Autoantigen Ro52 alpha was recently identified as an E3 ubiquitin ligase. Its splicing variant Ro52 beta, which lacks a leucine zipper, has not been characterized yet. We therefore characterized Ro52 beta in contrast to Ro52 alpha. Our biochemical assays revealed that both Ro52a and Ro52 beta function as E3 ubiquitin ligases and self-ubiquitinate in cooperation with UbcH5B in vitro. In addition, both Ro52 alpha and Ro52 beta are ubiquitinated when overexpresscd with ubiquitin in HEK293T cells, Suggesting that both function as E3 ligases and self-ubiquitinate in vivo. However, cytological studies revealed that Ro52 alpha mainly localizes to the cytoplasmic rod-like structures, whereas Ro52 beta diffusely localizes to both the cytoplasm and the nucleus. Since the leucine zipper plays a role in the homodimerization and heterodimerization of Ro52 alpha, the dimerization might be required for the localization of Ro52 alpha to the rod-like Structures. On the basis of these results, Ro52 alpha and Ro52 beta appear to ubiquitinate their particular substrates at different locations. (c) 2005 Elsevier Inc. All rights reserved.