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.
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.
The formation of embryonic muscle fibers determines the amount of postnatal muscles and is regulated by a variety of signaling pathways and transcription factors.Previously, by using chromatin immunoprecipitation-sequencing and RNA-Seq techniques, we identified a large number of genes that are regulated by H3K27me3 in porcine embryonic skeletal muscles. Among these genes, we found that ITGB6 is regulated by H3K27me3. However, its function in muscle development is unknown. In this study, we first verified that ITGB6 was differentially regulated by H3K27me3 and that its expression levels were upregulated in porcine skeletal muscles at embryonic Days 33, 65, and 90. Then, we performed gain-or loss-of-function studies on porcine skeletal muscle satellite cells to study the role of ITGB6 in porcine skeletal muscle development. The proliferation of porcine skeletal muscle satellite cells was studied through real-time polymerase chain reaction, Cell Counting Kit-8, 5-ethynyl-2′deoxyuridine staining, Western blot, and flow cytometry analyses. We found that the ITGB6 gene was regulated by H3K27me3 during muscle development and had an inhibitory effect on the proliferation of porcine skeletal muscle satellite cells.
The proper methylation status of histones is essential for appropriate cell lineage and organogenesis. EZH2, a methyltransferase catalyzing H3K27me3, has been abundantly studied in human and mouse embryonic development. The pig is an increasing important animal model for molecular study and pharmaceutical research. However, the transcript variant and temporal expression pattern of EZH2 in the middle and late porcine fetus are still unknown. Here, we identified the coding sequence of the EZH2 gene and characterized its expression pattern in fetal tissues of Duroc pigs at 65- and 90-day postcoitus (dpc). Our results showed that the coding sequence of EZH2 was 2241 bp, encoding 746 amino acids. There were 9 amino acid insertions and an amino acid substitution in this transcript compared with the validated reference sequence in NCBI. EZH2 was ubiquitously expressed in the fetal tissues of two time points with different expression levels. These results validated a different transcript in pigs and characterized its expression profile in fetal tissues of different gestation stages, which indicated that EZH2 played important roles during porcine embryonic development.
How to maximize the use of the genetic merits of the high-ranking boars (also called superior ones) is a considerable question in the pig breeding industry, considering the money and time spent on selection. Somatic cell nuclear transfer (SCNT) is one of the potential ways to answer the question, which can be applied to produce clones with genetic resources of superior boar for the production of commercial pigs. For practical application, it is essential to investigate whether the clones and their progeny keep behaving better than the “normal boars”, considering that in vitro culture and transfer manipulation would cause a series of harmful effects to the development of clones. In this study, 59,061 cloned embryos were transferred into 250 recipient sows to produce the clones of superior Pietrain boars. The growth performance of 12 clones and 36 non-clones and the semen quality of 19 clones and 28 non-clones were compared. The reproductive performance of 21 clones and 25 non-clones were also tested. Furthermore, we made a comparison in the growth performance between 466 progeny of the clones and 822 progeny of the non-clones. Our results showed that no significant difference in semen quality and reproductive performance was observed between the clones and the non-clones, although the clones grew slower and exhibited smaller body size than the non-clones. The F1 progeny of the clones showed a greater growth rate than the non-clones. Our results demonstrated through the large animal population showed that SCNT manipulation resulted in a low growth rate and small body size, but the clones could normally produce F1 progeny with excellent growth traits to bring more economic benefits. Therefore, SCNT could be effective in enlarging the merit genetics of the superior boars and increasing the economic benefits in pig reproduction and breeding.
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