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.
Highlights (Up to four bullet points. The length of each highlight cannot exceed 85 characters, including spaces) ! Stress induces phase-separated TDP-43 NBs to alleviate cytotoxicity ! The two RRMs interact with different RNAs and act distinctly in the assembly of TDP-43 NBs ! LncRNA NEAT1 promotes TDP-43 LLPS and is upregulated in stressed neurons ! The ALS-causing D169G mutation is NB-defective and forms pTDP-43 cytoplasmic foci .
SUMMARYMutations in RNA-binding proteins localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregations, which are pathological hallmarks in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In this study, we investigate the function of PARylation, an important protein posttranslational modification known to be involved in regulating many cellular functions such as DNA repair, transcriptional regulation and cell death, in RNP-related neurodegeneration. We reveal that PARylation levels are a major regulator of the dynamic assembly-disassembly of the disease-related RNP granules. We show that hnRNP A1 and TDP-43 can both be PARylated and bind to PARylated proteins, and we further identify the PARylation site and the PAR-binding motif of hnRNP A1. In vitro, PARylation promotes the liquid-liquid phase separation (LLPS) of hnRNP A1; in vivo, PARylation regulates the translocation of hnRNP A1 to stress granules as well as its interaction with TDP-43. Finally, we establish that 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 support PARP as a potential therapeutic target for treating ALS and related diseases.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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