Circular RNAs (circRNAs) have been identified from various tissues and species, but their regulatory functions during developmental processes are not well understood. We examined circRNA expression profiles of two developmental stages of bovine skeletal muscle (embryonic and adult musculus longissimus) to provide first insights into their potential involvement in bovine myogenesis. We identified 12 981 circRNAs and annotated them to the Bos taurus reference genome, including 530 circular intronic RNAs (ciRNAs). One parental gene could generate multiple circRNA isoforms, with only one or two isoforms being expressed at higher expression levels. Also, several host genes produced different isoforms when comparing development stages. Most circRNA candidates contained two to seven exons, and genomic distances to back-splicing sites were usually less than 50 kb. The length of upstream or downstream flanking introns was usually less than 105 nt (mean≈11 000 nt). Several circRNAs differed in abundance between developmental stages, and real-time quantitative PCR (qPCR) analysis largely confirmed differential expression of the 17 circRNAs included in this analysis. The second part of our study characterized the role of circLMO7—one of the most down-regulated circRNAs when comparing adult to embryonic muscle tissue—in bovine muscle development. Overexpression of circLMO7 inhibited the differentiation of primary bovine myoblasts, and it appears to function as a competing endogenous RNA for miR-378a-3p, whose involvement in bovine muscle development has been characterized beforehand. Congruent with our interpretation, circLMO7 increased the number of myoblasts in the S-phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase. Moreover, it promoted the proliferation of myoblasts and protected them from apoptosis. Our study provides novel insights into the regulatory mechanisms underlying skeletal muscle development and identifies a number of circRNAs whose regulatory potential will need to be explored in the future.

/pdf/circular-rna-profiling-reveals-an-abundant-circlmo7-that-5pdaa9qvno.pdf

Circular RNA profiling reveals an abundant circLMO7 that regulates myoblasts differentiation and survival by sponging miR-378a-3p.

Maternal obesity in humans is associated with poor outcomes across the reproductive spectrum. Emerging evidence indicates that these defects are likely attributed to factors within the oocyte. Although various molecules and pathways may contribute to impaired oocyte quality, prevention of fertility issues associated with maternal obesity is a challenge. Using mice fed a high-fat diet (HFD) as an obesity model, we document spindle disorganization, chromosome misalignment, and elevated reactive oxygen species (ROS) levels in oocytes from obese mice. Oral administration of melatonin to HFD mice not only reduces ROS generation, but also prevents spindle/chromosome anomalies in oocytes, consequently promoting the developmental potential of early embryos. Consistent with this finding, we find that melatonin supplement during in vitro maturation also markedly attenuates oxidative stress and meiotic defects in HFD oocytes. Finally, by performing morpholino knockdown and acetylation-mimetic mutant overexpression assays, we reveal that melatonin ameliorates maternal obesity-induced defective phenotypes in oocytes through the SIRT3-SOD2-dependent mechanism. In sum, our data uncover the marked beneficial effects of melatonin on oocyte quality from obese females; this opens a new area for optimizing culture system as well as fertility management.

Melatonin protects against maternal obesity-associated oxidative stress and meiotic defects in oocytes via the SIRT3-SOD2-dependent pathway

Muscle development is regulated under a series of complicate processes, and non-coding RNAs, such as microRNAs (miRNAs) and circular RNAs (circRNAs), have been reported to play important roles in regulating myoblast proliferation and differentiation. We found that miR-107 expression was high in skeletal muscle of Qinchuan cattle. Overexpression of miR-107 inhibited bovine myoblasts differentiation and protected cells from apoptosis. Wnt3a was identified as a target of miR-107 by luciferase activity, real-time qPCR, and western blotting assays. Knockdown of Wnt3a inhibited bovine myoblasts differentiation and apoptosis, and this effect was similar to miR-107 overexpression. We also found circFGFR4 to promote myoblasts differentiation and to induce cell apoptosis. Via luciferase screening and RNA pull-down assays, circFGFR4 was observed to sponge miR-107. Overexpression of circFGFR4 increased the expression of Wnt3a, whereas this effect was abolished by miR-107. These results demonstrated that circFGFR4 binding miR-107 promotes cell differentiation via targeting Wnt3a in bovine primary myoblasts.

circFGFR4 Promotes Differentiation of Myoblasts via Binding miR-107 to Relieve Its Inhibition of Wnt3a.

Amyotrophic lateral sclerosis (ALS) is a clinical subtype of motor neurone disease (MND), a fatal neurodegenerative disease involving the loss of both the upper and lower motor neurones from the motor cortex, brainstem, and spinal cord. Identifying specific disease biomarkers would help to not only improve diagnostic delay but also to classify disease subtypes, monitor response to therapeutic drugs and track disease progression. miRNAs are small non-coding RNA responsible for regulating gene expression and ultimately protein expression and have been used as biomarkers for many cancers and neurodegenerative disorders. Investigating the detection of miRNAs in cerebrospinal fluid (CSF), the fluid that bathes the central nervous system (CNS) is a prime target for identifying potential biomarkers for ALS. This is the first study to investigate the expression of miRNAs in the CSF of ALS patients using small RNA sequencing. We detected 11 differentially expressed miRNAs in the CSF of sporadic ALS (sALS) patients related to neural and glial activity. Additionally, miRNAs involved in glucose metabolism and the regulation of oxidative stress were also identified. Detecting the presence of potential CSF derived miRNA biomarkers in sALS could open up a whole new area of knowledge to help gain a better understanding of disease pathophysiology. Additionally, with further investigation, the tracking of CSF miRNA over the disease course could be used to follow the disease progression and monitor the effect of novel therapeutics that could be personalized to an individual disease phenotype.

/pdf/small-rna-sequencing-of-sporadic-amyotrophic-lateral-1rqdvcjuf2.pdf

Small RNA Sequencing of Sporadic Amyotrophic Lateral Sclerosis Cerebrospinal Fluid Reveals Differentially Expressed miRNAs Related to Neural and Glial Activity.

Lipid metabolism is fundamental to life. In insects, it is critical, during reproduction, flight, starvation, and diapause. The coordination center for insect lipid metabolism is the fat body, which is analogous to the vertebrate adipose tissue and liver. Fat body contains various different cell types; however, adipocytes and oenocytes are the primary cells related to lipid metabolism. Lipid metabolism starts with the hydrolysis of dietary lipids, absorption of lipid monomers, followed by lipid transport from midgut to the fat body, lipogenesis or lipolysis in the fat body, and lipid transport from fat body to other sites demanding energy. Lipid metabolism is under the control of hormones, transcription factors, secondary messengers and posttranscriptional modifications. Primarily, lipogenesis is under the control of insulin-like peptides that activate lipogenic transcription factors, such as sterol regulatory element-binding proteins, whereas lipolysis is coordinated by the adipokinetic hormone that activates lipolytic transcription factors, such as forkhead box class O and cAMP-response element-binding protein. Calcium is the primary-secondary messenger affecting lipid metabolism and has different outcomes depending on the site of lipogenesis or lipolysis. Phosphorylation is central to lipid metabolism and multiple phosphorylases are involved in lipid accumulation or hydrolysis. Although most of the knowledge of insect lipid metabolism comes from the studies on the model Drosophila; other insects, in particular those with obligatory or facultative diapause, also have great potential to study lipid metabolism. The use of these models would significantly improve our knowledge of insect lipid metabolism.

A journey into the world of insect lipid metabolism.

Muscle development, or myogenesis, is a highly regulated, complex process. A subset of microRNAs (miRNAs) have been identified as critical regulators of myogenesis. Recently, miR-378a was found to be involved in myogenesis, but the mechanism of how miR-378a regulates the proliferation and differentiation of myoblasts has not been determined. We found that miR-378a-3p expression in muscle was significantly higher than in other tissues, suggesting an important effect on muscle development. Overexpression of miR-378a-3p increased the expression of MyoD and MHC in C2C12 myoblasts both at the level of mRNA and protein, confirming that miR-378a-3p promoted muscle cell differentiation. The forced expression of miR-378a-3p promoted apoptosis of C2C12 cells as evidenced by CCK-8 assay and Annexin V-FITC/PI staining results. Through TargetScan, histone acetylation enzyme 4 (HDAC4) was identified as a potential target of miR-378a-3p. We confirmed targeting of HDAC4 by miR-378a-3p using a dual luciferase assay and western blotting. Our RNAi analysis results also showed that HDAC4 significantly promoted differentiation of C2C12 cells and inhibited cell survival through Bcl-2. Therefore, we conclude that miR-378a-3p regulates skeletal muscle growth and promotes the differentiation of myoblasts through the post-transcriptional down-regulation of HDAC4.

https://www.tandfonline.com/doi/pdf/10.1080/15476286.2016.1239008

miR-378a-3p promotes differentiation and inhibits proliferation of myoblasts by targeting HDAC4 in skeletal muscle development

MicroRNAs (miRNAs), a class of single stranded, small (~22 nucleotides), non-coding RNAs, play an important role in muscle development. We focused on the role of the miR-30-5p family during bovine muscle development from previous high-throughput sequencing results and analyzed their expression profiles. MHC and MyoG mRNAs expression as well as their proteins were suppressed in differentiated C2C12 cells, suggesting the importance of miR-30-5p in muscle development. MBNL, the candidate target of miR-30-5p, is an alternative splicing regulation factor. MBNL1 and MBNL3 have opposite effects on muscle differentiation. Our results confirmed that miR-30a-5p and miR-30e-5p repress the expression of MBNL1, MBNL2 and MBNL3, whereas miR-30b-5p inhibits MBNL1 and MBNL2 expression. This provides direct evidence that MBNL expression can be flexibly regulated by miR-30-5p. Previous studies showed that MBNL1 promotes exon inclusion of two muscle-related genes (Trim55 and INSR). Through RNA splicing studies, we found that miR-30-5p had an effect on their alternative splicing, which means miR-30-5p via MBNL1 could be integrated into muscle signaling pathways in which INSR or Trim55 are located. In conclusion, miR-30-5p could inhibit muscle cell differentiation and regulate the alternative splicing of Trim55 and INSR by targeting MBNL. These results promote the understanding of the function of miRNAs in muscle development.

https://www.mdpi.com/1422-0067/17/2/182/pdf

miR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL

The posttranscriptional gene regulation mediated by microRNAs (miRNAs) plays an important role in various species. However, to date limited miRNAs have been reported between fat and muscle tissues in beef cattle. In this paper, 412 known and 22 novel miRNAs in backfat as well as 334 known and 10 novel miRNAs in longissimus thoracis were identified in the Chinese Qinchuan beef cattle. Bta-miR-199a-3p, -154c, -320a and -432 were expressed at higher levels in backfat tissue, while bta-miR-1, -133a, -206, and -378 were also significantly enriched in muscle tissue. Functional analysis revealed that fat-enriched miRNAs targeted PRKAA1/2, PPARA and PPARG genes to modulate lipid and fatty acid metabolism, and muscle-enriched miRNAs targeted CSRP3 gene to present function involved in skeletal and muscular system development. The results obtained may help in the design of new selection strategies to improve beef quality.

/pdf/comparative-transcriptome-analysis-reveals-significant-2byeczllqf.pdf

Comparative Transcriptome Analysis Reveals Significant Differences in MicroRNA Expression and Their Target Genes between Adipose and Muscular Tissues in Cattle

Identification of genetic variants, including SNPs (Single Nucleotide Polymorphisms), CNVs (Copy Number Variations) and alternative splicing, within functional genes has received increasing attention in animal science research. HGF (Hepatocyte Growth Factor) is a very important growth factor that works as a mitogen or a morphogen during tissue growth, development and regeneration. However, to date, the functions of genetic variants within the bovine HGF gene, particularly their effects on mRNA expression, have not been determined well. The present study aimed to perform association analysis between genetic variants and mRNA expression for the bovine HGF gene in Qinchuan cattle using various strategies, including PCR-RFLP (Restriction Fragment Length Polymorphism), qPCR (Quantitative Real-time quantitative PCR), TA cloning, DNA sequencing and bioinformatics analysis. A total of five SNPs were identified and only SV1 locus significantly affected HGF mRNA expression in fetal skeletal muscle (P < 0.05). Heterozygous genotype individuals showed significantly higher HGF expression (P < 0.05), which was significantly greater in the “CTCCAGGGTT” combined genotype than that in the “CCCCGGGGTT” combined genotype (P < 0.05). In addition, two alternative splicing variations, HGF-W and HGF-M, were identified, which resulted from alternative 3′ splice sites of exon 5, and HGF-W showed higher mRNA levels than HGF-M in all tissues. In summary, genetic variations within the HGF gene affected mRNA expression. These findings provide new insight into the molecular characteristics and functions of bovine HGF.

/pdf/effects-of-snps-and-alternative-splicing-within-hgf-gene-on-cp28n0z3ai.pdf

Bowen Zhang

Papers

miR-378a-3p promotes differentiation and inhibits proliferation of myoblasts by targeting HDAC4 in skeletal muscle development

miR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL

Comparative Transcriptome Analysis Reveals Significant Differences in MicroRNA Expression and Their Target Genes between Adipose and Muscular Tissues in Cattle

Effects of SNPs and alternative splicing within HGF gene on its expression patterns in Qinchuan cattle

A new H9 influenza virus mRNA vaccine elicits robust protective immunity against infection.