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Identification and characterization of circRNAs in the skin during wool follicle development in Aohan fine wool sheep.

ABSTRACT: BACKGROUND:Aohan fine wool sheep (AFWS) is a historically bred fine wool sheep, cultivated in China. The wool has excellent quality and good textile performance. Investigating the molecular mechanisms that regulate wool growth is important to improve wool quality and yield. Circular RNAs (circRNAs) are widely expressed non-coding RNAs that can act as competitive endogenous RNAs (ceRNAs) to bind to miRNAs. Although circRNAs have been studied in many fields, research on their activity in sheep wool follicles is limited. To understand the regulation of circRNAs in the growth of fine wool in sheep, we used RNA-Seq to identify circRNAs in sheep shoulder skin samples at three developmental stages: embryonic day 90 (E90d), embryonic day 120 (E120d), and at birth (Birth). RESULTS:We identified 8753 circRNAs and found that 918 were differentially-expressed. We then analyzed the classification and characteristic of the circRNAs in sheep shoulder skin. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we identified the source genes of circRNAs, which were mainly enriched in cellular component organization, regulation of primary metabolic processes, tight junctions, and the cGMP-PKG and AMPK signaling pathways. In addition, we predicted interactions between 17 circRNAs and eight miRNAs, using miRanda software. Based on the significant pathways, we speculate that circ_0005720, circ_0001754, circ_0008036, circ_0004032, circ_0005174, circ_0005519, and circ_0007826 might play an important role in regulating wool follicle growth in AFWS. Seven circRNAs were randomly selected to validate the RNA-Seq results, using qRT-PCR. CONCLUSION:Our results provide more information about circRNAs regulation of wool follicle development in AFWS, and establish a solid foundation for future research.

SUBMITTER: Zhao R

PROVIDER: S-EPMC7048093 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aohan fine-wool sheep (AFWS) is a high-quality fine-wool sheep breed that supplies wool and meat. Research is needed on the molecular mechanism behind intramuscular fat (IMF) deposition that greatly improves mutton quality. The widely expressed non-coding RNA is physiologically used in roles such as competitive endogenous RNA (ceRNA) that includes circular RNAs (circRNAs). Although circRNAs were studied in many fields, little research was devoted to IMF in sheep. We used the longissimus dorsi muscle of 2 and 12-month-old AWFS as research material to identify circRNAs related to IMF deposition in these sheep by RNA-seq screening for differentially expressed circRNAs in the two age groups. A total of 11,565 candidate circRNAs were identified, of which the 104 differentially expressed circRNAs in the two age groups were analyzed. Enrichment analysis was performed using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. The enriched pathways included lipid transport (GO:0006869), negative regulation of canonical Wnt signaling pathway (GO:0090090), fat digestion and absorption (ko04975), and sphingolipid metabolism (ko00600). The differentially expressed circRNAs included ciRNA455, circRNA9086, circRNA7445, circRNA4557, and others. The source genes involved in these pathways might regulate IMF deposition. We used the TargetScan and miRanda software for interaction analysis, and a network diagram of circRNA-miRNA interactions was created. CircRNA455-miR-127, circRNA455-miR-29a, circRNA455-miR-103, circRNA4557-mir149-5p, and circRNA2440-mir-23a might be involved in the IMF deposition process. The targeting relationship of circRNA4557-miR-149-5p was verified by a dual-luciferase reporter assay. The RT-qPCR results of seven randomly selected circRNAs were consistent with the sequencing results. This study provides additional information on circRNA regulation of IMF deposition in AFWS and is a useful resource for future research on this sheep breed.

Project description:BackgroundIntramuscular fat (IMF) content has become one of the most important indicators for measuring meat quality, and levels of IMF are affected by various genes. Long non-coding RNAs (lncRNAs) are widely expressed non-coding RNAs that play an important regulatory role in a variety of biological processes; however, research on the lncRNAs involved in sheep IMF deposition is still in its infancy. Aohan fine-wool sheep (AFWS), one of China's most important meat-hair, dual-purpose sheep breed, provides a great model for studying the role of lncRNAs in the regulation of IMF deposition. We identified lncRNAs by RNA sequencing in Longissimus thoracis et lumborum (LTL) samples of sheep at two ages: 2?months (Mth-2) and 12?months (Mth-12).ResultsWe identified a total of 26,247 genes and 6935 novel lncRNAs in LTL samples of sheep. Among these, 199 mRNAs and 61 lncRNAs were differentially expressed. We then compared the structural characteristics of lncRNAs and mRNAs. We obtained target genes of differentially expressed lncRNAs (DELs) and performed enrichment analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). We found that target mRNAs were enriched in metabolic processes and developmental pathways. One pathway was significantly enriched, namely tight junction. Based on the analysis of critical target genes, we obtained seven candidate lncRNAs that potentially regulated lipid deposition and constructed a lncRNA-mRNA co-expression network that included MSTRG.4051.3-FZD4, MSTRG.16157.3-ULK1, MSTRG.21053.3-PAQR3, MSTRG.19941.2-TPI1, MSTRG.12864.1-FHL1, MSTRG.2469.2-EXOC6 and MSTRG.21381.1-NCOA1. We speculated that these candidate lncRNAs might play a role by regulating the expression of target genes. We randomly selected five mRNAs and five lncRNAs to verify the accuracy of the sequencing data by qRT-PCR.ConclusionsOur study identified the differentially expressed mRNAs and lncRNAs during intramuscular lipid deposition in Aohan fine-wool sheep. The work may widen the knowledge about the annotation of the sheep genome and provide a working basis for investigating intramuscular fat deposition in sheep.

Project description:Murine primary hair follicle induction is driven by the communication between the mesenchyme and epithelium and mostly governed by signaling pathways including wingless-related integration site (WNT), ectodysplasin A receptor (EDAR), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF), as observed in genetically modified mouse models. Sheep skin may serve as a valuable system for hair research owing to the co-existence of sweat glands with wool follicles in trunk skin and asynchronized wool follicle growth pattern similar to that of human head hair follicles. However, the mechanisms underlying wool follicle development remain largely unknown. To understand how long non-coding RNAs (lncRNAs) and mRNAs function in primary wool follicle induction in carpet wool sheep, we conducted high-throughput RNA sequencing and revealed globally altered lncRNAs (36 upregulated and 26 downregulated), mRNAs (228 elevated and 225 decreased), and 80 differentially expressed novel transcripts. Several key signals in WNT (WNT2B and WNT16), BMP (BMP3, BMP4, and BMP7), EDAR (EDAR and EDARADD), and FGF (FGFR2 and FGF20) pathways, and a series of lncRNAs, including XLOC_539599, XLOC_556463, XLOC_015081, XLOC_1285606, XLOC_297809, and XLOC_764219, were shown to be potentially important for primary wool follicle induction. GO and KEGG analyses of differentially expressed mRNAs and potential targets of altered lncRNAs were both significantly enriched in morphogenesis biological processes and transforming growth factor-β, Hedgehog, and PI3K-Akt signaling, as well as focal adhesion and extracellular matrix-receptor interactions. The prediction of mRNA-mRNA and lncRNA-mRNA interaction networks further revealed transcripts potentially involved in primary wool follicle induction. The expression patterns of mRNAs and lncRNAs of interest were validated by qRT-PCR. The localization of XLOC_297809 and XLOC_764219 both in placodes and dermal condensations was detected by in situ hybridization, indicating important roles of lncRNAs in primary wool follicle induction and skin development. This is the first report elucidating the gene network of lncRNAs and mRNAs associated with primary wool follicle early development in carpet wool sheep and will shed new light on selective wool sheep breeding.

Project description:To investigate single nucleotide polymorphism (SNP) loci associated with yearling wool traits of fine-wool sheep for optimizing marker-assisted selection and dissection of the genetic architecture of wool traits, we conducted a genome-wide association study (GWAS) based on the fixed and random model circulating probability unification (FarmCPU) for yearling staple length (YSL), yearling mean fiber diameter (YFD), yearling greasy fleece weight (YGFW), and yearling clean fleece rate (YCFR) by using the whole-genome re-sequenced data (totaling 577 sheep) from the following four fine-wool sheep breeds in China: Alpine Merino sheep (AMS), Chinese Merino sheep (CMS), Qinghai fine-wool sheep (QHS), and Aohan fine-wool sheep (AHS). A total of 16 SNPs were detected above the genome-wise significant threshold (P = 5.45E-09), and 79 SNPs were located above the suggestive significance threshold (P = 5.00E-07) from the GWAS results. For YFD and YGFW traits, 7 and 9 SNPs reached the genome-wise significance thresholds, whereas 10 and 12 SNPs reached the suggestive significance threshold, respectively. For YSL and YCFR traits, none of the SNPs reached the genome-wise significance thresholds, whereas 57 SNPs exceeded the suggestive significance threshold. We recorded 14 genes located at the region of ±50-kb near the genome-wise significant SNPs and 59 genes located at the region of ±50-kb near the suggestive significant SNPs. Meanwhile, we used the Average Information Restricted Maximum likelihood algorithm (AI-REML) in the "HIBLUP" package to estimate the heritability and variance components of the four desired yearling wool traits. The estimated heritability values (h2) of YSL, YFD, YGFW, and YCFR were 0.6208, 0.7460, 0.6758, and 0.5559, respectively. We noted that the genetic parameters in this study can be used for fine-wool sheep breeding. The newly detected significant SNPs and the newly identified candidate genes in this study would enhance our understanding of yearling wool formation, and significant SNPs can be applied to genome selection in fine-wool sheep breeding.

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Identification and characterization of circRNAs in the skin during wool follicle development in Aohan fine wool sheep.

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