Landscape of tissue-specific RNA Editome provides insight into co-regulated and altered gene expression in pigs (Sus-scrofa)

Abstract

RNA editing generates genetic diversity in mammals by altering amino acid sequences, miRNA targeting site sequences, influencing the stability of targeted RNAs, and causing changes in gene expression. However, the extent to which RNA editing affect gene expression via modifying miRNA binding site remains unexplored. Here, we first profiled the dynamic A-to-I RNA editome across tissues of Duroc and Luchuan pigs. The RNA editing events at the miRNA binding sites were generated. The biological function of the differentially edited gene in skeletal muscle was further characterized in pig muscle-derived satellite cells. RNA editome analysis revealed a total of 171,909 A-to-I RNA editing sites (RESs), and examination of its features showed that these A-to-I editing sites were mainly located in SINE retrotransposons PRE-1/Pre0_SS element. Analysis of differentially edited sites (DESs) revealed a total of 4,552 DESs across tissues between Duroc and Luchuan pigs, and functional category enrichment analysis of differentially edited gene (DEG) sets highlighted a significant association and enrichment of tissue-developmental pathways including TGF-beta, PI3K-Akt, AMPK, and Wnt signaling pathways. Moreover, we found that RNA editing events at the miRNA binding sites in the 3'-UTR of HSPA12B mRNA could prevent the miRNA-mediated mRNA downregulation of HSPA12B in the muscle-derived satellite (MDS) cell, consistent with the results obtained from the Luchuan skeletal muscle. This study represents the most systematic attempt to characterize the significance of RNA editing in regulating gene expression, particularly in skeletal muscle, constituting a new layer of regulation to understand the genetic mechanisms behind phenotype variance in animals.Abbreviations: A-to-I: Adenosine-to-inosine; ADAR: Adenosine deaminase acting on RNA; RES: RNA editing site; DEG: Differentially edited gene; DES: Differentially edited site; FDR: False discovery rate; GO: Gene Ontology; KEGG: Kyoto Encyclopaedia of Genes and Genomes; MDS cell: musclederived satellite cell; RPKM: Reads per kilobase of exon model in a gene per million mapped reads; UTR: Untranslated coding regions.

Keywords: RNA editing; gene expression; hspa12b; miRNA; muscle-derived satellite cell; skeletal muscle.

Figure 2.

Differentially editing sites (DESs) between Luchuan and Duroc pigs. (A) Distribution of DESs in 3′-UTRs, 5′-UTRs, introns, missense, synonymous, and intergenic regions. Numbers above the bars are the numbers of the sites. See Additional file 1: Table S4 for all DESs identified in the present study. (B) Distribution of DESs across tissue types. The numbers above the bars are the number of sites. (C) Overall editing level across tissue types between Duroc and Luchuan. (D-E) The GO terms and Kyoto Encyclopaedia of Genes and Genomes pathways of all expressed genes harbouring DESs using ShinyGO v0.61 [33]. See Additional file 2: Table S6 and Table S7 for p-values of functional category enrichment analyses

Figure 1.

Overview of RNA editome profiling. (A) Flowchart for the identification of RESs across tissues from Duroc and Luchuan. The analyses are shown in red and green highlighted boxes. (B) Number of RNA editing variant types in the pig tissue transcriptome. (C) Total A-to-I RESs found in forward and reverse strands, respectively. (D) Distribution of RESs in major repetitive element families. (E) Distribution of RESs across the repetitive element types. (F) Distribution of RESs across different genomic regions. The numbers above the bars are the numbers of RESs. (G) The Venn diagram shows overlaps between RESs in this study and DREP database. (H) Validation of 80 RESs by Sanger sequencing and multiple alignments of reads. The validated RESs are represented by blue bars and genomic DNA mutations are represented by pink bars. The numbers inside the bars are the number of sites. In the Additional file: Table S3, validation results for these 80 RESs are reported

Figure 3.

The differentially edited genes (DEGs) in the pig skeletal muscle. (A) The overall editing level of DEGs located in the miRNA targeting regions. (B) The expression levels of TM2D1, HSPA12B, CCDC86, and EIF2AK2 were quantified by Reads Per Kilobase of the transcript, per Million mapped reads (RPKM). (C) The qRT-PCR results of TM2D1, HSPA12B, CCDC86, and EIF2AK2 in Luchuan and Duroc skeletal muscles. GAPDH was taken as an internal control. Significance in (A-C): *P < 0.05; **P < 0.01

Figure 4.

RNA editing in 3′-UTR regulates gene expression via miRNA binding sites. The relative expression level of (A), HSPA12B; (B), miR-181b; and (C), miR-205 in Luchuan and Duroc skeletal muscles. GAPDH was used as an internal control for HSPA12B and U6 for miR-181b and miR-205. Error bars indicate standard errors. (D-E) The qRT-PCR results of HSPA12B expression level in MDS cells with/without miR-181b/miR-205 transfection. GAPDH was utilized as an internal control. Error bars signify the standard errors. (F-G) The relative luciferase activity in HEK293T cells carrying edited HSPA12B-mutant-3′-UTR (HSPA12B-MUT-UTR) or not-edited HSPA12B 3′-UTR (HSPA12B-WT-UTR) in the miRNA targeting regions. Y-axis shows the relative luciferase activity. A two-tailed Wilcoxon test was used to evaluate the difference of luciferase activity using R. Error bars signify the standard errors. (H-I) Schematics of the predicted binding sites for miR-181b/miR-205 in the 3′-UTR of HSPA12B RNA editing site. The RNA mutations are shown in green letters. Significance in (A, and D-G): *P < 0.05; **P < 0.01; ***P < 0.001; ns: no significance

Figure 5.

The role of HSPA12B in MDS cell proliferation. (A-B) The mRNA expression level of HSPA12B in MDS cells after si-HSPA12B and HSPA12B-OE transfection. (C) The protein expression level of HSPA12B was accessed by western blot using GAPDH protein and PCNA (proliferation marker) as an internal control after si-HSPA12B transfection. Data collected from three independently replicated wells. (D) The HSPA12B protein expression level accessed by western blot using GAPDH protein as an internal control after HSPA12B-OE transfection. Data collected from three independently replicated wells. (E-F) The CCK-8 assay displayed absorbance at 450 nm in MDS cells after si-HSPA12B and HSPA12B-OE transfection. Data were collected and calculated as mean ± SD from three independently replicated wells. (G-H) The EdU positive cells in red after si-HSPA12B and HSPA12B-OE transfection. Nuclei were stained with DAPI (blue). Data were collected from three independently replicated wells. Scale bar, 100 µm. Significance in (A-B) and (E-F): **P < 0.01

Figure 6.

HSPA12B is identified as a functional target for miR-181b. (A) Co-localization of HSPA12B and miR-181b in MDS cells using RNA-FISH assay. Nuclei were stained with DAPI. HSPA12B was probed with Cy3 (red) while miR-181b was probed with FAM (green), Scale bar, 100 µm. (B) The miR-181b expression level in MDS cells after miR-181b (mimic) and anti-miR-181b (inhibitor) transfection as determined by qPCR analysis. Data are expressed as mean ± SD from three independent experiments. (C) The protein expression level of HSPA12B in MDS cells after miR-181b (mimic) and anti-miR-181b (inhibitor) transfection as determined by western blot. Data were collected from three independently replicated wells and the band intensity of HSPA12B protein expression was measured by ImageJ software. (D) HSPA12B and miR-181b relative expression levels in pig MDS cells, with each data point representing an individual sample. (E) The CCK-8 assay displayed absorbance at 450 nm in MDS cells after miR-181b (mimic) and anti-miR-181b (inhibitor) transfection. Data were collected and calculated as mean ± SD from three independently replicated wells. (F) The EdU positive cells in red after miR-181b (mimics) and anti-miR-181b transfection as determined by data collected from three independently replicated wells. Nuclei were stained with DAPI (blue). Scale bar, 100 µm. Significance in (B), (C), and (E): *p < 0.05; **P < 0.01; ***P < 0.001; ns: no significance