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Updated: Jan 29, 2026
Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols
Published on: June 6, 2017
Karyopherin Msn5 is involved in a novel mechanism controlling the cellular level of cell cycle regulators Cln2 and
Inma Quilis1,2, Francisco J Taberner1,2, Carlos A Martínez-Garay1,2
1a Departament de Bioquímica i Biologia Molecular , Universitat de València , Valencia , Spain.
Abstract:
The yeast β-karyopherin Msn5 controls the SBF cell-cycle transcription factor, responsible for the periodic expression of CLN2 cyclin gene at G1/S, and the nuclear export of Cln2 protein. Here we show that Msn5 regulates Cln2 by an additional mechanism. Inactivation of Msn5 causes a severe reduction in the cellular content of Cln2. This occurs by a post-transcriptional mechanism, since CLN2 mRNA level is not importantly affected in asynchronous cultures. Cln2 stability is not significantly altered in msn5 cells and inactivation of Msn5 causes a reduction in protein level even when Cln2 is stabilized. Therefore, the reduced amount of Cln2 in msn5 cells is mainly due not to a higher rate of protein degradation but to a defect in Cln2 synthesis. In fact, analysis of polysome profiles indicated that Msn5 inactivation causes a shift of CLN2 and SWI5 mRNAs from heavy-polysomal to light-polysomal and non-polysomal fractions, supporting a defect in Cln2 and Swi5 protein synthesis in the msn5 mutant. The analysis of truncated versions of Cln2 and of chimeric cyclins combining distinct domains from Cln2 and the related Cln1 cyclin identified an internal region in Cln2 from 181 to 225 residues that when fused to GFP is able to confer Msn5-dependent regulation of protein cellular content. Finally, we showed that a high level of Cln2 is toxic in the absence of Msn5. In summary, we described that Msn5 is required for the proper protein synthesis of specific proteins, introducing a new level of control of cell cycle regulators.
Insights
The yeast protein Msn5 is crucial for Cln2 protein synthesis, not just its export. Msn5 inactivation reduces Cln2 levels by impairing its synthesis, impacting cell cycle regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Yeast Genetics
Background:
- Msn5 is a yeast β-karyopherin known to regulate the SBF transcription factor and the nuclear export of Cln2 protein.
- The SBF transcription factor controls the periodic expression of the CLN2 cyclin gene, essential for cell cycle progression at G1/S.
Purpose of the Study:
- To investigate the regulatory role of Msn5 on Cln2 protein levels beyond its known function in nuclear export.
- To elucidate the post-transcriptional mechanisms by which Msn5 influences Cln2 cellular content.
Main Methods:
- Analysis of CLN2 mRNA and Cln2 protein levels in Msn5-inactivated yeast cells.
- Assessment of Cln2 protein stability and synthesis rates using polysome profiling.
- Identification of regulatory domains within Cln2 using truncated and chimeric cyclin constructs with GFP.
Main Results:
- Msn5 inactivation significantly reduces cellular Cln2 content via a post-transcriptional mechanism, without affecting CLN2 mRNA levels or protein stability.
- Polysome profile analysis revealed impaired synthesis of Cln2 and Swi5 proteins in msn5 mutants.
- A specific internal region of Cln2 (residues 181-225) was identified as conferring Msn5-dependent regulation.
- High Cln2 levels were found to be toxic in the absence of Msn5.
Conclusions:
- Msn5 plays a novel role in regulating the protein synthesis of specific targets, including cell cycle regulators like Cln2.
- This discovery introduces a new layer of post-transcriptional control over cell cycle progression mediated by Msn5.
- Msn5 is essential for efficient Cln2 and Swi5 protein synthesis, impacting yeast cell cycle dynamics.
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