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A non-conventional nuclear import pathway Sandra Korge1, Bert Maier1, Achim Kramer1 1Charité-Universitätsmedizin Berlin, Berlin, GERMANY Generating a 24 hour rhythm of the molecular circadian clock is influenced by transcriptional and translational regulation as well as post-translational processes as nucleocytoplasmic protein shuttling. As it is known for Period (PER), Cryptochrome (CRY) and other clock proteins to carry classical nuclear localization signals (NLS) little is known about Tnpo1 interacting domains. Using RNAi, we could show period lengthening upon knockdown of importin beta (Imp ? ), which recognizes classical NLS. On the opposite knockdown of transportin1 (Tnpo1), the M9 interacting carrier, results in period shortening. Therefore we investigated potential target clock proteins bioinformatically for M9 characteristic amino acid sequences such as the PY motif. We found potential M9-like sequences in BMAL1, PERs and CRYs as well as in Casein kinases and RevErb?. To study the influence of TNPO1 on clock protein localization, we performed a nuclear import assay, where putative M9-sequences of clock proteins are fused to a cyan fluorescent protein (CFP). The M9-CFP fusion protein will change its localization from cytoplasmic to nuclear, if the putative M9 sequence is sufficient to interact with a nuclear import carrier, potentially TNPO1. To clarify if the M9-CFP fusion protein is actively translocated by TNPO1, nuclear import was monitored upon Tnpo1 knockdown. We identified putative M9 sequences in CRY1, CRY2 as well as PER2 and were able to show their specific TNPO1 translocation. Furthermore fluorescence recovery after photobleach (FRAP) will give the opportunity to study the kinetics of the M9-CFP nuclear import in the presence or absence of TNPO1.