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RNA. Jan 2012; 18(1): 135–144.
PMCID: PMC3261735

MicroRNAs 296 and 298 are imprinted and part of the GNAS/Gnas cluster and miR-296 targets IKBKE and Tmed9

Abstract

Genomic imprinting is the phenomenon whereby a subset of genes is differentially expressed according to parental origin. Imprinted genes tend to occur in clusters, and microRNAs are associated with the majority of well-defined clusters of imprinted genes. We show here that two microRNAs, miR-296 and miR-298, are part of the imprinted Gnas/GNAS clusters in both mice and humans. Both microRNAs show imprinted expression and are expressed from the paternally derived allele, but not the maternal allele. They arise from a long, noncoding antisense transcript, Nespas, with a promoter more than 27 kb away. Nespas had been shown previously to act in cis to regulate imprinted gene expression within the Gnas cluster. Using microarrays and luciferase assays, IKBKE, involved in many signaling pathways, and Tmed9, a protein transporter, were verified as new targets of miR-296. Thus, Nespas has two clear functions: as a cis-acting regulator within an imprinted gene cluster and as a precursor of microRNAs that modulate gene expression in trans. Furthermore, imprinted microRNAs, including miR-296 and miR-298, impose a parental specific modulation of gene expression of their target genes.

Keywords: microRNA, miR-296, miR-298, genomic imprinting, antisense, GNAS/Gnas, microarray

INTRODUCTION

Genomic imprinting is the phenomenon whereby some genes are monoallelically expressed according to parental origin. Thus, some imprinted genes are paternally expressed and maternally repressed, and others are maternally expressed and paternally repressed. Imprinted genes tend to occur in clusters that can extend for over a megabase and contain more than 15 genes (http://www.har.mrc.ac.uk/research/genomic_imprinting). MicroRNAs (miRNAs) have been located in the vicinity of imprinted clusters. miRNAs are a group of small, ~18–25-nt long, endogenous, single-stranded noncoding RNAs. miRNAs are initially produced as part of a long polyadenylated primary transcript (Cai et al. 2004) that is processed to excise a stem–loop to form the premature miRNA (Lee et al. 2003), and this, in turn, is processed to the mature miRNA by a protein complex that includes the RNase III enzyme Dicer (Hutvágner et al. 2001; Ketting et al. 2001). miRNAs regulate gene expression at the post-transcriptional or translational level. Mammalian miRNAs can target coding sequences (Forman et al. 2008; Tay et al. 2008), but most target the 3′ UTR of mRNAs and repress gene expression by affecting the stability of the transcript or by altering the translational efficiency (Bartel 2004; Doench and Sharp 2004; Filipowicz 2005). At least one miRNA can target promoter sequences and induce gene expression (Place et al. 2008).

miRNAs have been mapped to more than half of the 13 well-defined imprinted gene clusters in the mouse. Multiple miRNAs have been found at two of these clusters, the Dlk1-Gtl2 cluster on chromosome 12 and the Sfmbt2 gene on proximal mouse chromosome 2. In the Dlk1-Gtl2 cluster, they occur in two groups, arise from noncoding RNA, and are maternally expressed (Seitz et al. 2003, 2004). At the protein-coding Sfmbt2 gene, they are located within introns and are presumably paternally expressed (Zhang et al. 2010). Much smaller numbers of miRNAs have been found at other clusters. Two miRNAs have been found at the Igf2 cluster on distal chromosome 7, one of which, mir-483, is located within the paternally expressed protein-coding gene Igf2, and the other, mir-675, arises from the noncoding maternally expressed H19 transcript (Cai and Cullen 2007; Landgraf et al. 2007). Two miRNAs, mir-489 and mir-653, have been found in the Peg10 cluster on proximal chromosome 6; three miRNAs—mir-29a, mir-29b, and paternally expressed mir-335—have been found in a second cluster on proximal chromosome 6; mir-344 in the PW/AS cluster on chromosome 7 (Royo and Cavaille 2008) cluster; and the brain-specific, paternally expressed mir-184 in the Rasgrf1 cluster (Nomura et al. 2008).

Two miRNAs, mir-296 and mir-298, have been identified (Griffiths-Jones et al. 2006) in the vicinity of the imprinted Gnas/GNAS clusters in mouse distal chromosome 2 and human chromosome 20q13.3. The Gnas/GNAS clusters are similarly organized in both species with multiple alternatively spliced protein-coding transcripts arising from three different promoter regions and first exons that splice onto exon 2 of Gnas/GNAS (Figs. 1A, 2A) and two noncoding transcripts (Hayward et al. 1998a,b; Peters et al. 1999, 2006). The noncoding transcripts are only expressed from the paternal allele. One, Nespas/NESPAS, runs antisense to its sense counterpart Nesp/NESP (Hayward and Bonthron 2000; Holmes et al. 2003) and regulates its expression (Williamson et al. 2011). Nespas/NESPAS is also the candidate precursor of miR-296 and miR-298 because both miRNAs are in an antisense orientation.

FIGURE 1.
Characterization of mouse miR-296 and miR-298. (A) Overview of the mouse Gnas cluster (not to scale). Features of the maternal and paternal alleles are shown above and below the line. Transcripts arising from the first exons of protein-coding transcripts, ...

The mouse orthologs of these miRNAs—mmu-miR-296 and mmu-miR-298—were first found in differentiated embryonic stem (ES) cells and reported to be ES-cell-specific (Houbaviy et al. 2003). Subsequent studies have shown that mmu-miR-296 is up-regulated during ES differentiation (Tay et al. 2008; Sun et al. 2011) and targets the pluripotency gene Nanog (Tay et al. 2008) and thus has a role in promoting differentiation. Twelve other targets of mmu-miR-296 have been verified (Miranda et al. 2006). mmu-miR-296 is expressed during prenatal mouse development (Tang et al. 2007; Chiang et al. 2010), and in adult testis and brain and cancer cell lines (Landgraf et al. 2007; Chiang et al. 2010). mmu-miR-296 is also known to be down-regulated by high glucose treatment of a pancreatic β-cell line (Tang et al. 2009). mmu-miR-298 was found to be relatively highly expressed in newborn ovary (Ahn et al. 2010).

No expressed copies of the human ortholog hsa-miR-298 were found from sequencing 250 small RNA libraries, so its expression appears to be limited (Landgraf et al. 2007). However, hsa-miR-296 is expressed in several tissues including ES cells (Suh et al. 2004; Lakshmipathy et al. 2007; Landgraf et al. 2007; Dentelli et al. 2010; Mao et al. 2010; Scagnolari et al. 2010; Mayor-Lynn et al. 2011), and its expression is altered in a several different tumors (Wurdinger et al. 2008; Corbetta et al. 2010; Hong et al. 2010; Wei et al. 2011). It was shown to be a growth promoter and inhibitor of apoptosis in HeLa cells (Cheng et al. 2005) and has a clear role in tumor angiogenesis through targeting HGS hepatocyte growth factor–regulated tyrosine kinase substrate (Wurdinger et al. 2008). It also targets HMGA1, high motility group At-hook protein 1, whose overexpression in prostate cancer is correlated with low levels of hsa-miR-296 (Wei et al. 2011). Recently it has been shown to play a role in ion transport through targeting HWNK4, human with-no-lysine kinase-4 (Mao et al. 2010).

We show here that mir-296 and mir-298 are part of the Gnas/GNAS clusters and that they have imprinted expression and are paternally expressed and maternally repressed. We also identify and verify novel targets for both mmu-miR-296 and hsa-miR-296.

RESULTS AND DISCUSSION

Paternally expressed microRNAs in the Gnas/GNAS imprinted clusters

Analysis of the FANTOM full-length cDNA collection had shown that transcription of the paternally expressed Nespas transcript extends to the F1 clone ~30 kb from its promoter (Holmes et al. 2003). The F1 clone lies 46 bp downstream from mmu-miR-296 and 503 bp downstream from mmu-miR-298 (BAC sequence AL593857.10). Thus, both microRNAs, miR-296 and miR-298, lie within the Nespas transcription unit and are part of the Gnas cluster (Fig. 1A–C). Most canonical miRNA genes produce one dominant mature miRNA, usually from the 5′ arm of the pre-miRNA hairpin, but some, such as miR-296, produce mature miRNAs in appreciable amounts from both the 5′ and 3′ arms. However, in most, but not all, tissues, miR-296-5p is more highly expressed than miR-296-3p (Chiang et al. 2010), and work on expression reported below relates to miR-296-5p.

To determine whether mmu-miR-296-5p showed imprinted paternal expression like Nespas, we analyzed RNA from uniparental disomies for distal mouse chromosome 2. We used 14.5 days postcoitum (dpc) mouse embryos with either two paternal but no maternal copies of the Gnas cluster [PatDp(dist2)] or two maternal but no paternal copies [MatDp(dist2)] and litter-matched wild-type controls. By Northern blot, miR-296-5p was seen in the PatDp(dist2) embryo and the wild-type embryo but not the MatDp(dist2) embryo (Fig. 1D). This result was confirmed by real-time PCR, which showed that expression of miR-296-5p was elevated in PatDp(dist2) embryos but reduced in MatDp(dist2) compared with their wild-type sibs (Fig. 1E). Real-time PCR also showed imprinted expression of mmu-miR-298 because there was increased expression in PatDp(dist2) embryos and reduced expression in the MatDp(dist2) embryos compared with the wild-type sibs (Fig. 1E).

Therefore, we have established that mouse miR-296-5p and miR-298 show imprinted expression and are paternally expressed and maternally repressed. The nearest known paternally expressed promoter was the Nespas promoter, more than 27 kb away from miR-296-5p. Previously, a targeted deletion of the Nespas promoter had been generated (Williamson et al. 2006). Real-time PCR analysis showed that miR-296-5p expression was almost completely ablated in embryos carrying a paternally inherited copy of the Nespas promoter deletion (Nesptm1Jop) compared with wild-type sibs (Fig. 1F), but that expression of miR-296-5p was not affected when this deletion was maternally inherited (Fig. 1F). Thus, the Nespas promoter can give rise to a transcript that can function as a primary miRNA precursor of miR-296-5p and miR-298.

The human GNAS cluster has a similar architecture to the mouse Gnas cluster (Fig. 2A). NESPAS transcription extends through hsa-miR-298 and hsa-miR-296 as determined by an RNA expression tiling array analysis (I Vlatkovic and DP Barlow, pers. comm.). To determine whether hsa-miR-296 showed imprinted paternal expression like NESPAS, we analyzed RNA from two human lymphoblastoid cell lines. One was a parthenogenetic cell line with two maternal copies of the GNAS cluster and no paternal copies (LCL-FD) (Hayward and Bonthron 2000), and the other was a control cell line with one maternal and one paternal copy of the GNAS cluster (LCL-2). Real-time PCR on RNA extracted from these two cell lines showed that miR-296 was much reduced in the parthenogenetic lymphoblastoid cell line (Fig. 2B), indicating that hsa-miR-296 is an imprinted, maternally repressed miRNA.

FIGURE 2.
Expression of miR-296 in humans. (A) Overview of the human GNAS cluster. Features of the maternal and paternal alleles are shown above and below the line. Transcripts arising from the first exons of protein coding transcripts, NESP, GNASXL, and GNAS (filled ...

Thus, we have shown that miR-296 and miR-298 lie within the Nespas/NESPAS transcription units and therefore are members of the Gnas/GNAS clusters. Both miRNAs are poorly conserved and appear to be specific to mammals (Targetscan). Nespas is a noncoding macroRNA with a promoter in the imprinting control region (ICR), a region common to imprinted gene clusters that brings about parental-specific silencing throughout the cluster. The paternally expressed gene Nespas has a cis-acting role in regulating imprinted gene expression within the Gnas cluster, and either transcription of Nespas or the Nespas transcript itself mediates down-regulation of its sense counterpart Nesp through chromatin modification (Williamson et al. 2011). Two other paternally expressed macroRNA genes, Airn and Kcnq1ot1, also have key roles in imprinted gene silencing in their respective clusters, the Igf2r and Kcnq1 clusters, but neither host miRNAs. Nespas has now been shown to have a second function as a precursor of miR-296 and miR-298 that target genes outside the Gnas/GNAS clusters and thus regulate gene expression in trans.

Expression of miR-296-5p

The analysis of the imprinting and regulatory status of miR-296-5p showed that miR-296 was expressed in the mouse embryo at 14.5 dpc and 15.5 dpc. Further Northern blot analysis extended this time from at least 13.5 dpc to 16.5 dpc. The Gnas locus is known to have significant effects on postnatal physiology, and much analysis of the Gnas cluster has focused on neonates. Thus, we decided to look at the expression levels of miR-296-5p in various neonatal mouse tissues. Expression of miR-296-5p was seen in liver, brown adipose tissue (BAT), heart, and brain (data not shown). Lack of a consistently expressed endogenous control prevented a full comparison of expression levels between the neonatal tissues, but BAT showed lower miR-296-5p expression than both brain and heart.

Target sites

To identify new potential target sites in humans and mice, screens for changes in gene expression in HeLa cells and NIH-3T3 cells following transfection of miR-296-5p were carried out using the RNG-MRC Human set 25K microarray. This microarray is an oligonucleotide array consisting of 25,000 target genes. We checked that transfection of miRNAs into cells worked in our hands by showing that PTK1, a target gene of miR-1, was knocked down in HeLa cells transfected with miR-1 (data not shown).

Targets of hsa-miR-296-5p

Using HeLa cells, 62 potential targets of hsa-miR-296-5p were found from the microarray screen, of which 58 were up-regulated at least twofold (Supplemental Table 1) and four were down-regulated at least twofold. The four down-regulated genes were MRPS14 (mitochondrial ribosome protein subunit 14), TBCA (tubulin-specific chaperone A), SLC13S5 (solute carrier family 13 [sodium-dependent citrate transporter], member 5) and CARD14 (caspase recruitment domain family, member 14). MRPS14, TBCA, and SLC13S5 were investigated further.

MRPS14 and TBCA were confirmed as targets of miR-296 by real-time PCR analysis, but SLC13S5 was not (Fig. 3A; data not shown). TBCA showed an 83% reduction, and MRPS14 showed a 63% reduction following transfection of miR-296-5p. However, no target sites for miR-296-5p in the 3′ UTR, 5′ UTR, or the coding regions of MRPS14 or TBCA could be identified by bioinformatics analysis. We hypothesized that this was because MRPS14 and TBCA are secondary targets of hsa-miR-296-5p and decided to look for the primary target gene. Both MRPS14 and TBCA are reported to interact with IKBKE, inhibitor of the κ light polypeptide gene enhancer in B-cells, kinase epsilon, using Ingenuity software (Fig. 3B; Ewing et al. 2007). A target site for hsa-miR-296-5p was found in the 3′ UTR of IKBKE using the regulatory elements and motifs finder RegRNA (Huang et al. 2006) and Targetscan and confirmed using the RNAHybrid tool (Fig. 3C; Rehmsmeier et al. 2004; Kruger and Rehmsmeier 2006).

FIGURE 3.
miR-296 interacts directly with IKBKE to regulate TBCA and MRPS14 expression. (A) TBCA and MRPS14 levels were reduced in HeLa cells following transfection with hsa-miR-296 but not by a scrambled control as analyzed by Real-time PCR. Expression was normalized ...

To test for interaction between miR-296-5p and IKBKE, luciferase constructs were made in which the potential target site for miR-296-5p in the IKBKE 3′ UTR or a mutated target site was cloned into the pGL3 luciferase vector immediately 5′ of the firefly luciferase coding regions to provide the pGL3-IKBKE-3′UTR construct and the pGL3-IKBKE-3′UTR-mut construct. Each construct was then cotransfected with pre-miR-296-5p or a randomly generated negative control Pre-miR into 293A or HeLa cells. In addition, in HeLa cells, each construct was cotransfected with anti-miR-296-5p. Cotransfection of the pGL3-IKBKE-3′UTR construct containing the potential target site and pre-miR-296-5p into either 293A or HeLa cells resulted in decreased luciferase production of the reporter construct compared with the randomly generated negative control (highly significant in 293A cells, P < 0.01) (Fig. 3D,E). Cotransfection of the pGL3-IKBKE-3′UTR construct containing the potential target site and the anti-miR-296-5p increased luciferase production of the reporter construct (Fig. 3D,E). In contrast, when cotransfections were carried out with the pGL3-IKBKE-3′UTR-mut construct, with a mutated target site, neither pre-miR-296-5p nor anti-miR-296-5p had any significant effect on luciferase production from the mutant construct compared with the negative control (Fig. 3D,E). Together these results show that miR-296-5p can act on IKBKE by a target site in the 3′ UTR and suggest that MRPS14 and TBCA are downstream from IKBKE. IKBKE appears to be a primate-specific target for further bioinformatics analysis using RegRNA failed to identify target sites for mmu-miR-296-5p and rno-miR-296 (both of which have identical mature sequences to hsa-miR-296-5p; miRBase) in mouse and rat Ikbke 3′ UTRs.

Although IKBKE was one of the genes on the microarray, it was not identified as a target. The reasons for this are unclear, but it is known that a number of miRNA targets are regulated in the main by translational repression (Selbach et al. 2008). Thus, one possibility is that miR-296-5p represses the translation of IKBKE mRNA into protein, but not the abundance of IKBKE mRNA. If so, IKBKE would not be identified as a target in the microarray screen.

Targets of mmu-miR-296-5p

Using NIH-3T3 cells, 13 potential targets of mmu-miR-296-5p were found from the microarray screen, all of which were down-regulated 1.5-fold to twofold (Supplemental Table 2). One of these, Tmed9, had a target site in the 3′ UTR and was investigated further. It was confirmed as a target by real-time PCR analysis (Fig. 4A). It was also confirmed by luciferase assay in which a luciferase construct was used that contained the potential Tmed9 3′-UTR target site inserted into the PGL3 vector immediately 5′ of the firefly luciferase-coding region. Cotransfection of this construct with pre-miR-296-5p decreased luciferase production of the reporter vector containing the Tmed9 miR-296-5p response element compared with cotransfection with the randomly generated Pre-miR negative control (Fig. 4B). Together these results show that miR-296-5p can act on Tmed9 by a target site in the Tmed9 3′ UTR (Fig. 4C). From bioinformatics analysis, Tmed9 is a predicted target of miR-296-5p in rat and primates.

FIGURE 4.
miR-296 interacts with Tmed9. (A) Expression of Tmed9 was reduced in 3T3 cells transfected with miR-296-5p but not by a scrambled control as analyzed by real-time PCR. Expression was normalized to Gapdh; error bars indicate RQ minimum and RQ maximum; ...

Gene network

IKBKE along with MRPS14 and TBCA and Tmed9 have not previously been associated but can be linked together in a network together with TRAF6 (TNF receptor-associated factor 6), involved in signaling (Ingenuity Systems). IKBKE, a member of the IKK family, has a known role in innate immunity, but less is known about MRPS14, TBCA, and Tmed9.

MRPS14 encodes a structural constituent of the ribosome, TBCA is involved in protein folding and the generation of microtubules, and Tmed9 is a member of a family of genes encoding transport proteins located in the endoplasmic reticulum and the Golgi. Our findings indicate that IKBKE can be linked to MRPS14 and TBCA, as do protein interaction studies (Ewing et al. 2007).

IKBKE is involved in many signaling pathways, including Toll-like receptor signaling and signal transduction resulting in induction of apoptosis. The level of apoptosis in HeLa cells is increased when miR-296 is inhibited (Cheng et al. 2005), a finding that is in keeping with our observation that inhibition of miR-296 leads to increased levels of IKBKE. Interestingly, IKBKE is also a predicted target of hsa-mir-298 (Targetscan) and so may be regulated by both microRNAs that arise from the imprinted antisense Nespas transcript.

Imprinting at a distance

The finding that both miR-296 and miR-298 show imprinted paternal expression has important ramifications for downstream genes that will have a parental-specific regulation imposed on them regardless of whether they themselves are biallelically expressed or imprinted. How influential this parental-specific regulation can be may depend in part at least on the number of other microRNAs that target these genes. None of the target genes of miR-296-5p identified thus far, including the ones verified in the present study, IKBKE and Tmed9, and secondary targets TBCA and MRPS14 are located in a known imprinted region, and none are known to show imprinted expression. With the discovery that mmu-miR-296 is imprinted, the influence of the Gnas/GNAS cluster is widened to include genes potentially throughout the genome because an individual miRNA can target and regulate many genes. This effect of “imprinting at a distance” is also true for the target genes of mmu-miR-298 such as β-amyloid precursor protein converting enzyme 1 (BACE1) (Boissonneault et al. 2009). BACE1 does not map to any known imprinted cluster, and there is no published information to suggest that it is expressed from one parental allele but not the other, so again this is likely to be a gene that has an imprinted regulation imposed on it through regulation by an imprinted miRNA.

MATERIALS AND METHODS

RNA extraction

RNA >200 nt was isolated using the RNeasy Mini Kit (QIAGEN). RNA <200 nt was isolated using the miRVana isolation kit (Ambion) according to the manufacturer's instructions, and then was further cleaned by ethanol precipitation.

Northern blotting

Northern blots were carried out using 30 ng of RNA <200 nt in length on a 12.5% denaturing acrylamide gel. The probe used to identify mature miR-296-5p was a polynucleotide kinase dATP 5′-end-labeled LNA oligonucleotide (Exiqon); the probe to identify let-7c was a polynucleotide kinase dATP-labeled oligonucleotide.

Quantitative PCR

RNA >200 nt was used in a reverse transcription reaction using the High Capacity Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Amplification was performed using a 7500 Fast machine according to the manufacturer's instructions. The primers and probes for amplification and analysis were TaqMan assays (Applied Biosystems).

Quantitative real-time PCR for microRNAs

RNA <200 nt was used in a reverse transcription reaction using the TaqMan microRNA reverse transcription kit (Applied Biosystems) and a probe contained in the Taqman assay, either Sno202, U6, miR-296-5p, miR-298, or let-7c. Amplification was performed using a 7500 Fast machine (Applied Biosystems) according to the manufacturer's instructions.

Quantitative real-time PCR analysis

To analyze the data produced by the quantitative real-time PCR analysis, relative quantification was used. The threshold value was determined using the default setting, and all experiments were performed in triplicate or quadruplicate. let-7c, Sno202, and U6 were used as the control genes for small RNA experiments with small RNAs; 18S was used as the control gene in other experiments. Data were analyzed using the 7500 software (Applied Biosystems), relative quantification was determined by the calculation 2ΔΔCT. The error bars for the bar graph were calculated using the RQ minimum, calculated as 2 − [ΔΔCT + T × SE(ΔCT)], and the RQ maximum, calculated as 2 − [ΔΔCT − T × SE(ΔCT)]. Prior to use, all assays were verified as between 90% and 110% efficiency.

Cell culture and transient transfection

A normal female lymphoblastoid cell line (LCL-2) and a parthenogenetic LCL (LCL-FD) (kindly provided by Doug Higgs and David Bonthron, respectively) (Hayward and Bonthron 2000) were maintained in RPMI 1640 plus glutamine (GIBCO) with 10% fetal bovine serum (Invitrogen) and 0.5% antibiotic/antimyotic (Invitrogen). HeLa, 293A, and NIH-3T3 cells were maintained in DMEM plus Glutamax (GIBCO) with 10% fetal bovine serum (Invitrogen) and 0.5% antibiotic/antimyotic (Invitrogen). For the transfections, cells were plated into six-well plates at a density of 1.5 × 105 cells per well. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions with 26 nM pre-miR-296-5p (Ambion), 26 nM random sequence Pre-miR (Ambion Pre-miR miRNA Precursor Negative Control), 26 nM miR-1 (Ambion), or 26 nM anti-miR-296-5p (Ambion). Cotransfections of one of the small RNAs described above along with 800 ng of a pGL3 construct (Promega) containing a putative target site or a mutated putative target site were also performed. All transfections were performed in a minimum of triplicate.

Microarray analysis

Total RNA was prepared from HeLa cells transfected with miR-1, miR-296-5p, or a randomly generated negative Pre-miR. For each transfection, equal amounts of RNA from three technical replicates were pooled. Double-stranded cDNA was then synthesized from 1 μg of total RNA using the SMART PCR cDNA synthesis Kit (Clontech), and purified using QIAGEN Qiaquick clean-up columns. One microgram of purified cDNA was labeled with 2 μL of Cye dye (GE Healthcare) using the Bioprime labeling kit (Invitrogen). After incubating for 3 h at 37°C, the labels were purified using ProbeQuant G50 micro columns (GE Healthcare). Each of the microRNAs were cohybridized with the pool of randomly generated negative Pre-miR. Three arrays were hybridized per comparison and a dye swap was also included. The labels were resuspended in 40 μL of hybridization buffer (40% deionized formamide; 5× Denhart's; 5× SSC; 1 mM Na pyrophosphate; 50 mM Tris at pH 7.4; 0.1% SDS) and hybridized onto a RNG-MRC Human set 25K microarray printed on GE Codelink slide (http://www.har.mrc.ac.uk/services/MPC/microarray/), overnight at 48°C in a water bath using the Corning hybridization chambers. After hybridization, the arrays were washed initially in 2× SSC until the coverslip had come off, then 5 min with vigorous shaking in 0.1× SSC, 0.1% SDS, and then finally in 0.1× SSC for 2 min with vigorous shaking. The arrays were then spun dry and scanned. The arrays were scanned using a ProScanArray HT (PerkinElmer) at seven different photomultiplicator gain settings from 40 to 70. The images were then processed using ImaGene 6.0.1 (Bio Discovery). Global gene expression was examined in the miR-1 and miR-296-5p RNA pools against a randomly generated negative Pre-miR control on an oligonucleotide array consisting of 25,000 target genes. The extracted multiple-scan data sets were processed using Mavi 2.6.0 (MWG Biotech AG). The data were then loaded into GeneSpring GX (Agilent technologies) for data normalization. Lists of genes that were twofold increased or decreased in miR-1 or miR-296-5p compared with the randomly generated negative Pre-miR control across all three arrays were produced.

Total RNA was also prepared from mouse NIH-3T3 cells transfected with either miR-296-5p or a randomly generated negative Pre-miR, and a microarray experiment was performed as described above.

Plasmid construction

Oligonucleotides encompassing a 27-bp portion of the IKBKE 3′ UTR that contained the putative miR-296-5p binding site (5′-CTAGTCTAGACCAAGGCCAGTGCCAGTGTCTTGGGGCCCCTTCTAGACGT-3′ and 5′-ACGTCTAGAAGGGGCCCCAAGACACTGGCACTGGCCTTGGTCTAGACTAG-3′) or that contained the target site but with the seed region mutated to an EcoRI site (5′-CTAGTCTAGACCAAGGCCAGTGCCAGTGTCTTGAATTCTCTTCTAGACGT-3′ and 5′-ACGTCTAGAAGAGAATTCAAGACACTGGCATGGCCTTGGTCTAGACTAG-3′) or that encompassed the Tmed9 3′-UTR putative miR-296-5p binding site (5′-TCTAGAAAGGGTGTGGAGGGGTTGCAGTGCACAAAAGGGGCCCATCTAGA-5′ and 5′-TCTAGATGGGCCCCTTTTGTGCACTGCAACCCCTCCACACCCTTTCTAGA-3′) were obtained from Sigma-Aldrich. The oligos were annealed, digested with XbaI, and ligated into the pGL3 control vector (Promega) in the multiple cloning region immediately 5′ of the luciferase gene to form the pGL3-IKBKE-3′UTR construct and the pGL3-IKBKE-3′UTR-mut construct. The sequence and orientation of the insertion were checked by sequencing (GeneService).

Luciferase assay

Quantification of Renilla and firefly luciferase was performed on a Luminoskan Ascent Luminometer (Thermo Labsystems) and using the duel-luciferase reporter assay system (Promega). Firefly luciferase expression was normalized for transfection efficiency by comparison to the Renilla luciferase expression.

Mouse crosses

The animal studies were carried out under the guidance issued by the Medical Research Council in “Responsibility in the Use of Animals for Medical Research” (July 1993) and under Home Office Project Licence Numbers 30/1518, 30/2065, and 30/1074. The inbred strain C3H/HeH or the (C3H/HeH × 101/H) F1 hybrid was used for production of some wild-type embryos. Embryos carrying a targeted deletion of the Nespas promoter (Nespas+/Nespastm1Jop or Nespastm1Jop/Nespas+ [maternally derived allele listed first]) were generated as previously described. Embryos with paternal or maternal disomy of distal chromosome two were generated as previously described (Wroe et al. 2000).

Statistical methods

All comparisons were made using an unpaired two-tailed Student's t-test. Statistical significance was taken when P < 0.05. Results are expressed as mean ± (RQ maximum)/(RQ minimum) for all real-time PCR analysis or mean ± SEM for all other analysis.

SUPPLEMENTAL MATERIAL

Supplemental material is available for this article.

ACKNOWLEDGMENTS

We thank Doug Higgs (WIMM, Oxford, UK) and David Bonthron (LIMM, Leeds, UK) for the provision of cell lines, Irena Vlatcovic and Denise Barlow for access to their unpublished results, Christine Williamson for useful discussions, and Lynn Jones and Diane Napper for animal husbandry.

Authors' contributions: J.E.R. and J.P. designed the research; J.E.R., S.A.E., and D.W. performed the research; J.E.R., D.W., P.U., and J.P. analyzed data; and J.E.R., S.A.E., D.W., and J.P. wrote the paper.

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.029561.111.

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