Novel splicing variant of mouse Orc1 is deficient in nuclear translocation and resistant for proteasome-mediated degradation.
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 beta-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.
Miyake
Yasuyuki
Y
Cellular Physiology Laboratory, RIKEN Discovery Research Institute, Wako, Saitama 351-0198, Japan.
Mizuno
Takeshi
T
Yanagi
Ken-ichiro
K
Hanaoka
Fumio
F
D021581:Active Transport, Cell Nucleus; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D019556:COS Cells; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005091:Exons; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D041681:NIH 3T3 Cells; D051738:Origin Recognition Complex; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014018:Tissue Distribution; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C497228:Orc1 protein, mouse; C497229:Orc3 protein, mouse; D051738:Origin Recognition Complex; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D001616:beta-Galactosidase; D046988:Proteasome Endopeptidase Complex