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J. Biol. Chem., Vol. 280, Issue 13, 12643-12652, April 1, 2005

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Novel Splicing Variant of Mouse Orc1 Is Deficient in Nuclear Translocation and Resistant for Proteasome-mediated Degradation^*

Yasuyuki Miyake¶, Takeshi Mizuno||, Ken-ichiro Yanagi||, and Fumio Hanaoka||**

From the Cellular Physiology Laboratory, RIKEN Discovery Research Institute and ^||CREST, Japan Science and Technology Corporation, Wako, Saitama 351-0198, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan

DNA replication is controlled by the stepwise assembly of the pre-replicative complex and the replication apparatus. Loading of the origin recognition complex (ORC) onto the chromatin is a prerequisite for the assembly of the pre-replicative complex. To define the physiological functions of the mammalian ORC, we cloned ORC subunit cDNAs from mouse NIH3T3 cells and found novel variant forms of Orc1, Orc2, and Orc3 each derived from alternative RNA splicing. The variant form of Orc1, Orc1B, lacks 35 amino acid residues in exon 5; the variant of Orc2, Orc2B, lacks 48 amino acid residues in exon 2. In the Orc3 variant, Orc3B, only 1 amino acid residue is deleted in exon 15. Reverse transcription-PCR analysis showed that the full-length Orc1–3 subunits, Orc1A, Orc2A, and Orc3A, as well as Orc2B and Orc3B, were widely expressed in various mouse cell lines and mouse tissues. In contrast, Orc1B was only expressed in the thymus and at an early embryonic stage. Overexpression of these Orc subunits in cultured cells revealed that Orc1A, Orc2A, Orc3A, Orc2B, and Orc3B are localized in the nucleus, whereas Orc1B remains exclusively in the cytoplasm. Moreover, fusion of the 35 amino acids spliced fragment from mOrc1A with -galactosidase resulted in its translocation into the nucleus. When Orc1B is expressed transiently, its degradation occurs in a proteasome-independent manner, whereas Orc1A is rapidly degraded by the ubiquitin-proteasome pathway. Taken together, we conclude that mouse Orc1, Orc2, and Orc3 each exist in two alternative-splicing variants and that naturally occurring Orc1B lacks a functional domain that is essential for nuclear translocation and proteasome-dependent degradation.

Received for publication, November 24, 2004 , and in revised form, January 3, 2005.

^* This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and grants from the Bioarchitect Research Project and the Chemical Biology Project of RIKEN. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB190255, AB190256, and AB190257.

^¶ Supported by the fellowship of Center of Excellence (COE) from the Japan Society for the Promotion of Science.

^** To whom correspondence should be addressed: Tel.: 81-6-6879-7975; Fax: 81-6-6877-9382; E-mail: fhanaoka{at}fbs.osaka-u.ac.jp.

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