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 antioxidant protein that protects cells from apoptosis induced by redox agents. The 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, being required for yeast growth and capable of promoting cell growth during serum starvation. Here, we report the genomic structure of the SAG gene that consists of four exons and three introns. We also report the characterization of a SAG splicing variant (SAG-v), that contains an additional exon (exon 2; 264 bp) not present in wildtype SAG. The inclusion of exon 2 disrupts 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 sequence at the C terminus. The entire RING-finger domain of SAG was not translated because of several inframe stop codons within the exon 2. The SAG-v protein 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 rescue 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/Hrt1 and 30% or 88% with SAG, respectively. Both genes are intronless with two inframe stop codons.
Swaroop
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Cancer Molecular Sciences, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105, USA.
Gosink
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Sun
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Y
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis; D001483:Base Sequence; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D018076:DNA, Complementary; D016166:Free Radical Scavengers / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000378:metabolism*; D010641:Phenotype; D011544:Pseudogenes; D016601:RNA-Binding Proteins; D044843:SKP Cullin F-Box Protein Ligases; D014018:Tissue Distribution; D044767:Ubiquitin-Protein Ligases; D016335:Zinc Fingers
D000975:Antioxidants; D018076:DNA, Complementary; D016166:Free Radical Scavengers; D016601:RNA-Binding Proteins; C118307:RNF7 protein, human; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D010453:Peptide Synthases