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Assessing the effect of the CLPG mutation on the microRNA catalogue of skeletal muscle using high-throughput sequencing

ABSTRACT: The callipyge phenotype is a monogenic muscular hypertrophy that is only expressed in heterozygous sheep receiving the CLPG mutation from their sire. The wild-type phenotype of CLPG/CLPG animals is thought to result from translational inhibition of paternally expressed DLK1 transcripts by maternally expressed miRNAs. To identify the miRNA responsible for this trans-effect, we used high-throughput sequencing to exhaustively catalogue miRNAs expressed in skeletal muscle of sheep of the four CLPG genotypes. We have identified 747 miRNA species of which 110 map to the DLK1-GTL2 or callipyge domain. We demonstrate that the latter are imprinted and preferentially expressed from the maternal allele. We show that the CLPG mutation affects their level of expression in cis (~3.2-fold increase) as well as in trans (~1.8-fold increase). In CLPG/CLPG animals, miRNAs from the DLK1-GTL2 domain account for ~20% of miRNAs in skeletal muscle.We show that the CLPG genotype affects the levels of A to I editing of at least five pri-miRNAs of the DLK1-GTL2 domain, but that levels of editing of mature miRNA are always minor. We present suggestive evidence that the miRNAs from the domain target the ORF of DLK1 thereby causing the trans-inhibition underlying polar overdominance. We highlight the limitations of high-throughput sequencing for digital gene expression profiling as a result of biased and inconsistent amplification of specific miRNAs.

ORGANISM(S): Ovis aries Rattus norvegicus Human gammaherpesvirus 8 Betapolyomavirus hominis Mus musculus Human alphaherpesvirus 1 JC polyomavirus Betapolyomavirus macacae Human immunodeficiency virus 1 human gammaherpesvirus 4 Homo sapiens Human betaherpesvirus 5

PROVIDER: GSE24146 | GEO | 2010/09/24

SECONDARY ACCESSION(S): PRJNA130023

REPOSITORIES: GEO

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Project description:The callipyge phenotype is a monogenic muscular hypertrophy that is only expressed in heterozygous sheep receiving the CLPG mutation from their sire. The wild-type phenotype of CLPG/CLPG animals is thought to result from translational inhibition of paternally expressed DLK1 transcripts by maternally expressed miRNAs. To identify the miRNA responsible for this trans-effect, we used high-throughput sequencing to exhaustively catalogue miRNAs expressed in skeletal muscle of sheep of the four CLPG genotypes. We have identified 747 miRNA species of which 110 map to the DLK1-GTL2 or callipyge domain. We demonstrate that the latter are imprinted and preferentially expressed from the maternal allele. We show that the CLPG mutation affects their level of expression in cis (~3.2-fold increase) as well as in trans (~1.8-fold increase). In CLPG/CLPG animals, miRNAs from the DLK1-GTL2 domain account for ~20% of miRNAs in skeletal muscle.We show that the CLPG genotype affects the levels of A to I editing of at least five pri-miRNAs of the DLK1-GTL2 domain, but that levels of editing of mature miRNA are always minor. We present suggestive evidence that the miRNAs from the domain target the ORF of DLK1 thereby causing the trans-inhibition underlying polar overdominance. We highlight the limitations of high-throughput sequencing for digital gene expression profiling as a result of biased and inconsistent amplification of specific miRNAs. These data were used to confirm the expression levels found by the high-throughput sequencing analysis. A total of 8 samples (two animals per callipyge genotype) were analysed using the miRCury LNA microRNA array (Exiqon). On each array, the test sample labelled with Hy3 was hybridized on one channel, and a pool made with the 8 test samples (labelled with Hy5) was used on the other channel as a reference for normalization. The callipyge mutation (C) is a SNP localised in an imprinted domain. Thus, heterozygous animals will have different phenotypes. The following 4 genotypes were analyzed in this study: NN: Wild-type (+mat/+pat) CN: Maternal heterozygous (Cmat/+pat) NC: Paternal heterozygous (+mat/Cpat) CC: Homozygous mutant (Cmat/Cpat)

Project description:MicroRNAs (miRNAs) are a class of small non-coding RNAs that function as post-transcriptional regulators of gene expression. Many miRNAs are expressed in the developing brain and regulate multiple aspects of neural development including neurogenesis, dendritogenesis and synapse formation. Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by mutations in the gene encoding Methyl-CpG binding protein 2 (MECP2). While Mecp2 is known to act as a global transcriptional regulator, miRNAs that are directly regulated by Mecp2 in the brain are not known. Using massively parallel sequencing methods, we have identified miRNAs whose expression is altered in cerebella of Mecp2-null mice before and after the onset of severe neurological symptoms. In vivo genome-wide analyses indicate that promoter regions of a significant fraction of dys-regulated miRNA transcripts, including a large polycistronic cluster of brain-specific miRNAs, are DNA methylated and directly bound by Mecp2. Functional analysis demonstrates that the 3’ untranslated region (UTR) of messenger RNA encoding Brain-derived neurotrophic factor (Bdnf) can be targeted by multiple miRNAs aberrantly up-regulated in absence of Mecp2. Taken together, these results suggest that dys-regulation of miRNAs may contribute to RTT pathoetiology, and also provide a valuable resource to further investigate the role of miRNAs in RTT. Chromatin extracted from postnatal 6-8 week old cerebellar (CB) tissues of wild-type (WT) or Mecp2-null (KO) male mice was immunoprecipitated with indicated antibodies and analyzed by a NimbleGen mouse 385K genomic tiling microarray (the array set26 of a 37-array set, which covers the mouse chromosome 12 that includes the entire Dlk1-Gtl2 imprinting domain). Whole cell extract (WCE) was used as input controls for ChIP or MeDIP/WCE experiments. For ChIP/ChIP experiments, immunoprecipitated DNA from WT and KO CB was directly compared on the same microarrays. DNA methylation profiles in WT CB were also analyzed by methylated DNA immunoprecipitation (MeDIP) followed by hybridization to the same genomic tiling microarrays (MeDIP-chip).

Project description:Background: Adenosine deaminases that act on RNA (ADARs) bind to double-stranded and structured RNAs and deaminate adenosines to inosines. This A to I editing is widespread and required for normal life and development. Besides mRNAs and repetitive elements, ADARs can target miRNA precursors. Editing of miRNA precursors can affect processing efficiency and alter target specificity. Interestingly, ADARs can also influence miRNA abundance independent of RNA-editing. In mouse embryos where editing levels are low, ADAR2 was found to be the major ADAR protein that affects miRNA abundance. Here we extend our analysis to adult mouse brains where high editing levels are observed. Results: Using Illumina deep sequencing we compare the abundances of mature miRNAs and editing events within them, between wild-type and ADAR2 knockout mice in the adult mouse brain. Reproducible changes in abundance of specific miRNAs are observed in ADAR2 deficient mice. Most of these quantitative changes seem unrelated to A to I editing events. However, many A to G transitions in cDNAs prepared from mature miRNA sequences, reflecting A to I editing events in the RNA, are observed with frequencies reaching up to 80%. About half of these editing events are primarily caused by ADAR2 while a few miRNAs show increased editing in the absence of ADAR2, suggesting preferential editing by ADAR1. Moreover, novel, previously unknown editing events were identified in several miRNAs. In general 64% of all editing events are located within the seed region of mature miRNAs. In one of these cases retargeting of the edited miRNA could be verified in reporter assays. Also, altered processing efficiency upon editing near a processing site could be experimentally verified. Conclusions: ADAR2 can significantly influence the abundance of certain miRNAs in the brain. Only in a few cases changes in miRNA abundance can be explained by miRNA editing. Thus, ADAR2 binding to miRNA precursors, without editing them, may influence their processing and thereby abundance. ADAR1 and ADAR2 have both overlapping and distinct specificities for editing of miRNA editing sites. Over 60% of editing occurs in the seed region possibly changing target specificities for many edited miRNAs. Examination of the effect of ADAR2 on mature miRNA abundance and sequence in adult mouse brain.

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Assessing the effect of the CLPG mutation on the microRNA catalogue of skeletal muscle using high-throughput sequencing

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