A Truncated Form of the p27 Cyclin-Dependent Kinase Inhibitor Translated from Pre-mRNA Causes G₂-Phase Arrest

ABSTRACT

Pre-mRNA splicing is an indispensable mechanism for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest at the G₁ and G₂/M phases, and this is thought to be one of the reasons for the potent antitumor activity of splicing inhibitors. However, the molecular mechanisms underlying the cell cycle arrest have many unknown aspects. In particular, the mechanism of G₂/M-phase arrest caused by splicing inhibition is completely unknown. Here, we found that lower and higher concentrations of pladienolide B caused M-phase and G₂-phase arrest, respectively. We analyzed protein levels of cell cycle regulators and found that a truncated form of the p27 cyclin-dependent kinase inhibitor, named p27*, accumulated in G₂-arrested cells. Overexpression of p27* caused partial G₂-phase arrest. Conversely, knockdown of p27* accelerated exit from G₂/M phase after washout of splicing inhibitor. These results suggest that p27* contributes to G₂/M-phase arrest caused by splicing inhibition. We also found that p27* bound to and inhibited M-phase cyclins, although it is well known that p27 regulates the G₁/S transition. Intriguingly, p27*, but not full-length p27, was resistant to proteasomal degradation and remained in G₂/M phase. These results suggest that p27*, which is a very stable truncated protein in G₂/M phase, contributes to G₂-phase arrest caused by splicing inhibition.

KEYWORDS:

• G₂/M
• pladienolide B
• mRNA splicing
• cell cycle
• p27

Declaration of Interests

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

We are grateful to Kei Kikuchi, Midori Shima, Shiori Nakada, and Kanjiro Nakagawa for technical assistance. We thank Yukiko Yoshida (Tokyo Metropolitan Institute of Medical Science) for HA-Skp2 plasmid. Additionally, we thank Keiichi Nakayama (Kyushu University) for Flag-KPC1 and HA-KPC2 plasmids. This research was funded by JSPS KAKENHI (JP21H02423 to D.K.), Takeda Science Foundation (to D.K.), Tamura Science & Technology Foundation (to D.K.), the Kato Memorial Bioscience Foundation (to D.K.), The Ichiro Kanehara Foundation (to D.K.), and Suzuken Memorial Foundation (to D.K.). Finally, we thank Edanz for editing a draft of the manuscript.