Enhancement of bZIP60 function through C-terminal region translated after splicing in Arabidopsis

The unfolded protein response (UPR) is a central regulatory pathway that ensures the proper function of the endoplasmic reticulum (ER) through efficient protein folding and quality control. In Arabidopsis, bZIP60 mRNA is activated by an IRE1-mediated unconventional splicing that excises a 23-nucleotide intron, resulting in the spliced form (bZIP60s mRNA) that encodes the active bZIP60 transcription factor lacking a transmembrane domain. In this study, we investigated the functional role of the spliced form-specific C-terminal extension, hereafter referred to as ORF2. Transient expression assays in Arabidopsis mesophyll protoplasts demonstrated that full-length bZIP60s potently activates the BiP3 promoter compared to a truncated variant lacking ORF2. Fusion of ORF2 to transcription factors unrelated to the UPR did not enhance their transcriptional potency, underscoring its specialized role in the context of bZIP60s. Furthermore, mutation in a conserved nuclear localization signal within ORF2 decreased promoter activation by bZIP60s. Fusion of ORF2 to GFP enhanced the nuclear localization of GFP. Our results suggest that ORF2 is critical for the full transcriptional activity of bZIP60s to ensure an efficient UPR.