Regulation of ascidian Rel by its alternative splice variant

The Rel/NF-kappaB family of transcription factors play key roles in morphogenesis and immune responses. We reported previously that As-rel1 and As-rel2 of the ascidian Halocynthia roretzi are involved in notochord formation. The As-rel1 protein is a typical Rel/NF-kappaB family member, whereas the As-rel2 protein is a novel truncated product of As-rel1 that lacks a nuclear localization signal and the unique C-terminal region. Here, we present conclusive evidence that As-rel1 and As-rel2 are generated from a single gene by alternative splicing. We analyzed the roles of As-rel2 using cells transfected with As-rel1 or As-rel2 or both. As-rel1 was localized in the nucleus and As-rel2 in the cytoplasm when they were transfected individually. In contrast, when they were transfected simultaneously, both were localized in the nucleus because of the association of As-rel2 with As-rel1. In this case, the transcriptional activity of As-rel1 was suppressed by As-rel2. Ascidian IkappaB was found to sequester As-rel1 in the cytoplasm and suppress its transcriptional activity when As-rel1 and IkappaB were transfected simultaneously. In contrast, when As-rel1 and IkappaB were transfected together with As-rel2, As-rel1 was transported into the nucleus and its transcriptional activity was rescued from inhibition by IkappaB, whereas As-rel2 remained localized in the cytoplasm, suggesting IkappaB sequestration in the cytoplasm by As-rel2. From these findings, we conclude that the alternative splice variant, As-rel2, regulates the nuclear localization and transcriptional activity of As-rel1.