The circadian deadenylase nocturnin interacts with KSRP and localizes in the cytoplasm outside of P-bodies and stress granules

GARBARINO PICO E; NIU S; GREEN CB

Bariloche, Argentina

Congreso; "Gene Expression and RNA Processing" y "Gene Expression and RNA Processing"; 2007

International Centre for Genetic Engineering and Biotechnology (ICGEB) - Fac. de Ciencias Exactas y Naturales, UBA - IFIBYNE-CONICET - ANPCyT

Nocturnin (Noc), a rhythmically expressed deadenylase, is thought to play an important role in circadian clock output pathways by post-transcriptionally regulating clock-related genes. Mouse NOC exists in multi-protein complexes and in order to identify its 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 containing mRNA (ARE-mRNA) binding protein that regulates mRNA turnover. We verified the interaction by co-IP and showed in gel filtration analysis that endogenous KSRP and NOC-FLAG co-fractionate in complexes between 100 and 200 kDa. Since KSRP is involved also in nuclear splicing, we studied the mNOC subcellular localization to determine where this interaction takes place. ICC experiments showed that mNOC is a cytoplasmic protein. As expected, co-immunostaining of both proteins showed overlap only in cytoplasm of HEK293 cells. Together, these results prove that KSRP is a NOC binding partner. Considering that many of the factors involved in mRNA decay and translational silencing localize in stress granules (SGs) and processing bodies (PBs), we analyzed whether NOC co-localizes in these cytoplasmic subdomains. NOC was not detected in SGs or PBs under any of the conditions tested.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 KSRP, which results in the deadenylation of these messages, and consequently, their degradation or translational silencing. This process appears to take place outside of SGs or PBs, sites proposed for silent-mRNA triage and storage.