SRSF3-TRIM28-MDC1 prevents DNA damage caused by R-loops in fatty liver disease in mice
Panyisha Wu, Manasi Das, Yanting Wang, Yichun Ji, Yuli Wu, Deepak Kumar, Lily J. Jih, Nicholas J.G. Webster
Panyisha Wu, Manasi Das, Yanting Wang, Yichun Ji, Yuli Wu, Deepak Kumar, Lily J. Jih, Nicholas J.G. Webster
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Research Article Endocrinology Hepatology

SRSF3-TRIM28-MDC1 prevents DNA damage caused by R-loops in fatty liver disease in mice

Abstract

Serine-rich splicing factor 3 (SRSF3) is crucial for the metabolic functions of the liver. The genetic deletion of SRSF3 in mouse hepatocytes impairs hepatic lipid and glucose metabolism and leads to fibrosis and formation of hepatocellular adenoma that progresses to hepatocellular carcinoma. SRSF3 protein is proteosomally degraded in metabolic-dysfunction associated fatty liver disease (MAFLD) and metabolic-dysfunction-associated steatohepatitis (MASH). We show here that depleting SRSF3 protein in hepatocytes promoted R-loop accumulation and increased DNA damage in the liver. Prevention of SRSF3 degradation in vivo protected hepatocytes from DNA double-strand breaks in mice with MASH. This protection extended to other DNA-damaging agents such as camptothecin, palmitic acid, or hydrogen peroxide when tested on HepG2 cells in vitro. SRSF3 interacted with TRIM28 and MDC1, which are components of the ATM DNA-damage repair complex, and knockdown of any of these 3 proteins reduced the expression of the other 2 proteins, suggesting they form a functional complex. Lastly, by preventing degradation of SRSF3, we were able to reduce tumors in a diethyl-nitrosamine–induced (DEN-induced) model of cirrhotic HCC. These findings suggest that maintenance of SRSF3 protein stability is crucial for preventing DNA damage and protecting liver from early metabolic liver disease and progression to HCC.

Authors

Panyisha Wu, Manasi Das, Yanting Wang, Yichun Ji, Yuli Wu, Deepak Kumar, Lily J. Jih, Nicholas J.G. Webster

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Figure 3

Knockdown of SRSF3 causes R-loop accumulation.

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Knockdown of SRSF3 causes R-loop accumulation. (A) Dot blot of R-loops u...
(A) Dot blot of R-loops using antibody S9.6 in HEK293 cells treated with control siRNA (si-Control) or SRSF3 siRNA (si-SRSF3). Bots were stripped and reblotted for double-stranded DNA (dsDNA). Graph shows quantification of R-loop levels normalized to dsDNA (n = 2/group). (B) Dot blot of R-loops in genomic DNA from Flox and SRSF3-KO hepatocytes (SRSF3-HKO). Graph shows quantification of R-loop levels normalized to dsDNA (n = 3/group). (C) HEK293 cells were transfected by GFP, Flagged-SRSF3-WT, or Flagged-SRSF3-K11R plasmids for 48 hours, before being treated with 0.1 μM CPT or DMSO (vehicle control) for 1 hour and R-loops detected by dot blot. Graph shows quantification of R-loop levels normalized to dsDNA (n = 3/group). (D) HepG2 cells were infected by AAV8 expressing GFP or Flagged-SRSF3-K11R, before being treated with 0.1 μM CPT or DMSO for 1 hour, and R-loops were detected by dot blot. Graph shows quantification of R-loop levels normalized to dsDNA (n = 3/group). (E) Dot blot of R-loops in genomic DNA from livers of lean mice on normal chow (control) or mice on a Western (MASH) diet 7 weeks after infection with AAV8 expressing GFP (MASH-GFP), WT SRSF3 (MASH-WT), or the degradation-resistant K11R-mutant SRSF3 (MASH-K11R). Graph shows quantification of R-loop levels normalized to dsDNA (n = 3/group). (F) Immunoblotting of SRSF3, γH2ax from HEK293 cells with or without SRSF3 knockdown. Five units of RNase H protein were transfected in selected wells for 4 hours to digest R-loops. Graph shows quantification of SRSF3 and γH2ax protein levels normalized to β-actin (n = 3/group). (G) Immunofluorescent staining for γH2ax in HEK293 cells treated as above. DAPI was stained to visualize the nuclei. ce staining for TRIM28 and SRSF3. In all cases DAPI was used to visualize the nuclei. Scale bar: 10 μm. Original magnification, ×630. Graph shows quantification of γH2ax+ nuclei/field (n = 3/group). All quantified results are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA.