RNF13 protein regulates endoplasmic reticulum stress induced apoptosis in dopaminergic SH-SY5Y cells by enhancing IRE1α stability.

Any interruption in the folding capacity of endoplasmic reticulum (ER) may result in inducing ER stress which would initiate an adaptive signaling mechanism called Unfolded Protein Response (UPR) in order to restore homeostasis, failing to which would initiate signaling pathway leading to death of the cell. Finding new mediators could help better understand the molecular mechanisms of ER stress-induced apoptosis. Our lab initiated a genetic screen method using retroviral insertion mutation system to look for genes whose inactivation would confer resistance to apoptosis. In our previous findings, Ring finger protein 13 (RNF13) was identified whose down-regulation conferred survival against ER stress-induced apoptosis. Our previous results also showed important role of RNF13 in apoptotic signaling in 293T cells as a result of strong RNF13-IRE1α interaction. In current study, using SH-SY5Y cells, overexpression of RNF13 in apoptosis assays and RT-PCR analysis has shown to induce apoptosis as well as splicing of X-box binding Protein 1 (XBP1) confirming its role in ER stress mediated cell death in this cell line as well. Western blot analysis has revealed that overexpression of both N-terminal as well as C-terminal tagged RNF13 resulted in activation of c-Jun N-terminal kinase (JNK) in SH-SY5Y cells. Our co-immunoprecipitation assays in SH-SY5Y cells also showed a strong interaction of RNF13 with IRE1α. Finally, Cycloheximide chase experiment exhibited that RNF13-IRE1α interaction increased the stability of IRE1α. Altogether, our data suggest that RNF13 may act as an important regulator of IRE1α, mediating ER stress-mediated apoptosis in neuroblastoma SH-SY5Y cells.