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.

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.

MicroRNAs play essential roles in biological processes by regulating gene expression at the post-transcriptional level. Our previous studies suggested the role of miR-26a in porcine fat accumulation. Here, through gain- and loss-of-function analyses, we first showed that miR-26a increased the proliferation of porcine preadipocytes by promoting cell division and that miR-26a inhibited the preadipocyte differentiation. Next, acyl-CoA dehydrogenase, medium chain (ACADM) was revealed to promote the proliferation and differentiation of preadipocytes for the first time. Then, it was revealed that miR-26a regulates adipogenesis by directly binding to the 3′ untranslated region of ACADM and the long-chain acyl-Co A synthetase 1 (ACSL1) gene, a previously known regulator of adipogenesis. Finally, RNA-sequencing, performed on preadipocytes overexpressing miR-26a, identified 337 differentially expressed genes in the early stage of adipogenesis; among them, nine genes were characterized as potential targets of miR-26a. The 337 genes were mainly involved in Gene Ontology terms related to cell division, indicating that cell cycle progression was also a major event regulated by miR-26a during adipogenesis. We provide novel data for understanding the molecular mechanisms underlying adipogenesis, which will contribute to controlling fat accumulation in animals.

The cAMP response element-binding protein (CREB), a basic leucine zipper transcription factor, is involved in the activation of numerous genes in a variety of cell types. The CREB gene is rich in alternative splicing (AS) events. However, studies on the AS of CREB genes in pigs are limited, and few reports have compared the roles of isoforms in activating gene expression. Here, five AS transcripts, V1–5, were characterized by RT-PCR and two, V3 and V5, were new identifications. Both V1 and V2 have all the functional domains of the CREB protein, with similar tissue expression profiles and mRNA stability, suggesting that they have similar roles. The transcriptional transactivation activities of four isoforms encoding complete polypeptides were analyzed on the expression of the B-cell CLL/lymphoma 2-like protein 2 and the poly (A)-binding protein, nuclear 1 genes with a dual-luciferase reporter system, and differential activities were observed. Both V1 and V2 have promoting effects, but their roles are gene-specific. V3 has no effect on the promoter of the two genes, while V4 functions as a repressor. The mechanisms underlying the differential roles of V1 and V2 were analyzed with RNA-seq, and the genes specifically regulated by V1 and V2 were identified. These results will contribute to further revealing the role of CREB and to analyzing the significance of AS in genes.