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

GARBARINO PICO E; NIU S; GREEN CB

Tucumán

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

SAIB

Mouse nocturnin (mNOC) is a circadian regulated deadenylase. mRNA deadenylation induces transcript degradation or translational silencing. In order to identify mNOC binding partners, we performed a large-scale immunoprecipitation (IP) screen. The mass spectroscopy analysis of co-IP proteins revealed KSRP (KH-homology splicing regulatory protein) as a potential mNOC binding protein. KSRP is an AU-rich element (ARE) containing mRNA-binding protein that regulates mRNA turnover. We verified this interaction by co-IP. We utilized three anti-KSRP antibodies, two of them detected two KSRP forms with apparent molecular weigh of 75 and 60 kDa, whereas the other one (monoclonal), only the 75 kDa form. mNOC co-IP and co-fractionate in gel filtration with the 60 kDa form. Since KSRP is also involved in nuclear splicing, we studied the mNOC and KSRP60 subcellular localization to determine where this interaction takes place. ICC and biochemical fractionation showed that both proteins co-localize in cytoplasm, whereas KSRP75 is in the nucleus. We confirmed the identity of KSRP60 by repeating the mass spectroscopy analysis. Our results suggest that NOC may play a role in regulating ARE-mRNA metabolism. Our current working hypothesis is that mNOC is recruited to ARE-mRNAs by KSRP60, which results in the deadenylation of these messages, and consequently, their degradation or translational silencing.