Mutations in RNA-binding proteins (RBPs) localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregation, which is a pathological hallmark of various neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. In this study, we investigate the function of poly(ADP-ribosyl)ation (PARylation), an important PTM involved in DNA damage repair and cell death, in RNP granule-related neurodegeneration. We reveal that PARylation levels are a major regulator of the assembly-disassembly dynamics of RNP granules containing disease-related RBPs, hnRNP A1 and TDP-43. We find that hnRNP A1 can both be PARylated and bind to PARylated proteins or poly(ADP-ribose) (PAR). We further uncover that PARylation of hnRNP A1 at K298 controls its nucleocytoplasmic transport, whereas PAR-binding via the PAR-binding motif (PBM) of hnRNP A1 regulates its association with stress granules. Moreover, we reveal that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, both genetic and pharmacological inhibition of PARP mitigates hnRNP A1- and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, our findings suggest a novel and crucial role for PARylation in regulating the dynamics of RNP granules, and that dysregulation in PARylation and PAR levels may contribute to ALS disease pathogenesis by promoting protein aggregation.
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We thank the BDSC and the Kyoto DGRC for providing fly strains, L. Pan for the pET32M.3C plasmid, B. Yang and X. Duan for help in in vitro PARylation assay, S. Qiu and S. Zhang for technical supports, and J. Yuan, A. Li and members of the Zhang lab, the Jiang lab, the Liu lab, and the Fang lab for helpful discussion and critical reading of the manuscript. This study was supported by grants from the National Key R&D Program of China (No. 2016YFA0501902), the National Natural Science Foundation of China (No. 81671254 and No. 31471017) to Y.F., (No. 91853112 and No. 31470748) to C.L., (No. 21778063 and No. 91753114) to H.J., (No. 31500665 and No. 31671428) to Y.Z., and funding from the Shanghai Science and Technology Committee (No. 18ZR1448300) to A.D.
Y.F. and Y.D. have filed an international patent via the PCT entitled “Application of PARP1 Inhibitors for Treating Patients with Amyotrophic Lateral Sclerosis”. The remaining authors declare no conflict of interests.
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Duan, Y., Du, A., Gu, J. et al. PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins. Cell Res 29, 233–247 (2019) doi:10.1038/s41422-019-0141-z
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