RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs.

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.

Integrated Analysis of DNA Methylation and Gene Expression in Porcine Placental Development.

Proper placental development is crucial for the conceptus to grow and survive, because the placenta is responsible for transporting nutrients and oxygen from the pregnant female to the developing fetus. However, the processes of placental morphogenesis and fold formation remain to be fully elucidated. In this study, we used whole-genome bisulfite sequencing and RNA sequencing to produce a global map of DNA methylation and gene expression changes in placentas from Tibetan pig fetuses 21, 28, and 35 days post-coitus. Substantial changes in morphology and histological structures at the uterine-placental interface were revealed via hematoxylin-eosin staining. Transcriptome analysis identified 3959 differentially expressed genes (DEGs) and revealed the key transcriptional properties in three stages. The DNA methylation level in the gene promoter was negatively correlated with gene expression. We identified a set of differentially methylated regions associated with placental developmental genes and transcription factors. The decrease in DNA methylation level in the promoter was associated with the transcriptional activation of 699 DEGs that were functionally enriched in cell adhesion and migration, extracellular matrix remodeling, and angiogenesis. Our analysis provides a valuable resource for understanding the mechanisms of DNA methylation in placental development. The methylation status of different genomic regions plays a key role in establishing transcriptional patterns from placental morphogenesis to fold formation.

Comprehensive analysis of pre-mRNA alternative splicing regulated by m6A methylation in pig oxidative and glycolytic skeletal muscles.

BACKGROUND: Different types of skeletal myofibers exhibit distinct physiological and metabolic properties that are associated with meat quality traits in livestock. Alternative splicing (AS) of pre-mRNA can generate multiple transcripts from an individual gene by differential selection of splice sites. N6-methyladenosine (m6A) is the most abundant modification in mRNAs, but its regulation for AS in different muscles remains unknown.  RESULTS: We characterized AS events and m6A methylation pattern in pig oxidative and glycolytic muscles. A tota1 of 1294 differential AS events were identified, and differentially spliced genes were significantly enriched in processes related to different phenotypes between oxidative and glycolytic muscles. We constructed the regulatory network between splicing factors and corresponding differential AS events and identified NOVA1 and KHDRBS2 as key splicing factors. AS event was enriched in m6A-modified genes, and the methylation level was positively correlated with the number of AS events in genes. The dynamic change in m6A enrichment was associated with 115 differentially skipping exon (SE-DAS) events within 92 genes involving in various processes, including muscle contraction and myofibril assembly. We obtained 23.4% SE-DAS events (27/115) regulated by METTL3-meditaed m6A and experimentally validated the aberrant splicing of ZNF280D, PHE4DIP, and NEB. The inhibition of m6A methyltransferase METTL3 could induce the conversion of oxidative fiber to glycolytic fiber in PSCs. CONCLUSION: Our study suggested that m6A modification could contribute to significant difference in phenotypes between oxidative and glycolytic muscles by mediating the regulation of AS. These findings would provide novel insights into mechanisms underlying muscle fiber conversion.

Comparative evaluation of production performances of cloned pigs derived from superior Duroc boars.

Since pig was successfully cloned in 2000, somatic cell nuclear transfer (SCNT) became a promising technique in preserving and expanding the genetics of superior boars. Assessing the safety, growth performance, and reproductive performance of cloned pigs and their progeny is critical for their wide application. In this study, three superior Duroc boars were used to construct 61,736 SCNT-cloned embryos. The semen quality and reproductive performance of the cloned Duroc pigs and the growth performances of their progeny were evaluated. Results showed that the cloned pigs derived from superior boars produced semen with normal quality and exhibited similar reproductive performance as the donor boars, whose progenies showed greater growth performance than those derived from non-cloned pigs under the same feed condition. The results shed light on the application of cloning technology in the conservation and expansion of the genetic resources of Duroc pigs.

Long non-coding RNA Gm10561 promotes myogenesis by sponging miR-432.

Skeletal myogenesis is a highly ordered process finely regulated by various factors. Long non-coding RNAs play an important regulatory role in myogenesis via multiple mechanisms. In this study, we identified the lncRNA Gm10561, which was upregulated during myogenic differentiation and is highly expressed in skeletal muscle. Knockdown of Gm10561 inhibited the proliferation and differentiation of C2C12 myoblasts in vitro and muscle growth in vivo. Overexpression of Gm10561 promoted the proliferation and differentiation of both C2C12 myoblasts and porcine muscle satellite cells. Notably, lncRNA Gm10561 is localized in the cytoplasm and competitively bound to miR-432, which directly targets MEF2C and E2F3. It was confirmed that lncRNA Gm10561 regulates the proliferation and differentiation of myoblasts by acting as a sponge of miR-432 to modulate MEF2C and E2F3 expression. Thus, the lncRNA-Gm10561-miR-432-MEF2C/E2F3 axis plays an important role in myogenesis.

Comprehensive Analysis of Long Noncoding RNA Modified by m6A Methylation in Oxidative and Glycolytic Skeletal Muscles.

N6-methyladenosine (m6A) is the most common modification in eukaryotic RNAs. Accumulating evidence shows m6A methylation plays vital roles in various biological processes, including muscle and fat differentiation. However, there is a lack of research on lncRNAs' m6A modification in regulating pig muscle-fiber-type conversion. In this study, we identified novel and differentially expressed lncRNAs in oxidative and glycolytic skeletal muscles through RNA-seq, and further reported the m6A-methylation patterns of lncRNAs via MeRIP-seq. We found that most lncRNAs have one m6A peak, and the m6A peaks were preferentially enriched in the last exon of the lncRNAs. Interestingly, we found that lncRNAs' m6A levels were positively correlated with their expression homeostasis and levels. Furthermore, we performed conjoint analysis of MeRIP-seq and RNA-seq data and obtained 305 differentially expressed and differentially m6A-modified lncRNAs (dme-lncRNAs). Through QTL enrichment analysis of dme-lncRNAs and PPI analysis for their cis-genes, we finally identified seven key m6A-modified lncRNAs that may play a potential role in muscle-fiber-type conversion. Notably, inhibition of one of the key lncRNAs, MSTRG.14200.1, delayed satellite cell differentiation and stimulated fast-to-slow muscle-fiber conversion. Our study comprehensively analyzed m6A modifications on lncRNAs in oxidative and glycolytic skeletal muscles and provided new targets for the study of pig muscle-fiber-type conversion.