A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing
TDP-43 is an RNA-binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C-terminal domain which mediates ALS inclusions, TDP-43 has a globular N-terminal domain (NTD). Here, we show that TDP-43 NTD assembles into head-to-tail linear chains and that phosphomimetic substitution at S48 disrupts TDP-43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro, fluidizes liquid–liquid phase separated nuclear TDP-43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head-to-tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher-order polymerization. These data provide structural detail for the established mechanistic role of the well-folded TDP-43 NTD in splicing and link this function to LLPS. In addition, the fusion-tag solubilized, recombinant form of TDP-43 full-length protein developed here will enable future phase separation and in vitro biochemical assays on TDP-43 function and interactions that have been hampered in the past by TDP-43 aggregation.
Source: EMBO Journal - Category: Molecular Biology Authors: Wang, A., Conicella, A. E., Schmidt, H. B., Martin, E. W., Rhoads, S. N., Reeb, A. N., Nourse, A., Ramirez Montero, D., Ryan, V. H., Rohatgi, R., Shewmaker, F., Naik, M. T., Mittag, T., Ayala, Y. M., Fawzi, N. L. Tags: Neuroscience, Protein Biosynthesis & Quality Control, RNA Biology Articles Source Type: research