Abstract
BackgroundHuman cyclin A2 is a key regulator of S phase progression and entry into mitosis. Alternative splice variants of the G1 and mitotic cyclins have been shown to interfere with full-length cyclin functions to modulate cell cycle progression and are therefore likely to play a role in differentiation or oncogenesis. The alternative splicing of human cyclin A2 has not yet been studied.Methodology/Principal FindingsSequence-specific primers were designed to amplify various exon–intron regions of cyclin A2 mRNA in cell lines and human tissues. Intron retaining PCR products were cloned and sequenced and then overexpressed in HeLa cells. The subcellular localization of the splice variants was studied using confocal and time-lapse microscopy, and their impact on the cell cycle by flow cytometry, immunoblotting and histone H1 kinase activity. We found a splice variant of cyclin A2 mRNA called A2V6 that partly retains Intron 6. The gene expression pattern of A2V6 mRNA in human tissues was noticeably different from that of wild-type cyclin A2 (A2WT) mRNA. It was lower in proliferating fetal tissues and stronger in some differentiated adult tissues, especially, heart. In transfected HeLa cells, A2V6 localized exclusively in the cytoplasm whereas A2WT accumulated in the nucleus. We show that A2V6 induced a clear G1/S cell cycle arrest associated with a p21 and p27 upregulation and an inhibition of retinoblastoma protein phosphorylation. Like A2WT, A2V6 bound CDK2, but the A2V6/CDK2 complex did not phosphorylate histone H1.Conclusion/SignificanceThis study has revealed that some highly differentiated human tissues express an intron-retaining cyclin A2 mRNA that induced a G1/S block in vitro. Contrary to full-length cyclin A2, which regulates cell proliferation, the A2V6 splice variant might play a role in regulating nondividing cell states such as terminal differentiation or senescence.
Highlights
Cyclins play an essential role in progression through the eukaryotic cell cycle, acting as regulatory subunits of cyclindependent kinases (CDKs)
It is interesting to note that the expression of A2V6 mRNA was noticeably lower than that of A2WT mRNA in the proliferating fetal tissues
It has been demonstrated that an endoplasmic reticulum (ER)-associated cyclin A2 mutant triggers CDK activation [12], over-duplicates centrosome, increases ploidy, and transforms cells [13]
Summary
Cyclins play an essential role in progression through the eukaryotic cell cycle, acting as regulatory subunits of cyclindependent kinases (CDKs). Types A and B cyclins are responsible for the onset of mitosis, and through their degradation, the exit from mitosis. Their activities are determined by changes in their subcellular localization during successive phases of the cell cycle [1]. Cyclin A2 achieves its regulatory activity predominantly, if not exclusively, in the nucleus from the G1/S transition to mitosis, participating in the entry into, and progress through, S phase, DNA replication, centrosome duplication, and the entry into mitosis [2,3,4]. The alternative splicing of human cyclin A2 has not yet been studied
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