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.

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.

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.

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.

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.