Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties
Grese ZR, Bastos AC, Mamede LD, French RL, Miller TM and Ayala YM
Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties
Grese ZR, Bastos AC, Mamede LD, French RL, Miller TM and Ayala YM
TDP-43 is an RNA-binding protein that forms ribonucleoprotein condensates via liquid-liquid phase separation (LLPS) and regulates gene expression through specific RNA interactions. Loss of TDP-43 protein homeostasis and dysfunction are tied to neurodegenerative disorders, mainly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Alterations of TDP-43 LLPS properties may be linked to protein aggregation. However, the mechanisms regulating TDP-43 LLPS are ill-defined, particularly how TDP-43 association with specific RNA targets regulates TDP-43 condensation remains unclear. We show that RNA binding strongly promotes TDP-43 LLPS through sequence-specific interactions. RNA-driven condensation increases with the number of adjacent TDP-43-binding sites and is also mediated by multivalent interactions involving the amino and carboxy-terminal TDP-43 domains. The physiological relevance of RNA-driven TDP-43 condensation is supported by similar observations in mammalian cellular lysate. Importantly, we find that TDP-43-RNA association maintains liquid-like properties of the condensates, which are disrupted in the presence of ALS-linked TDP-43 mutations. Altogether, RNA binding plays a central role in modulating TDP-43 condensation while maintaining protein solubility, and defects in this RNA-mediated activity may underpin TDP-43-associated pathogenesis.
TDP-43 dysfunction results in R-loop accumulation and DNA replication defects
Wood M, Quinet A, Lin YL, Davis AA, Pasero P, Ayala YM and Vindigni A
TDP-43 dysfunction results in R-loop accumulation and DNA replication defects
Wood M, Quinet A, Lin YL, Davis AA, Pasero P, Ayala YM and Vindigni A
TAR DNA-binding protein 43 (TDP-43; also known as TARDBP) is an RNA-binding protein whose aggregation is a hallmark of the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 loss increases DNA damage and compromises cell viability, but the actual function of TDP-43 in preventing genome instability remains unclear. Here, we show that loss of TDP-43 increases R-loop formation in a transcription-dependent manner and results in DNA replication stress. TDP-43 nucleic-acid-binding and self-assembly activities are important in inhibiting R-loop accumulation and preserving normal DNA replication. We also found that TDP-43 cytoplasmic aggregation impairs TDP-43 function in R-loop regulation. Furthermore, increased R-loop accumulation and DNA damage is observed in neurons upon loss of TDP-43. Together, our findings indicate that TDP-43 function and normal protein homeostasis are crucial in maintaining genomic stability through a co-transcriptional process that prevents aberrant R-loop accumulation. We propose that the increased R-loop formation and genomic instability associated with TDP-43 loss are linked to the pathogenesis of TDP-43 proteinopathies.This article has an associated First Person interview with the first author of the paper.
TDP-43 α-helical structure tunes liquid-liquid phase separation and function
Conicella AE, Dignon GL, Zerze GH, Schmidt HB, D’Ordine AM, Kim YC, Rohatgi R, Ayala YM, Mittal J and Fawzi NL
TDP-43 α-helical structure tunes liquid-liquid phase separation and function
Conicella AE, Dignon GL, Zerze GH, Schmidt HB, D’Ordine AM, Kim YC, Rohatgi R, Ayala YM, Mittal J and Fawzi NL
Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.
RNA-Based Therapies for Neurodegenerative Diseases
Ayala YM and Nguyen AD
RNA-Based Therapies for Neurodegenerative Diseases
Ayala YM and Nguyen AD
Most neurodegenerative disorders afflict the ageing population and are often incurable. Therefore, therapeutic development is a major focus in biomedical research. We highlight a new class of drugs, RNA molecules, to control gene expression and decrease neurotoxicity. Their efficacy is shown in pre-clinical studies, clinical trials and in cases of approved patient treatment. As the number of RNA-based strategies increases, so does the promise of targeting more disease-associated genes through a variety of different mechanisms.
TDP-43 Oligomerization and Phase Separation Properties Are Necessary for Autoregulation
Koehler LC, Grese ZR, Bastos ACS, Mamede LD, Heyduk T and Ayala YM
TDP-43 Oligomerization and Phase Separation Properties Are Necessary for Autoregulation
Koehler LC, Grese ZR, Bastos ACS, Mamede LD, Heyduk T and Ayala YM
Loss of TDP-43 protein homeostasis and dysfunction, in particular TDP-43 aggregation, are tied to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is an RNA binding protein tightly controlling its own expression levels through a negative feedback loop, involving TDP-43 recruitment to the 3′ untranslated region of its own transcript. Aberrant TDP-43 expression caused by autoregulation defects are linked to TDP-43 pathology. Therefore, interactions between TDP-43 and its own transcript are crucial to prevent TDP-43 aggregation and loss of function. However, the mechanisms that mediate this interaction remain ill-defined. We find that a central RNA sequence in the 3′ UTR, which mediates TDP-43 autoregulation, increases the liquid properties of TDP-43 phase separation. Furthermore, binding to this RNA sequence induces TDP-43 condensation in human cell lysates, suggesting that this interaction promotes TDP-43 self-assembly into dynamic ribonucleoprotein granules. In agreement with these findings, our experiments show that TDP-43 oligomerization and phase separation, mediated by the amino and carboxy-terminal domains, respectively, are essential for TDP-43 autoregulation. According to our additional observations, CLIP34-associated phase separation and autoregulation may be efficiently controlled by phosphorylation of the N-terminal domain. Importantly, we find that specific ALS-associated TDP-43 mutations, mainly M337V, and a shortened TDP-43 isoform recently tied to motor neuron toxicity in ALS, disrupt the liquid properties of TDP-43-RNA condensates as well as autoregulatory function. In addition, we find that M337V decreases the cellular clearance of TDP-43 and other RNA binding proteins associated with ALS/FTD. These observations suggest that loss of liquid properties in M337V condensates strongly affects protein homeostasis. Together, this work provides evidence for the central role of TDP-43 oligomerization and liquid-liquid phase separation linked to RNA binding in autoregulation. These mechanisms may be impaired by TDP-43 disease variants and controlled by specific cellular signaling.