Exploring the potential interaction between seven in absentia homolog-1a and retinoid receptors

Seven in Absentia Homolog 1a (Siah-la) is a protein that has been shown to be essential for spermatogenesis in mice. Siah-la functions as an E3 ubiquitin ligase, a part of the ubiquitinproteasome pathway. Siah-1a mRNA levels have been shown to increase in the presence of the ligands for Retinoic Acid Receptor Alpha (RARA), indicating that RARA may be partially responsible for Siah-la regulation and expression. Since RARA is known to be degraded via the ubiquitin-proteasome pathway and Siah-la is an E3 ubiquitin ligase, the potential interaction between Siah-la and the proteins in the RARA transcriptional complex was investigated. In the models initially proposed for this study, Siah-1a could interact directly with the RARA or its heterodimer receptor partner, Retinoid X Receptor Alpha (RXRA), or indirectly with the coregulator molecules such as Nuclear Corepressor (NCoR) or Coactivator (CoA), bridging between Siah-la and the RARAJRXRA heterodimer. Interestingly, NCoR has recently been found to contain a putative Siah-l binding site. We initially focused on the role of RARA or RXRA as the possible binding proteins for Siah-l a by performing immunoprecipitation experiments. Protein samples were collected from mouse Sertoli cell line (MSC-I cells) and immunoprecipitated using anti-Siah-l antibody. The immunoprecipitates were then separated on a Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel and immunoblotted with either anti-RARA or anti-RXRA antibodies. In reverse, protein samples from MSC-I cells were immunoprecipitated with anti-RARA or anti-RXRA antibodies and immunoblotted with anti-Siah-l antibody. Our results showed that anti-Siah-l antibody can immunoprecipitate detectable amounts of RARA. In addition, more RARA was immunoprecipitated in the absence of AM580 compared to the amount immunoprecitated in the presence of AM580. However, in reverse, both anti-RARA and anti-RXRA antibodies failed to immunoprecipitate an appreciable amount of Siah-I. These results suggest that the interaction between Siah-l and RARA or RXRA is either weak or is mediated by additional proteins such as NCoR or CoA. From our results, we propose that RARA, Siah-la, and NCoR are associated together in the absence ofretinoic acid. In the presence ofretinoic acid, NCoR and Siah-l come off RARA, with Siah-l shepherding NCoR for ubiquitination and proteasome degradation. After transcription by the activated RARA/RXRA dimer, the active transcriptional complex is also a target of Siah-l, for ubiquitination and proteasome degradation. The experiments to determine whether NCoR or CoA bridges between Siah-l and the RARA/RXRA complex are in progress.Honors College, Washington State UniversityLogsdon, Marcie Exploring the potential interaction between seven in absentia homolog-1a and retinoid receptors, Washington State University Honors College thesis, Spring 2006, 26 p