ALS-linked mutations in ubiquilin-2 reduce interaction with heterogeneous nuclear ribonucleoproteins

Abstract

ALS-linked mutations in ubiquilin-2 and some members of the heterogeneous nuclear ribonucleoprotein (hnRNPs) family are implicated in ALS. Most mutations in ubiquilin-2 are missense mutations that occur in and around a PXX repeat motif located in the central domain of the protein. The PXX motif is not present in other ubiquilins, suggesting it might have some specialized function. However, neither the function of the PXX motif nor the mechanism by which mutations in ubiquilin-2 lead to ALS is known. The present study was designed to screen a yeast two-hybrid library using the central domain of ubiquilin-2 to identify proteins whose binding to ubiquilin-2 was affected by ALS-mutations in ubiquilin-2. Three such interactors were identified and all were members of the hnRNP family--hnRNPA1, hnRNPA3 and hnRNPU. The interacting region in each of these proteins was their C-terminal glycine rich-domain, which is the domain most frequently mutated in other RNA-binding proteins also known to be linked to ALS, such as TDP-43 and FUS/TLS. The subsequent experiments focused my studies on hnRNPA1 because a mutation (D262V) in this protein has been linked to ALS. Immunoprecipitation and pulldown studies confirmed that ubiquilin-2 interacts with wild type (WT) hnRNPA1 proteins. However, binding of ubiquilin-2 proteins containing any of five different ALS mutations (P497H, P497S, P506T, P509S, P525S) to WT hnRNPA1 was weaker than that of WT hnRNPA1 as compared with WT ubiquilin-2. Moreover, hnRNPA1 containing the D262V mutations did not bind WT ubiquilin-2. Overexpression of the ubiquilin-2 mutants in NSC-34 cells increased cell death and, for several of the mutants, this coincided with increased translocation of hnRNPA1 to the cytoplasm. Knockdown of ubiquilin-2 decreased the cytoplasmic accumulation of hnRNPA1 protein, in part because it increased turnover of the protein. The discoveries that ubiquilin-2 interacts with hnRNP proteins and that mutations in either protein disrupt that interaction suggest a connection between proteostasis and RNA metabolism. Based on the results presented here, it is also tempting to speculate that decreased interaction between ALS-linked mutations of ubiqiuilin-2 and hnRNPA1 may contribute to the neurodegeneration characteristic of this catastrophic condition.