Does mPER2 protein oscillate without its coding mRNA cycling?: post-transcriptional regulation by cell clock.
Does the mammalian oscillatory protein mPER2 show the rhythm without its coding mRNA cycling? Here we answer this question by inserting a single copy of exogenous mPer2 gene to a NIH3T3 fibroblasts cell line, using Flp-In system. We generated the stable cell lines which constantly express mRNAs coding either N-terminal FLAG-tagged full length mPER2 (FLAG-mPER2(full)) or its C-terminal deleted form (FLAG-mPER2(1-1068)), which lacks the binding site to mCRY proteins, under the control of human EF-1alpha promoter. Although serum shock induced the rhythm of endogenous clock machinery in these cell lines, it did not initiate the rhythm of exogenously inserted FLAG-mPer2 genes at the mRNA level. In contrast, FLAG-mPER2(full) proteins showed the rhythm without their coding mRNA cycling. Since cells expressing FLAG-mPER2(1-1068) also showed the rhythm of FLAG-mPER2(1-1068) proteins, the direct binding of mCRY and mPER2 seems not necessary for this protein oscillation. This system clearly demonstrates that the intracellular endogenous clock system has an ability to modify the mPer2 gene post-transcriptionally to make mPER2 proteins oscillate without its coding mRNA cycling.
Fujimoto
Yoshito
Y
Division of Molecular Brain Science, Department of Brain Sciences, Kobe University Graduate School of Medicine, Chuo-ku, Kobe 650-0017, Japan.
Yagita
Kazuhiro
K
Okamura
Hitoshi
H
D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D002478:Cells, Cultured; D002940:Circadian Rhythm / Q000235:genetics; D006801:Humans; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D056950:Period Circadian Proteins; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C421902:PER2 protein, human; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; D012333:RNA, Messenger; D014157:Transcription Factors