NAT10-mediated ac4C modification of Lipin1 mRNA contributes to the pathogenesis of PWMI
Xinyu Li, Meng Zhang, Yanan Liu, Chunjie Guo, Yiwei Liu, Lei Han, Zhaowei Feng, Xiue Wei, Ruiqin Yao
Xinyu Li, Meng Zhang, Yanan Liu, Chunjie Guo, Yiwei Liu, Lei Han, Zhaowei Feng, Xiue Wei, Ruiqin Yao
View: Text | PDF
Research Article Cell biology Neuroscience

NAT10-mediated ac4C modification of Lipin1 mRNA contributes to the pathogenesis of PWMI

Abstract

Preterm white matter injury (PWMI) is a leading cause of cerebral palsy and chronic neurological disabilities in premature infants. It is characterized by defects in oligodendrocyte precursor cell (OPC) differentiation and dysmyelination. Currently, there are no effective therapeutic strategies available in clinical practice. Lipid homeostasis plays a crucial role in myelin development, yet the function of Lipin1 — a key phosphatidic acid phosphatase involved in phospholipid synthesis — remains unclear. In this study, we identified a significant downregulation of Lipin1 in OPCs from PWMI mice, which impaired OPC differentiation and myelin formation. Conversely, Lipin1 overexpression in these mice promoted OPC maturation and enhanced myelin development. We found evidence that N-acetyltransferase 10 (NAT10) acts as a regulator of Lipin1 expression through RNA pull-down and mass spectrometry. NAT10-mediated N4-acetylcytidine (ac4C) modification enhanced Lipin1 mRNA stability and translation, and NAT10 knockdown in OPCs impaired myelination, highlighting its crucial role in Lipin1-mediated myelination. Our study revealed that the downregulation of Lipin1 impaired OPC differentiation and myelination in PWMI, with NAT10-mediated ac4C modification playing a critical role in regulating Lipin1 expression. These findings highlight Lipin1 and NAT10 as promising therapeutic targets for treating myelination defects in PWMI, warranting further investigation into their potential in preterm birth–related neurological disorders.

Authors

Xinyu Li, Meng Zhang, Yanan Liu, Chunjie Guo, Yiwei Liu, Lei Han, Zhaowei Feng, Xiue Wei, Ruiqin Yao

×

Figure 6

NAT10 regulated the ac4C acetylation of Lipin1 mRNA in OPCs.

Options: View larger image (or click on image) Download as PowerPoint
NAT10 regulated the ac4C acetylation of Lipin1 mRNA in OPCs. (A) RNA pul...
(A) RNA pull-down assay to verify the interaction between Lipin1 acetylation sites and NAT10 after constructing ac4C-site mutation plasmids. (B and C) In RIP experiments, Western blotting confirmed the expression of NAT10, and RT-qPCR measured the relative mRNA abundance of Lipin1. (D–F) Western blot analysis of NAT10 and Lipin1 protein levels in primary cultured OPCs and statistical analysis. Representative of 3 independent experiments. (G) RT-qPCR analysis of Lipin1 mRNA levels in primary cultured OPCs. (H) ac4C-RIP analysis of Lipin1 mRNA levels modified by ac4C in OPCs after NAT10 knockdown. (I) Cells treated with actinomycin D for 0, 2, and 4 hours to inhibit total RNA transcription, and RT-qPCR analysis of relative Lipin1 mRNA expression in mice. *siCtrl vs. siNAT10-1; #siCtrl vs. siNAT10-2. (J and K) Sucrose density gradient centrifugation assay to validate the regulation of Lipin1 translation efficiency by NAT10. The position of mRNA in the sucrose gradient reflects its translation status: mRNA coprecipitating with ribonucleoproteins (RNPs) or ribosomal subunits indicates lack of translation, while coprecipitation with polysomes suggests active translation. Statistics: 2-tailed Student’s t test (C), 1-way ANOVA with LSD post hoc analysis (E–H and K), and 2-way ANOVA with Tukey’s multiple-comparison test (I). *siCtrl vs. siNAT10-1; #siCtrl vs. siNAT10-2. n = 3. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ##P < 0.01, ###P < 0.001.