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.
Short title (<50 characters): Phase-separated TDP-43 NBs mitigate stress 20 Keywords: TDP-43, nuclear body, RNP granule, phase separation, lncRNA NEAT1 21 Word count: ~6500 words (excluding the title page, abstract, references and figure legends)Recent studies indicate that liquid-liquid phase separation (LLPS) of RNA-binding protein 72 (RBPs) drives the assembly of liquid droplet (LD)-like, membraneless RNP granules in the 73 cytoplasm and nucleoplasm (25)(26)(27)(28)(29). Several ALS-related RBPs including TDP-43, FUS, hnRNP 74 A1, hnRNP A2 and TIA1 are shown to phase separate in vitro (14,(30)(31)(32)(33)(34)(35). The intrinsically 75 disordered LCD domains of the RBPs are thought to mediate the LLPS (28, 31,(36)(37)(38) and 76 posttranslational protein modifications play an important role in determining the biophysical and 77 biological properties of the phase behavior of the RBPs (34,(39)(40)(41)(42)(43)(44)(45)(46). In addition to the LCD, 78 recent studies reveal that RNA is of vital importance in regulating the LLPS (47,48), which can 79 either suppress or promote phase separation depending on the contents and concentrations of 80 the RNAs (25,(49)(50)(51). 81In this study, we find that various cellular stresses induce TDP-43 to form distinct, highly 82 dynamic and reversible NBs, which not only attenuate the cytotoxicity of TDP-43 in mammalian 83 cells but also ameliorate neurodegeneration and behavioral deficits in a Drosophila model of ALS. 84Further investigation with super-resolution microscopy reveals the unexpected opposing 85 functions of the two RRMs in maintaining the core-shell architecture of TDP-43 NBs. 86Furthermore, we demonstrate a crucial role of the paraspeckle scaffolding RNA nuclear-enriched 87 abundant transcript 1 (NEAT1) in promoting TDP-43 LLPS in vitro, which is compromised by the 88 ALS-causing mutation D169G, leading to a defect in forming TDP-43 NBs. Together, our findings 89 demonstrate how stress induces the assembly of TDP-43 NBs in the generally "suppressive" 90 nucleoplasm environment and suggest the involvement of dysfunctional TDP-43 NBs in ALS 91 pathogenesis. 92Phase-separated TDP-43 NBs mitigate stress_CWang Page 6 of 44 RESULTS 93 Arsenic stress induces dynamic and reversible TDP-43 NBs 94TDP-43 protein is predominantly localized to the nucleus but can shuttle between the nucleus 95 and the cytoplasm. In response to cellular stress, TDP-43 is recruited to cytoplasmic SGs (11). 96Interestingly, in an earlier related work of our group (46), we noticed that although arsenite, a 97 commonly used experimental reagent to raise cellular stress (52), induced TDP-43 + cytoplasmic 98SGs, the majority of TDP-43 signal remained in the nucleus. Furthermore, in a great portion of 99 the cells, the nuclear TDP-43 lost the normal diffused pattern and instead exhibited a distinct 100 granular appearance (Movie S1 and Fig. 1, A and B). 101 A membraneless nuclear structure fulfills the requirements of NBs if it is: (1) microscopically 102 visible, (2) enriched with specific nuclear fac...
RNA-binding proteins (RBPs) and RNAs can form dynamic, liquid droplet-like cytoplasmic condensates, known as stress granules (SGs), in response to a variety of cellular stresses. This process is driven by liquid–liquid phase separation, mediated by multivalent interactions between RBPs and RNAs. The formation of SGs allows a temporary suspension of certain cellular activities such as translation of unnecessary proteins. Meanwhile, non-translating mRNAs may also be sequestered and stalled. Upon stress removal, SGs are disassembled to resume the suspended biological processes and restore the normal cell functions. Prolonged stress and disease-causal mutations in SG-associated RBPs can cause the formation of aberrant SGs and/or impair SG disassembly, consequently raising the risk of pathological protein aggregation. The machinery maintaining protein homeostasis (proteostasis) includes molecular chaperones and co-chaperones, the ubiquitin-proteasome system, autophagy, and other components, and participates in the regulation of SG metabolism. Recently, proteostasis has been identified as a major regulator of SG turnover. Here, we summarize new findings on the specific functions of the proteostasis machinery in regulating SG disassembly and clearance, discuss the pathological and clinical implications of SG turnover in neurodegenerative disorders, and point to the unresolved issues that warrant future exploration.
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