SAG/ROC2/Rbx2/Hrt2, a component of SCF E3 ubiquitin ligase: Genomic structure, a splicing variant, and two family pseudogenes

We have recently cloned and characterized an evolutionarily conserved gene, Sensitive to Apoptosis Gene (SAG), which encodes a redox-sensitive antioxi dant protein that protects cells from apoptosis induced by redox agents. Th e SAG protein was later found to be the second family member of ROC/Rbx/Hrt , a component of the Skp1-cullin-F box protein (SCF) E3 ubiquitin ligase, b eing required for yeast growth and capable of promoting cell growth during serum starvation. Here, we report the genomic structure of the SAG gene tha t consists of four exons and three introns. We also report the characteriza tion of a SAG splicing variant (SAG-v), that contains an additional exon (e xon 2; 264 bp) not present in wildtype SAG. The inclusion of exon 2 disrupt s the SAG ORF and gives rise to a protein of 108 amino acids that contains the first 59 amino acids identical to SAG and a 49-amino acid novel sequenc e at the C terminus. The entire RING-finger domain of SAG was not translate d because of several inframe stop codons within the exon 2. The SAG-v prote in was expressed in multiple human tissues as well as cell lines, but at a much lower level than wildtype SAG. Unlike SAG, SAG-v was not able to rescu e yeast cells from lethality in a ySAG knockout, nor did it bind to cullin- 1 or have ligase activity, probably because of the lack of the RING-finger domain. Finally, we report the identification of two SAG family pseudogenes , SAGP1 and SAGP2, that share 36% or 47% sequence identity with ROC1/Rbx1/H rt1 and 30% or 88% with SAG, respectively. Both genes are intronless with t wo inframe stop codons.