Identification and characterization of long non-coding RNA in prenatal and postnatal skeletal muscle of sheep.

lncRNAs are a class of transcriptional RNA molecules of >200 nucleotides in length. However, the overall expression pattern and function of lncRNAs in sheep muscle is not clear. Here, we identified 1566 lncRNAs and 404 differentially expressed lncRNAs in sheep muscle from prenatal (110 days of fetus) and postnatal (2 to 3 years old of adult sheep) developmental stages by using RNA-seq technology. Several lncRNAs were identified by using RT-PCR and DNA sequencing. The expression levels of several lncRNAs were confirmed by qRT-PCR. We analyzed the effect of lncRNAs that act cis to the target genes. lncRNA targeting genes were involved in signaling pathways associated with growth and development of muscle by GO and KEGG enrichment analysis. Through our study, we provide a comprehensive expression profile of muscle lncRNAs in sheep, which provides valuable resources for further understanding genetic regulation of muscle growth and development from the perspective of lncRNA.

Expression profiles of microRNAs in skeletal muscle of sheep by deep sequencing.

OBJECTIVE: MicroRNAs are a class of endogenous small regulatory RNAs that regulate cell proliferation, differentiation and apoptosis. Recent studies on miRNAs are mainly focused on mice, human and pig. However, the studies on miRNAs in skeletal muscle of sheep are not comprehensive. METHODS: RNA-seq technology was used to perform genomic analysis of miRNAs in prenatal and postnatal skeletal muscle of sheep. Targeted genes were predicted using miRanda software and miRNA-mRNA interactions were verified by quantitative real-time polymerase chain reaction. To further investigate the function of miRNAs, candidate targeted genes were enriched for analysis using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment. RESULTS: The results showed total of 1,086 known miRNAs and 40 new candidate miRNAs were detected in prenatal and postnatal skeletal muscle of sheep. In addition, 345 miRNAs (151 up-regulated, 94 down-regulated) were differentially expressed. Moreover, miRanda software was performed to predict targeted genes of miRNAs, resulting in a total of 2,833 predicted targets, especially miR-381 which targeted multiple muscle-related mRNAs. Furthermore, GO and KEGG pathway analysis confirmed that targeted genes of miRNAs were involved in development of skeletal muscles. CONCLUSION: This study supplements the miRNA database of sheep, which provides valuable information for further study of the biological function of miRNAs in sheep skeletal muscle.

Expression profiles of circular RNAs in sheep skeletal muscle.

OBJECTIVE: Circular RNAs (circRNAs) are a newfound class of non-coding RNA in animals and plants. Recent studies have revealed that circRNAs play important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports about muscle development-related circRNAs in livestock. METHODS: RNA sequencing analysis was employed to identify and annotate circRNAs from longissimus dorsi of sheep. Reverse transcription followed by real-time quantitative (q) polymerase chain reaction (PCR) analysis verified the presence of these circRNAs. Targetscan7.0 and miRanda were used to analyse the interaction of circRNA-microRNA (miRNA). To investigate the function of circRNAs, an experiment was conducted to perform enrichment analysis hosting genes of circRNAs using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. RESULTS: About 75.5 million sequences were obtained from RNA libraries of sheep skeletal muscle. These sequences were mapped to 729 genes in the sheep reference genome. We identified 886 circRNAs, including numerous circular intronic RNAs and exonic circRNAs. Reverse transcription PCR (RT-PCR) and DNA sequencing analysis confirmed the presence of several circRNAs. Real-Time RT-PCR analysis exhibited resistance of sheep circRNAs to RNase R digestion. We found that many circRNAs interacted with muscle-specific miRNAs involved in growth and development of muscle, especially circ776. The GO and KEGG enrichment analysis showed that hosting genes of circRNAs was involved in muscle cell development and signaling pathway. CONCLUSION: The study provides comprehensive expression profiles of circRNAs in sheep skeletal muscle. Our study offers a large number of circRNAs to facilitate a better understanding of their roles in muscle growth. Meanwhile, we suggested that circ776 could be analyzed in future study.

Cloning and functional analysis of sheep U6 promoters.

Gene silencing mediated by small interfering RNA has become a powerful biological tool for the regulation of gene expression. In order to develop an effective short hairpin RNA (shRNA) expression vector, specifically for use in sheep species, we have identified two sheep U6 promoters based on the highly conserved polymerase III promoter elements. Promoter activity was measured by U6 promoter-driven shRNA to suppress enhanced green fluorescent protein (EGFP) expression. The knock down assay demonstrated that the two sheep U6 promoters and mouse U6 promoter induced a similar level of EGFP knockdown. These results suggest that the two sheep U6 promoters could efficiently drive shRNA expression for gene silencing and may have applications in RNAi-based sheep research.

Comprehensive Expression Profiling Analysis of Pituitary Indicates that circRNA Participates in the Regulation of Sheep Estrus.

The pituitary gland is the most important endocrine organ that mainly regulates animal estrus by controlling the hormones synthesis. There is a significant difference between the estrus state and anestrus state of sheep pituitary system. Here, we studied the circular RNA (circRNA) expression profiles of the anterior pituitary of estrus and anestrus sheep using RNA-seq technology. Through this study, we identified a total of 12,468 circRNAs and 9,231 differentially expressed circRNAs in the estrus and anestrus pituitary system of sheep. We analyzed some differentially expressed circRNAs by reverse transcription quantitative-PCR (RT-qPCR), and some circRNAs were demonstrated using RNase-R+ resistance experiments. CircRNAs involving the regulation of estrus-related terms and pathways are enriched by using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. In addition, we also predicted partial microRNA-circRNA interaction network for circRNAs that regulate sheep estrus. Overall, this study explored a potential substantial role played by circRNAs involved in pituitary regulation on sheep estrus and proposed new questions for further study.

Comprehensive analysis of circRNAs expression profiles in different periods of MDBK cells infected with bovine viral diarrhea virus.

CircRNAs play an important regulatory role in the regulation of disease. However, we have a limited understanding of the role of circRNAs in the host's complex protective and pathological mechanisms of BVDV infection. Transcriptome analysis of circRNAs in host cells after BVDV infection may allow us to understand the biological functions of circRNAs in the regulation of BVDV infection. Here, we identified a total of 19,118 circRNAs from the MBDK cells (at 12 h, 24 h, and 48 h post-infection) infected with BVDV by using RNA-seq technology. We confirmed several circRNAs using RT-PCR and DNA sequencing, and qRT-PCR analysis was performed to identify several circRNAs expression and circRNAs resistance to RNase R digestion. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that the host genes of differentially expressed circRNAs were involved in the regulation of cell proliferation, apoptosis, cycle and viral infection related signaling pathways. These results indicate that circRNA in host cells plays a broad regulatory role after BVDV infection and provides a valuable resource for studying circRNA biology in host cells after BVDV infection.

Expression profiles of MicroRNAs from multiple lumbar spine in sheep.

The formation of the spine is a critical stage of mammalian development. The increase of the number of individual axons affects its performance, especially in meat production. To understand the role of miRNAs in sheep vertebrae development, the purpose of this article is to screen candidate microRNAs (miRNAs) associated with sheep spine development. MicroRNAs (miRNAs) are a rich family of small regulatory RNAs that negatively regulate gene expression at the post-transcriptional level. In this study, we used high-throughput sequencing techniques to analyze the microRNAs (miRNAs) expression profiles of L6 (6 lumbar vertebrae) and L7 (7 lumbar vertebrae) in sheep. A total number of 223 miRNAs were detected in the two libraries, and a total of 150 and 148 conserved miRNAs were obtained in L6 and L7, respectively. A total of 5 miRNAs expression differences in L6 compared to L7 (P < 0.05). Of the five obviously differently expressed miRNAs, four miRNAs were down-regulated in the L6 of sheep, and one was up-regulated. In order to further explore the functions of these miRNAs, we predicted the target genes of these differently expressed miRNAs, and obtained 1298 target genes. At the same time, NDRG2 gene, targeted by novel miR-391, which possible plays an important role in the development of the spine. Linkage-integration analysis method was used to construct the interaction network of spinal-associated miRNA and its hypothesized target. In summary, this study provides valuable resources for the transcriptome of multiple vertebral traits in sheep.

Genome-wide analysis of circular RNAs in prenatal and postnatal muscle of sheep.

Circular RNAs (circRNAs), a type of non-coding RNA with circular structure, were generated by back splicing and widely expressed in animals and plants. Recent studies have shown that circRNAs extensively participate in cell proliferation, cell differentiation, cell autophagy and other biological processes. However, the role and expression of circRNAs in the development and growth of muscle have not been studied in sheep. In our study, we first used RNA-seq to study the circRNAs in prenatal and postnatal longissimus dorsi muscle of sheep. A total of 6113 circRNAs were detected from the RNA-seq data. Several circRNAs were identified using reverse transcription PCR, DNA sequencing and RNase R digestion experiments. The expression levels of several circRNAs in prenatal and postnatal muscle were confirmed by Real-Time RT-PCR. The gene ontology (GO) and KEGG enrichment analysis of the host gene of the circRNAs showed that these circRNAs were mainly involved in the growth and development of muscle related signaling pathways. These circRNAs might sponge microRNAs (miRNAs) in predicted circRNA-miRNA-mRNA networks. The circRNAs expression profiles in muscle provided an important reference for the study of circRNAs in sheep.