Identification and characterization of two splicing variants of human Noxa.
Noxa is a pro-apoptotic Bcl-2 homology 3 (BH3)-only containing protein. Here we report the identification of two splicing variants of the human Noxa gene, which consists of three exons and two introns. Alternative splicing of exon 2 yields three transcripts. Transcript-1 joins exons 1 and 3 to encode Noxa of 54 amino acids. Transcript-2, consisting of exon-1, partially spliced exon-2, and exon-3, encodes NSV-1 (Noxa splicing variant-1) of 136 amino acids, whereas transcript-3, containing all three exons, encodes NSV-2 of 70 amino acids, which is 100% identical to the first 70 amino acids of NSV-1. All three transcripts, controlled by the same promoter with two p53 consensus-binding sites, were inducible by p53. Although NSV-1 and NSV-2 mRNA were readily detectable by RT-PCR in multiple human cancer cell lines, no endogenous variant proteins were detected under physiological or stressed conditions. Even under forced expression, both variants were barely detectable without proteasome inhibitor, MG132. The protein half-life of the two variants is approximately 40 to 60 mins with MG132, suggesting they are rapidly degraded via proteasome-dependent and independent pathways. Unlike Noxa, BH3-less variants failed to potentiate apoptosis induced by etoposide. Thus, Noxa variants are unlikely to play a role in apoptosis regulation.
Wang
Zhen
Z
Division of Cancer Biology, Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA.
Sun
Yi
Y
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D045744:Cell Line, Tumor; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000970:Antineoplastic Agents; D007976:Leupeptins; C498702:PMAIP1 protein, human; D020033:Protein Isoforms; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde