An Intrinsically Disordered Low Complexity Domain is Required for U1‐70K Self‐association

Abstract

The RNA binding protein U1‐70K aggregates via an unknown mechanism in Alzheimer's disease (AD). Interestingly, AD brain homogenates can induce the aggregation of soluble U1‐70K from control brain or recombinant U1‐70K (rU1‐70K), rendering it detergent‐insoluble. The intrinsically disordered C‐terminus of U1‐70K is necessary for aggregation and harbors two low‐complexity (LC) domains, LC1 and LC2. Similar LC domains have been shown to polymerize into amyloid‐like aggregates in vitro, and the RNA binding proteins TDP‐43 and FUS, which also harbor LC domains, form detergent‐insoluble amyloid‐like aggregates in neurodegenerative disease. Our objective is to determine if the LC domains of U1‐70K are necessary for self‐association. Addressing this question may provide mechanistic insight into the aggregation of U1‐70K in AD. To investigate this question we performed co‐immunoprecipitations of full‐length rU1‐70K and various mutants lacking either the LC1, LC2, or both LC domains. These assays revealed that the LC1 domain (amino acids 231‐308) of rU1‐70K is necessary and sufficient for association with native U1‐70K. Similarly, we utilized bio‐layer interferometry to measure the binding affinity of the LC1 domain with a synthetic peptide corresponding to amino acids 252‐270 of U1‐70K. These data provide evidence for a direct self‐association between regions of the LC1 domain. Finally, by immunocytochemistry we show that rU1‐70K mutants lacking the LC1 domain have an impaired ability to form nuclear granule structures. Together these data indicate that the disordered LC1 domain is necessary for U1‐70K self‐association and subnuclear compartmentalization.Sources of Research Support:NIRG‐12‐242297 (NTS) and T32‐NS007480 (IB)