TDP-43 is a Master Regulator of Paraspeckle Condensation

Abstract

The paraspeckle is a stress-inducible membraneless organelle (MLO) assembled on an architectural lncRNA, NEAT1_2. Paraspeckles are widely suppressed in normal tissues but are rapidly yet transiently upregulated under stress; how this tight control is achieved is not fully understood. This MLO is dysregulated in multiple human diseases, from cancer to neurodegeneration. Here we demonstrate that TDP-43 cooperates with FUS to regulate the spectrum of NEAT1_2/paraspeckle assemblies. We find that TDP-43 disperses paraspeckles when in excess, in an RNA-binding dependent manner, and this phenotype can be replicated using in vitro FUS condensates and rescued by FUS supplementation. In stressed cells, TDP-43 is depleted from the nucleoplasm via its sequestration into de novo nuclear condensates, promoting paraspeckle assembly. In TDP-43 sufficient cells, paraspeckle suppression can be bypassed via TDP-43 condensation on NEAT1_2 UG repeats and microphase separation with FUS. TDP-43-containing and -depleted paraspeckles have different properties, with the latter being more dynamic and less prone to form clusters. Genetic analysis in a cohort of ~8000 individuals revealed that longer NEAT1_2 UG repeats associated with paraspeckle suppression positively correlate with disease severity in a neurodegenerative disease amyotrophic lateral sclerosis (ALS). To conclude, we demonstrate that TDP-43 acts as a molecular switch between the paraspeckle low- and high-assembly states and fine-tunes their properties via repeat-RNA binding, sub-MLO condensation and cross-MLO shuttling – a mechanism dysregulated in neurodegeneration.

Note:

Funding Information: The work was supported by the UKRI Future Leaders Fellowship (MR/W004615/1), MRC (MR/W028522/1) and BBSRC (BB/V014110/1 and BB/V014528/1) grants, MND Association grant (Shelkovnikova/Oct17/968-799) and MND Scotland PhD studentship (2022/MNDS/PHD/8045SHEL).

Conflict of Interests: E.G.P.S. is an employee of Fida Biosystems. All other authors have no conflicting interests to declare.

Ethical Approval: All participants gave written informed consent. The institutional review board of the University Medical Center Utrecht approved this study.

Keywords: TDP-43, paraspeckle, NEAT1, FUS, UG-repeat, ALS, membraneless organelle, condensate, stress response