ALS-linked mutations in ubiquilin-2 or hnRNPA1 reduce interaction between ubiquilin-2 and hnRNPA1

Amyotrophic lateral sclerosis (ALS)-linked mutations in UBQLN2 and some members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) family cause ALS. Most mutations in UBQLN2 are missense mutations that occur in and around a PXX repeat motif located in the central domain of the encoded protein. However, neither the function of the PXX motif nor the mechanism by which mutations in UBQLN2 cause ALS is known. We screened a yeast two-hybrid library using the central domain of ubiquilin-2 hoping to identify proteins whose binding is affected by the UBQLN2 mutations. Three such interactors were identified-hnRNPA1, hnRNPA3 and hnRNPU-all members of the hnRNP family. The interacting region in each of these proteins was their glycine-rich domain, the domain most frequently mutated in hnRNP-related proteins that cause ALS. We focused on hnRNPA1, because a mutation in the protein causes ALS. We confirmed the interaction between wild-type (WT) ubiquilin-2 and hnRNPA1 proteins in vitro and in cells. In contrast, all five ALS mutations in ubiquilin-2 that we examined had reduced binding with WT hnRNPA1. In addition, hnRNPA1 carrying the D262V missense mutation that causes ALS failed to bind WT ubiquilin-2. Overexpression of ubiquilin-2 containing the ALS mutations increased cell death and, for several of the mutants, this correlated with increased translocation of hnRNPA1 to the cytoplasm. Knockdown of ubiquilin-2 led to increased turnover of hnRNPA1, indicating ubiquilin-2 functions to stabilize hnRNPA1. The discovery that ubiquilin-2 interacts with hnRNP proteins and that mutation in either protein disrupts interaction suggests a connection between proteostasis and RNA metabolism.