BioProject

TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4, and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing. Overall design: Hence, we hypothesized that the SIRT4-mediated deacetylation of U2AF2 participates in the regulation of these processes. RNA-sequencing was performed to identify the changes in gene expression and alternative splicing in adenovirus-mediated SIRT4OE-transfected cells or adenovirus-Ctrl-transfected cells after TGF-β1 stimulation (Fig. 6A). Surprisingly, SIRT4 stimulation upregulated several genes (P < 0.01; Table EV1) that are involved in mRNA processing and the TGF-β signaling pathway. A total of 142 differentially expressed genes (P < 0.0005) were identified (Fig. 6A). Meanwhile, 248 genes showed differential intron retention (FDR < 0.05; Table EV2) (Fig. 6A). This analysis revealed four genes that were common between the differentially expressed genes and the differentially spliced genes (Fig. 6A). Ccn2, one of these four genes, ranked third among the all upregulated genes (P < 0.0005) after SIRT4 overexpression.