Meat quality is one of the most important economic traits in pig breeding and production. Intramuscular fat (IMF) is a major factor that improves meat quality. To better understand the alternative splicing (AS) events underlying meat quality, long-read isoform sequencing (Iso-seq) was used to identify differential (D)AS events between the longissimus thoracis (LT) and semitendinosus (ST), which differ in IMF content, together with short-read RNA-seq. Through Iso-seq analysis, we identified a total of 56,789 novel transcripts covering protein-coding genes, lncRNA, and fusion transcripts that were not previously annotated in pigs. We also identified 456,965 AS events, among which 3930 were DAS events, corresponding to 2364 unique genes. Through integrative analysis of Iso-seq and RNA-seq, we identified 1174 differentially expressed genes (DEGs), among which 122 were DAS genes, i.e., DE-DAS genes. There are 12 overlapped pathways between the top 20 DEGs and DE-DAS genes, as revealed by KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, indicating that DE-DAS genes play important roles in the differential phenotype of LT and ST. Further analysis showed that upregulated DE-DAS genes are more important than downregulated ones in IMF deposition. Fatty acid degradation and the PPAR (peroxisome proliferator-activated receptor) signaling pathway were found to be the most important pathways regulating the differential fat deposition of the two muscles. The results update the existing porcine genome annotations and provide data for the in-depth exploration of the mechanisms underlying meat quality and IMF deposition.

Chimeric RNA was considered a special marker of cancer. However, recent studies have demonstrated that chimeric RNAs also exist in non-cancerous cells and tissues. Here, we analyzed and predicted jointly 49 chimeric RNAs by Star-Fusion and FusionMap. One chimeric RNA, we named TNNI2-ACTA1, and its eight transcript variants were identified by reverse transcriptase–polymerase chain reaction. The overexpression of TNNI2-ACTA1 V1 inhibited the proliferation of porcine skeletal muscle satellite cells through down-regulating the mRNA expression levels of cell cycle–related genes cyclinD1. However, as parental genes, there is no such effect in the TNNI2 and ACTA1. To explore the underlying mechanism for this phenomenon, we used RNA-seq to profile the transcriptomes of PSCs with overexpression. Compared with the negative control group, 1,592 differentially expressed genes (DEGs) were upregulated and 1,077 DEGs downregulated in TNNI2 group; 1,226 DEGs were upregulated and 902 DEGs downregulated in ACTA1 group; and 13 DEGs were upregulated and 16 DEGs downregulated in TNNI2-ACTA1 V1 group, respectively. Compared with the parental gene groups, three specific genes were enriched in the TNNI2-ACTA1 V1 group (NCOA3, Radixin, and DDR2). These three genes may be the key to TNNI2-ACTA1 V1 regulating cell proliferation. Taken together, our study explores the role of chimeric RNAs in normal tissues. In addition, our study as the first research provides the foundation for the mechanism of chimeric RNAs regulating porcine skeletal muscle growth.

cis-Splicing of adjacent genes (cis-SAGe) has been involved in multiple physiological and pathological processes in humans. However, to the best of our knowledge, there is no report of cis-SAGe in adipogenic regulation. In this study, a cis-SAGe product, BCL2L2–PABPN1 (BP), was characterized in fat tissue of pigs with RT-PCR and RACE method. BP is an in-frame fusion product composed of 333 aa and all the functional domains of both parents. BP is highly conserved among species and rich in splicing variants. BP was found to promote proliferation and inhibit differentiation of primary porcine preadipocytes. A total of 3074/44 differentially expressed mRNAs (DEmRs)/known miRNAs (DEmiRs) were identified in porcine preadipocytes overexpressing BP through RNA-Seq analysis. Both DEmRs and target genes of DEmiRs were involved in various fat-related pathways with MAPK and PI3K-Akt being the top enriched. PPP2CB, EGFR, Wnt5A and EHHADH were hub genes among the fat-related pathways identified. Moreover, ssc-miR-339-3p was found to be critical for BP regulating adipogenesis through integrated analysis of mRNA and miRNA data. The results highlight the role of cis-SAGe in adipogenesis and contribute to further revealing the mechanisms underlying fat deposition, which will be conductive to human obesity control.

Long-chain acyl-CoA synthetase 1 (ACSL1) plays an important role in fatty acid metabolism and fat deposition. The transcription of the ACSL1 gene is regulated specifically among cells and physiological processes, and transcriptional regulation of ACSL1 in adipogenesis remains elusive. Here, we characterize transcription factors (TFs) associated with adipogenesis in the porcine ACSL1 gene. CCAAT-enhancer binding protein (C/EBP)α, a well-known adipogenic marker, was found to enhance the expression of the ACSL1 gene via binding two tandem motifs in the promoter. Further, we demonstrate that ACSL1 mediates C/EBPα effects on adipogenesis in preadipocytes cultured from subcutaneous fat tissue of pigs via gain- and loss-of-function analyses. The cAMP-response element binding protein, another TF involved in adipogenesis, was also identified in the regulation of ACSL1 gene expression. Additionally, single nucleotide polymorphisms (SNPs) were screened in the promoter of ACSL1 among four breeds including the Chinese indigenous Min, and Duroc, Berkshire, and Yorkshire pigs through sequencing of PCR products. Two tightly linked SNPs, −517G>T and −311T>G, were found exclusively in Min pigs. The haplotype mutation decreases promoter activity in PK-15 and ST cells, and in vivo the expression of ACSL1, illustrating a possible role in adipogenesis regulated by C/EBPα/ACSL1 axis. Additionally, a total of 24 alternative splicing transcripts were identified, indicating the complexity of alternative splicing in the ACSL1 gene. The results will contribute to further revealing the regulatory mechanisms of ACSL1 during adipogenesis and to the characterization of molecular markers for selection of fat deposition in pigs.