AK003491. (A) The Dlk1-Dio3 domain contains three paternally expressed protein-coding genes, Dlk1 (EU434917), Rtl1/Peg11 (EU434918), and Dio3 (AK146060), and several maternally expressed noncoding RNA genes, Mico1 (EF648170), Mico1-os (EF648171), Meg3/Gtl2 (Y13832), Anti-Rtl1 (EU434921), Meg8 (EU434919), Irm (AF498294), AK050713, AK053394, and Meg9/Mirg (EU616813). IG-DMR is the imprinting control region in this domain. Arrows indicate transcriptional orientations. Exons are dark colored while introns are light. (B) AK003491, AK038654 and AK141773 overlap with one another, and are located in the intron of Rtl1/Peg11, which is transcribed in the opposite orientation. The indicated primer sets, F1/R1, F2/R2 and F3/R3, were used for imprinting analysis, synthesis of RNA probes and real-time PCR, respectively. The red bar indicates the position of the SNP used in imprinting analysis. Several miRNAs are processed from anti-Rtl1 and regulate Rtl1/Peg11 in trans.

Recently, in the region between Dlk1 and Gtl2, two overlapping ~2 kb transcripts, Mico1 (EF648170) and Mico1-os (EF648171), were shown to be expressed from opposite strands exclusively from the maternal chromosome. They most likely represent noncoding RNAs, and their expression is tissue-specific and shows diurnal and circadian oscillations (Labialle et al., 2008). In the region between Meg8 and Meg9/Mirg, AK050713 and AK053394 were found to be maternally expressed in primary mouse embryonic fibroblasts (Hagan et al., 2009). In this study, we focused on a Riken mRNA, AK003491, located in the intergenic region between Meg3/Gtl2 and Meg8 (Fig. 1A). Until now, its imprinting status and expression pattern were unknown. Here, we demonstrate that AK003491 is maternally expressed and provide detailed expression analysis for AK003491 during mouse embryonic development and after birth.

MATERIALS AND METHODS

Mouse strains

C57BL/6J and ICR mice were purchased from the Beijing Laboratory Animal Research Center (Beijing, China). Presence of vaginal plugs the morning after mating was defined as E0.5. Animal experiments were performed in compliance with the “Rules for experimental animals” published by the Chinese Government.

DNA and RNA isolation, and cDNA synthesis

Genomic DNA was isolated from mouse tissues by proteinase K and RNase A digestion, followed by phenol chloroform extraction and ethanol precipitation. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using 2 μg total RNA in a 25 μl reaction volume, containing 5 μl 5 × M-MLV reaction buffer, 1 μl oligo d(T)₁₈ primers (0.5 μg/μl, Takara, Dalian, China), 1.25 μl dNTP (10 mM each), 0.5 μl RNase inhibitor (40 units/μl), and 1 μl (200 units) M-MLV reverse transcriptase (Promega, Madison, WI, USA), with incubation at 42℃ for 60 min.

SNP identification and imprinting analysis

Genomic DNA samples from parents and F1 embryos were amplified with primers and directly sequenced to identify SNPs between C57BL/6J (B6) and ICR mice. For AK003491, an SNP (B6, C; ICR, T) was found at chr12:110833929 (mm9). For imprinting analysis, total RNA was isolated from E15.5 BIF1 (B6♀ × ICR♂) and IBF1 (ICR♀ × B6♂) brain, tongue, heart, lung, liver and kidney, and treated with DNase I (Roche, Mannheim, Germany) to exclude genomic DNA. The genomic DNA-free total RNA was reverse transcribed as described above. RT-PCR was performed using appropriate primers (F1: 5′-GTCGGTTGGAAAGGAGTGT-3′, R1: 5′-TTGTGAAATAGCAGGCATCT-3′). Amplified products were purified and directly sequenced using the ABI PRISM 3130 Genetic Analyzer with a BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

Preparation of embryos and in situ hybridization analysis

E15.5 embryos were fixed with 4% paraformaldehyde in PBS and incubated at 4℃ overnight. Fixed embryos were dehydrated and paraffin embedded. Sections (10 μm) were cut and adhered to slides. Prehybridization was performed in hybridization buffer (50% formamide, 5×SSC, 0.1% Tween 20, 50 μg/ml ssDNA, 50 μg/ml heparin) at 65℃ for 1 h. Hybridization was performed at 65℃ overnight in hybridization buffer with probes added (final concentration 500 ng/ml). Signals were detected using an anti-digoxigenin antibody conjugated to alkaline phosphatase, with final detection using NBT/BCIP Solution (Roche). To generate antisense and sense RNA probes, AK003491 RT-PCR products were amplified using appropriate primers (F2: 5′-TCACATCTCAATGCCACTC-3′, R2: 5′-CCTGTACCAGACGAAATCA-3′), and subcloned into pBluescript^® II KS (+) vector (Stratagene) via T-A cloning. After sequencing confirmation, plasmids were linearized and used as templates for in vitro transcription to synthesize probes using a DIG RNA labeling kit (Roche).

Quantitative real-time RT-PCR

Total RNA samples were isolated in triplicate from E15.5 brain, tongue, heart, lung, liver and kidney tissues, and reverse transcribed as described above. Real-time PCR was performed using the Applied Biosystems 7500 Real-Time PCR System with SYBR^® Green PCR Master Mix (Applied Biosystems) and the following AK003491 primers: (F3: 5′-GAAAGAGGAGAAATTGGG-3′, R3: 5′-GTTTTCAGCACATAGTAGG-3′). Results were normalized using Gapdh (F: 5′-AAATGGTGAAGGTCGGTGTGA-3′, R: 5′-CCGTTGAATTTGCCGTGAGTG-3′). Reaction specificity was controlled by post-amplification melting curve analysis, and the relative standard curve method was used to determine the AK003491 expression levels.

Semiquantitative RT-PCR

To determine the AK003491 expression pattern after birth, total RNA samples were isolated from various tissues at postnatal day 1 and day 30. PCR amplification was performed for AK003491 and Gapdh using the same primers as above.

RESULTS

AK003491 is a noncoding RNA located within the intron of Rtl1/Peg11

To find novel imprinted genes in the intergenic region of the mouse Dlk1-Dio3 domain, we performed a detailed search for expressed sequences using the UCSC Genome Browser (http://genome.ucsc.edu/), and identified three Riken mRNAs, AK003491, AK038654 and AK141773 (Fig. 1B). All three mRNAs map into the intron of Rtl1/Peg11 (EU434918), a protein-coding gene transcribed exclusively from the paternal chromosome in the opposite orientation (Hagan et al., 2009). Moreover, all three mRNAs are unspliced, overlap with one another, and are transcribed in the same direction as Meg3/Gtl2. Using the Open Reading Frame Finder (www.ncbi.nlm.nih.gov/gorf/gorf.html), no obvious open reading frames were identified in any of their sequences, suggesting they are noncoding RNAs like Meg3/Gtl2.

AK003491 is specifically expressed from the maternal allele

Allele-specific expression analysis of AK003491 was performed by directly sequencing RT-PCR products amplified from tissues from the mouse lines, BIF1 and IBF1. A C/T SNP was used to discriminate the two parental alleles. When strand-specific RT-PCR was performed, only sense strand transcripts were detected, indicating AK003491 is transcribed in the sense orientation (Fig. 2A); direct sequencing of the RT-PCR products showed AK003491 is maternally expressed (data not shown). When oligo dT primers were used for cDNA synthesis, monoallelic expression was identified in all tissues tested. As indicated from sequencing chromatograms, AK003491 is maternally expressed in E15.5 brain, tongue, heart, lung, liver and kidney (Fig. 2B). This imprinting pattern is the same as numerous other noncoding RNAs, from Meg3/Gtl2 to Mirg. We were unable to identify SNPs for AK038654 or AK141773 within the mouse strains in our laboratory, namely C57BL/6J, BALB/cJ, DBA/2J, FVB/NJ and ICR, therefore we could not investigate their imprinting status. However, as they overlap with AK003491, it is possible they are also maternally expressed.

Fig. 2.

Imprinting analysis of AK003491 by direct sequencing of RT-PCR products. (A) Strand-specific RT-PCR detected only sense strand transcripts. (B) As indicated by the SNP, AK003491 is expressed only from the maternal allele in brain, tongue, heart, lung, liver and kidney at E15.5.

Expression analysis of AK003491 by in situ hybridization

An essential step towards understanding the function of noncoding RNAs is to determine their expression pattern during development. As AK003491, AK038654 and AK141773 overlap with each other, we chose to focus on AK003491, and examined its expression pattern at E15.5 by performing in situ hybridization analysis. AK003491 expression signal was predominantly detected in the olfactory epithelium, forebrain, tongue, thymus, somites, lung and liver (Fig. 3). Using the corresponding sense probe under the same experimental conditions, we did not detect any signal (Fig. 3B), showing specificity of the antisense probe.

Fig. 3.

AK003491 expression analysis by in situ hybridization at E15.5. Signal is detected in the olfactory epithelium, Oe (C), forebrain, Fb (D), tongue, To (E), somites, So (F, G), thymus, Th (H), lung, Lu (I), and liver, Li (J). No signal was detected with the sense probe (B). Scale bars, 1 mm.

Real-time PCR analysis of AK003491

To quantify relative AK003491 expression levels in various tissues, and to validate our in situ hybridization results, we performed quantitative real-time RT-PCR analysis of AK003491 using E15.5 brain, tongue, heart, lung, liver and kidney. The results showed AK003491 was ubiquitously expressed, with the highest expression in the tongue (Fig. 4A). Moreover, expression levels in brain, lung and liver were greater than in heart and kidney. Despite maternal expression of AK003491 in all tested tissues, expression levels in each tissue are different, suggesting tissue-specific regulatory mechanisms may exist.

Fig. 4.

AK003491 expression analysis at E15.5, P1 and P30. (A) Quantitative real-time RT-PCR analysis of AK003491 in E15.5 brain, tongue, heart, lung, liver and kidney. Results were normalized using Gapdh. Data are presented as mean ± SD, from at least three mouse embryos. (B) Semiquantitative RT-PCR analysis of AK003491 at postnatal days 1 (P1) and 30 (P30). (C) AK003491 is maternally expressed in the brain at P30. Marker (M), brain (Br), tongue (To), thymus (Th), heart (He), lung (Lu), liver (Li), kidney (Ki), testis (Te), ovary (Ov), and negative control (NC).

AK003491 is highly expressed in adult brain

Semiquantitative RT-PCR at postnatal day 1 showed a similar expression pattern to E15.5, with the highest AK003491 expression in the tongue, moderate expression in brain, lung and liver, and lower expression in heart and kidney (Fig. 4B). However, at postnatal day 30, AK003491 expression is restricted to the brain (Fig. 4B), and is still maternally allele-specifically expressed (Fig. 4C), suggesting AK003491 functions in the adult brain. This is consistent with a previous report showing that in adult tissues, expression of noncoding RNAs, such as Meg3/Gtl2 and Anti-Rtl1, are restricted to the brain (Hagan et al., 2009).

DISCUSSION

We have shown that AK003491 within the mouse Dlk1-Dio3 domain is a novel maternally expressed noncoding RNA. Serial expression analysis shows AK003491 adopts a similar expression pattern (during embryonic development and postnatally) to that of other previously described noncoding RNAs within this domain (Tierling et al., 2006; Hagan et al., 2009; Gu et al., 2011; Zhang et al., 2011), and supports the notion that these noncoding RNAs, ranging from Meg3/Gtl2 to Meg9/Mirg, may form a large polycistronic transcription unit (da Rocha et al., 2008).

The exact transcriptional start sites and overlapping genomic organization of these maternally expressed noncoding RNAs are likely complex, as evident with the miR-433/127 locus, where the primary transcripts of miR-433 and miR-127 overlap in a compact space, in a 5’-3’ unidirectional manner (Song and Wang, 2008). Several miRNAs are processed from anti-Rtl1 and regulate Rtl1/Peg11 in trans by guiding RNA-induced silencing complex (RISC)-mediated cleavage of mRNA (Davis et al., 2005). It would be interesting to determine if AK003491, AK038654 and AK141773 function as primary miRNA transcripts and/or if they influence transcription or interact with transcripts of the paternally expressed Rtl1 gene.

The function of AK003491 is unknown, but several lines of evidence provide hints to its potential role. Recently, a RIP-seq method was developed to capture the Polycomb repressive complex 2 (PRC2) transcriptome in mouse embryonic stem cells (Zhao et al., 2010). The results show that Meg3/Gtl2 RNA directly, and specifically, binds PRC2. The locus of AK003491 is also included in the PRC2 transcriptome, suggesting AK003491 may function as a noncoding RNA by directly binding to the components of PRC2. Alternatively, AK003491 may function as a primary miRNA transcript, as the Dlk1-Dio3 domain contains one of the largest known miRNA clusters in mouse (Seitz et al., 2003, 2004). Indeed, AK003491 is located 418 bp downstream of the mir-136 precursor sequence, and there are continuous and overlapping ESTs between AK003491 and pre-mir-136, therefore it is feasible that AK003491 may be a partial sequence of the primary mir-136 transcript.

In conclusion, we have identified AK003491 as a novel maternally expressed noncoding RNA, and analyzed its expression pattern in mouse tissues from E15.5 to P30. AK003491 is highly expressed in forebrain, tongue, thymus, somites, lung and liver at E15.5, but is mainly restricted to the brain at postnatal day 30. Our results will contribute to further study of its regulatory mechanism and function.

ACKNOWLEDGMENT

This study was supported by grants from the National Natural Science Foundation of China (Nos. 31171383 and 31100934), and the Fundamental Research Funds for the Central Universities (No. HIT.NSRIF.2010027).

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