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Post-transcriptional homeostasis and regulation of MCM2-7 in mammalian cells.

Chuang CH, Yang D, Bai G, Freeland A, Pruitt SC, Schimenti JC - Nucleic Acids Res. (2012)

Bottom Line: Remarkably, depletion or mutation of one Mcm can decrease all Mcm levels.First, the Mcm4(Chaos3) allele, which disrupts MCM4:MCM6 interaction, triggers a Dicer1 and Drosha-dependent ≈ 40% reduction in Mcm2-7 mRNAs.The decreases in Mcm mRNAs coincide with up-regulation of the miR-34 family of microRNAs, which is known to be Trp53-regulated and target Mcms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Center for Vertebrate Genomics, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, USA.

ABSTRACT
The MiniChromosome Maintenance 2-7 (MCM2-7) complex provides essential replicative helicase function. Insufficient MCMs impair the cell cycle and cause genomic instability (GIN), leading to cancer and developmental defects in mice. Remarkably, depletion or mutation of one Mcm can decrease all Mcm levels. Here, we use mice and cells bearing a GIN-causing hypomophic allele of Mcm4 (Chaos3), in conjunction with disruption alleles of other Mcms, to reveal two new mechanisms that regulate MCM protein levels and pre-RC formation. First, the Mcm4(Chaos3) allele, which disrupts MCM4:MCM6 interaction, triggers a Dicer1 and Drosha-dependent ≈ 40% reduction in Mcm2-7 mRNAs. The decreases in Mcm mRNAs coincide with up-regulation of the miR-34 family of microRNAs, which is known to be Trp53-regulated and target Mcms. Second, MCM3 acts as a negative regulator of the MCM2-7 helicase in vivo by complexing with MCM5 in a manner dependent upon a nuclear-export signal-like domain, blocking the recruitment of MCMs onto chromatin. Therefore, the stoichiometry of MCM components and their localization is controlled post-transcriptionally at both the mRNA and protein levels. Alterations to these pathways cause significant defects in cell growth reflected by disease phenotypes in mice.

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Defective cell cycle in MCM-deficient cells and impact of WT or mutant MCM3 levels. (A) Flow cytometric analysis of cell cycle in unsynchronized MEFs. The values are plotted as a percentage of the measured average of Mcm4Chaos3/Chaos3 MEFs for each cell cycle stage (Y-axis), which is set to 100%. The MEFs were established from littermates. ‘C3’ = Mcm4Chaos3; ‘M#’ = Mcm#; ‘GT’ is shorthand for the mutant allele of that Mcm. Error bars represent SEM, derived from at least 14 independent experiments (‘N’ numbers indicated). ‘*’ = statistically significant to P < 0.05). (B) Cell cycle histograms of McmC3/C3 Mcm2+/− Mcm3+/− MEFs infected with lentiviruses expressing the either LacZ alone (‘CON’) or the indicated Mcm. Only infected cells were scored, based on expression of epitope tags. (C) Quantification of G2/M population data (gates are shown in ‘B’). The percentages of G2/M population were normalized against LacZ controls (set to 100%). Error bars represent SEM from three independent experiments.
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gks176-F5: Defective cell cycle in MCM-deficient cells and impact of WT or mutant MCM3 levels. (A) Flow cytometric analysis of cell cycle in unsynchronized MEFs. The values are plotted as a percentage of the measured average of Mcm4Chaos3/Chaos3 MEFs for each cell cycle stage (Y-axis), which is set to 100%. The MEFs were established from littermates. ‘C3’ = Mcm4Chaos3; ‘M#’ = Mcm#; ‘GT’ is shorthand for the mutant allele of that Mcm. Error bars represent SEM, derived from at least 14 independent experiments (‘N’ numbers indicated). ‘*’ = statistically significant to P < 0.05). (B) Cell cycle histograms of McmC3/C3 Mcm2+/− Mcm3+/− MEFs infected with lentiviruses expressing the either LacZ alone (‘CON’) or the indicated Mcm. Only infected cells were scored, based on expression of epitope tags. (C) Quantification of G2/M population data (gates are shown in ‘B’). The percentages of G2/M population were normalized against LacZ controls (set to 100%). Error bars represent SEM from three independent experiments.

Mentions: Consistent with this hypothesis, ectopically expressed mouse MCM3 co-IP’d XPO1 in HEK cells (Figure 4A), whereas a mutant version in which three leucines and one isoleucine within the predicted NES were changed to alanines (‘L4A’; Figure 4B) abolished MCM3:XPO1 interaction. Over-expression of MCM3 but not MCM3L4A in stably transfected (via lentivirus) HeLa cells, which are known to express very high levels of Mcms (40), caused a decrease of chromatin-bound MCM2, 4, 5, 6 and 7 (Figure 4C; thereby increasing the soluble/chromatin MCM ratio as plotted in Figure 4D) that was not due to an increase of cells arrested in G2/M (Supplementary Figure S1b). Flow cytometric analysis of MCM2 levels in nuclei, which were decreased in G1 cells, were consistent with the Western blot analyses (Supplementary Figure S1a). These changes in MCM2–7 localization had functional correlates with cell growth; over-expression of MCM3 markedly decreased colony formation compared to MCM3L4A (Figure 4C). Since CDK phosphorylation of scMcm3 regulates its nuclear transport ability (37), we also tested whether a presumed phosphorylation-dead version of MCM3 (MCM3S5A), in which five predicted MCM3 CDK phosphorylation sites were mutagenized (Figure 4B), lacked the ability to increase MCM2–7 loading and improve cell growth. This was not the case; MCM3S5A transfection had effects similar to the WT construct (Figures 4E, 5A and B). Furthermore, dephosphorylation of protein extracts did not disrupt MCM3:XPO1 interaction (Figure 4A), suggesting that the anti-licensing effects of MCM3 may occur via a mechanism not involving, or in addition to, XPO1 interaction. We posit that the anti-licensing activity of MCM3 might involve interaction of another protein with the leucine-rich domain.Figure 5.


Post-transcriptional homeostasis and regulation of MCM2-7 in mammalian cells.

Chuang CH, Yang D, Bai G, Freeland A, Pruitt SC, Schimenti JC - Nucleic Acids Res. (2012)

Defective cell cycle in MCM-deficient cells and impact of WT or mutant MCM3 levels. (A) Flow cytometric analysis of cell cycle in unsynchronized MEFs. The values are plotted as a percentage of the measured average of Mcm4Chaos3/Chaos3 MEFs for each cell cycle stage (Y-axis), which is set to 100%. The MEFs were established from littermates. ‘C3’ = Mcm4Chaos3; ‘M#’ = Mcm#; ‘GT’ is shorthand for the mutant allele of that Mcm. Error bars represent SEM, derived from at least 14 independent experiments (‘N’ numbers indicated). ‘*’ = statistically significant to P < 0.05). (B) Cell cycle histograms of McmC3/C3 Mcm2+/− Mcm3+/− MEFs infected with lentiviruses expressing the either LacZ alone (‘CON’) or the indicated Mcm. Only infected cells were scored, based on expression of epitope tags. (C) Quantification of G2/M population data (gates are shown in ‘B’). The percentages of G2/M population were normalized against LacZ controls (set to 100%). Error bars represent SEM from three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367205&req=5

gks176-F5: Defective cell cycle in MCM-deficient cells and impact of WT or mutant MCM3 levels. (A) Flow cytometric analysis of cell cycle in unsynchronized MEFs. The values are plotted as a percentage of the measured average of Mcm4Chaos3/Chaos3 MEFs for each cell cycle stage (Y-axis), which is set to 100%. The MEFs were established from littermates. ‘C3’ = Mcm4Chaos3; ‘M#’ = Mcm#; ‘GT’ is shorthand for the mutant allele of that Mcm. Error bars represent SEM, derived from at least 14 independent experiments (‘N’ numbers indicated). ‘*’ = statistically significant to P < 0.05). (B) Cell cycle histograms of McmC3/C3 Mcm2+/− Mcm3+/− MEFs infected with lentiviruses expressing the either LacZ alone (‘CON’) or the indicated Mcm. Only infected cells were scored, based on expression of epitope tags. (C) Quantification of G2/M population data (gates are shown in ‘B’). The percentages of G2/M population were normalized against LacZ controls (set to 100%). Error bars represent SEM from three independent experiments.
Mentions: Consistent with this hypothesis, ectopically expressed mouse MCM3 co-IP’d XPO1 in HEK cells (Figure 4A), whereas a mutant version in which three leucines and one isoleucine within the predicted NES were changed to alanines (‘L4A’; Figure 4B) abolished MCM3:XPO1 interaction. Over-expression of MCM3 but not MCM3L4A in stably transfected (via lentivirus) HeLa cells, which are known to express very high levels of Mcms (40), caused a decrease of chromatin-bound MCM2, 4, 5, 6 and 7 (Figure 4C; thereby increasing the soluble/chromatin MCM ratio as plotted in Figure 4D) that was not due to an increase of cells arrested in G2/M (Supplementary Figure S1b). Flow cytometric analysis of MCM2 levels in nuclei, which were decreased in G1 cells, were consistent with the Western blot analyses (Supplementary Figure S1a). These changes in MCM2–7 localization had functional correlates with cell growth; over-expression of MCM3 markedly decreased colony formation compared to MCM3L4A (Figure 4C). Since CDK phosphorylation of scMcm3 regulates its nuclear transport ability (37), we also tested whether a presumed phosphorylation-dead version of MCM3 (MCM3S5A), in which five predicted MCM3 CDK phosphorylation sites were mutagenized (Figure 4B), lacked the ability to increase MCM2–7 loading and improve cell growth. This was not the case; MCM3S5A transfection had effects similar to the WT construct (Figures 4E, 5A and B). Furthermore, dephosphorylation of protein extracts did not disrupt MCM3:XPO1 interaction (Figure 4A), suggesting that the anti-licensing effects of MCM3 may occur via a mechanism not involving, or in addition to, XPO1 interaction. We posit that the anti-licensing activity of MCM3 might involve interaction of another protein with the leucine-rich domain.Figure 5.

Bottom Line: Remarkably, depletion or mutation of one Mcm can decrease all Mcm levels.First, the Mcm4(Chaos3) allele, which disrupts MCM4:MCM6 interaction, triggers a Dicer1 and Drosha-dependent ≈ 40% reduction in Mcm2-7 mRNAs.The decreases in Mcm mRNAs coincide with up-regulation of the miR-34 family of microRNAs, which is known to be Trp53-regulated and target Mcms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Center for Vertebrate Genomics, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, USA.

ABSTRACT
The MiniChromosome Maintenance 2-7 (MCM2-7) complex provides essential replicative helicase function. Insufficient MCMs impair the cell cycle and cause genomic instability (GIN), leading to cancer and developmental defects in mice. Remarkably, depletion or mutation of one Mcm can decrease all Mcm levels. Here, we use mice and cells bearing a GIN-causing hypomophic allele of Mcm4 (Chaos3), in conjunction with disruption alleles of other Mcms, to reveal two new mechanisms that regulate MCM protein levels and pre-RC formation. First, the Mcm4(Chaos3) allele, which disrupts MCM4:MCM6 interaction, triggers a Dicer1 and Drosha-dependent ≈ 40% reduction in Mcm2-7 mRNAs. The decreases in Mcm mRNAs coincide with up-regulation of the miR-34 family of microRNAs, which is known to be Trp53-regulated and target Mcms. Second, MCM3 acts as a negative regulator of the MCM2-7 helicase in vivo by complexing with MCM5 in a manner dependent upon a nuclear-export signal-like domain, blocking the recruitment of MCMs onto chromatin. Therefore, the stoichiometry of MCM components and their localization is controlled post-transcriptionally at both the mRNA and protein levels. Alterations to these pathways cause significant defects in cell growth reflected by disease phenotypes in mice.

Show MeSH
Related in: MedlinePlus