The circadian deadenylase Nocturnin interacts with a novel form of the RNA-binding protein KSRP

GARBARINO PICO E, NIU S AND GREEN CB

Tucumán, Argentina

Congreso; XLV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

SAIB

CB-P32.THE CIRCADIAN DEADENYLASE NOCTURNININTERACTS WITH A NOVEL FORM OF THE RNABINDINGPROTEIN KSRPGarbarino Pico E1; Niu S2; Green CB21CIQUIBIC-Dpto. Química Biológica, Facultad CienciasQuímicas, UNCórdoba. 2University of Virginia. E-mail:garba@mail.fcq.unc.edu.arMouse nocturnin (mNOC) is a circadian regulated deadenylase.mRNA deadenylation induces transcript degradation ortranslational silencing. In order to identify mNOC binding partners,we performed a large-scale immunoprecipitation (IP) screen. Themass spectroscopy analysis of co-IP proteins revealed KSRP (KHhomology splicing regulatory protein) as a potential mNOC bindingprotein. KSRP is an AU-rich element (ARE) containing mRNAbinding protein that regulates mRNA turnover. We verified thisinteraction by co-IP. We utilized three anti-KSRP antibodies, two ofthem detected two KSRP forms with apparent molecular weigh of75 and 60 kDa, whereas the other one (monoclonal), only the 75 kDaform. mNOC co-IP and co-fractionate in gel filtration with the 60kDa form. Since KSRP is also involved in nuclear splicing, westudied the mNOC and KSRP60 subcellular localization todetermine where this interaction takes place. ICC and biochemicalfractionation showed that both proteins co-localize in cytoplasm,whereas KSRP75 is in the nucleus. We confirmed the identity ofKSRP60 by repeating the mass spectroscopy analysis.Our results suggest that NOC may play a role in regulating ARE-mRNAmetabolism. Our current working hypothesis is that mNOCis recruited to ARE-mRNAs by KSRP60, which results in thedeadenylation of these messages, and consequently, theirdegradation or translational silencing.