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. 2022 Feb 28;13(3):445.
doi: 10.3390/genes13030445.

Characterization of a Read-through Fusion Transcript, BCL2L2-PABPN1, Involved in Porcine Adipogenesis

Jiyuan Zhu  1 , Zewei Yang  1 , Wanjun Hao  1 , Jiaxin Li  1 , Liang Wang  2 , Jiqiao Xia  1 , Dongjie Zhang  2 , Di Liu  2 , Xiuqin Yang  1
Affiliations

Characterization of a Read-through Fusion Transcript, BCL2L2-PABPN1, Involved in Porcine Adipogenesis

Jiyuan Zhu et al. Genes (Basel). .

Abstract

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.

Keywords: BCL2L2-PABPN1; RNA-Seq; adipogenesis; chimeric RNA; cis-SAGe; genome-wide analysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Comparison of subcutaneous adipose tissues between Yorkshire and Min pigs. (A) Morphological analysis with HE staining. (B) Adipocyte area of subcutaneous adipose tissues. The bar is 200 μm. (C) Triglyceride contents in subcutaneous adipose tissues. The data are shown as mean ± standard error. * and ** indicate p < 0.05, and p < 0.01, respectively.
Figure 2
Figure 2
Characterization of porcine BCL2L2-PABPN1 (BP). (A) Score distribution of read-throughs identified. (B) Fragment distribution of BP in Min and Yorkshire pigs determined by ChimeraScan. (C) Confirmation of BP with RT-PCR and sequencing.
Figure 3
Figure 3
Characterization of porcine BCL2L2-PABPN1 (BP). (A) Genomic and mRNA structure of porcine BP. Boxes and lines indicate exons and introns, respectively. Dotted boxes in PABPN1 indicate that the sequences are first obtained here. Figures over the boxes indicate the length of the corresponding exons, while those in the boxes indicate exon No. Primer locations are shown with arrows. E, exon. I, intron. # indicates the position of poly(A) signal. Start and stop codons are shown under the boxes. (B) Motifs in the polypeptide of BP. (C) Identification of mechanisms underlying BP formation with RT-PCR. Templates were showed below. No RT, no reverse transcriptase control.
Figure 4
Figure 4
Effects of BCL2L2-PABPN1 (BP) on subcutaneous preadipocyte proliferation and differentiation. (A) Expression of BP during porcine preadipocyte proliferation determined with real-time PCR. (B) Expression of BP during porcine preadipocyte differentiation determined with real-time PCR. (C) Effect of BP on cell proliferation measured by CCK-8 assay. (D) Effects of BP on cell cycle measured with flow cytometry. (E) Oil red O staining of differentiated adipocyte. * and ** indicates p < 0.05 and p < 0.01, respectively, compared with control groups. The bar is 100 μm.
Figure 5
Figure 5
Characterization of differentially expressed mRNAs (DEmRs) induced by BCL2L2-PABPN1. (A) Volcano plot of DEmRs. (B) Validation of RNA-Seq data with real-time PCR method. (C) GO enrichment analysis of DEmRs. (D) KEGG pathway analysis of upregulated DEmRs. (E) KEGG pathway analysis of downregulated DEmRs.
Figure 5
Figure 5
Characterization of differentially expressed mRNAs (DEmRs) induced by BCL2L2-PABPN1. (A) Volcano plot of DEmRs. (B) Validation of RNA-Seq data with real-time PCR method. (C) GO enrichment analysis of DEmRs. (D) KEGG pathway analysis of upregulated DEmRs. (E) KEGG pathway analysis of downregulated DEmRs.
Figure 6
Figure 6
Characterization of differentially expressed mRNAs (DEmRs) involved in fat-related pathways. (A) Heatmap cluster of DEmRs involved in fat-related pathways. (B) The protein-protein interaction network of DEmRs involved in fat-related pathways. The size of the circle indicates the degree of interaction between the genes. The network was constructed with score > 0.9, FDR stringency = 1 percent, and disconnected nodes were hide.
Figure 7
Figure 7
Characterization of differentially expressed miRNAs (DEmiRs) induced by BCL2L2-PABPN1. (A) Length distribution of miRNAs among six samples. (B) Barplot profiling the expression levels of the miRNAs in each sample. (C) Heatmap cluster of known DemiRs. (D) Validation of Illumina data with real-time PCR. (E) GO enrichment of target genes of DEmiRs. 1, detection of chemical stimulus involved in sensory perception of smell; 2, G-protein coupled receptor signaling pathway; 3, homophilic cell adhesion via plasma membrane adhesion molecules; 4, small GTPase mediated signal transduction; 5, integral component of plasma membrane; 6, RNA polymerase II core promoter proximal region sequence-specific DNA binding. (F) KEGG enrichment of target genes of DEmiRs. (G) Fat-related miRNA-mRNA interaction network.
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