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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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Depletion of Mcm2–7 mRNAs in Mcm4C3/C3 cells is Dicer1 and Drosha-regulated, and associated with up-regulation of micro RNAs miR-34a-c. (A) The graphs showed qRT-PCR analysis of the indicated genes in Mcm4C3/C3 or WT MEFs that were treated with siRNA against Dicer1 (top) and Drosha (bottom) for 120 h. N = 3 replicates; SEM bars are shown. Data were standardized against β-actin. (B) qRT-PCR analysis of various miRNAs. The values are plotted as percentages of the levels of the indicated miRNAs in Mcm4C3/C3 (Chaos3) MEFs versus WT MEFs. The data represent analysis of 5 littermate pairs of MEFs. SEM bars are shown.
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gks176-F2: Depletion of Mcm2–7 mRNAs in Mcm4C3/C3 cells is Dicer1 and Drosha-regulated, and associated with up-regulation of micro RNAs miR-34a-c. (A) The graphs showed qRT-PCR analysis of the indicated genes in Mcm4C3/C3 or WT MEFs that were treated with siRNA against Dicer1 (top) and Drosha (bottom) for 120 h. N = 3 replicates; SEM bars are shown. Data were standardized against β-actin. (B) qRT-PCR analysis of various miRNAs. The values are plotted as percentages of the levels of the indicated miRNAs in Mcm4C3/C3 (Chaos3) MEFs versus WT MEFs. The data represent analysis of 5 littermate pairs of MEFs. SEM bars are shown.

Mentions: To determine if endogenous RNAi pathways control MCM mRNA levels, siRNA-mediated depletions of Dicer1 and Drosha were performed in Chaos3 and control MEFs. Dicer1 knockdown (68%) in Chaos3 MEFs was accompanied by increase in Mcm2, 3, 4, 5 and 7 by up to 1.6-fold (Figure 2A). Drosha knockdown (72%) caused 1.3- to 1.7-fold increases of Mcm RNAs. No changes were induced by Dicer or Drosha knockdown in WT cells. These data are consistent with the possibility that endogenous small RNA-mediated degradation mechanisms are responsible for Mcm mRNA depletion in Chaos3 cells. Interestingly, Dicer mRNA itself also increased upon Drosha knockdown (Figure 2A), suggesting a regulatory relationship as observed by others (29).Figure 2.


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)

Depletion of Mcm2–7 mRNAs in Mcm4C3/C3 cells is Dicer1 and Drosha-regulated, and associated with up-regulation of micro RNAs miR-34a-c. (A) The graphs showed qRT-PCR analysis of the indicated genes in Mcm4C3/C3 or WT MEFs that were treated with siRNA against Dicer1 (top) and Drosha (bottom) for 120 h. N = 3 replicates; SEM bars are shown. Data were standardized against β-actin. (B) qRT-PCR analysis of various miRNAs. The values are plotted as percentages of the levels of the indicated miRNAs in Mcm4C3/C3 (Chaos3) MEFs versus WT MEFs. The data represent analysis of 5 littermate pairs of MEFs. SEM bars are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks176-F2: Depletion of Mcm2–7 mRNAs in Mcm4C3/C3 cells is Dicer1 and Drosha-regulated, and associated with up-regulation of micro RNAs miR-34a-c. (A) The graphs showed qRT-PCR analysis of the indicated genes in Mcm4C3/C3 or WT MEFs that were treated with siRNA against Dicer1 (top) and Drosha (bottom) for 120 h. N = 3 replicates; SEM bars are shown. Data were standardized against β-actin. (B) qRT-PCR analysis of various miRNAs. The values are plotted as percentages of the levels of the indicated miRNAs in Mcm4C3/C3 (Chaos3) MEFs versus WT MEFs. The data represent analysis of 5 littermate pairs of MEFs. SEM bars are shown.
Mentions: To determine if endogenous RNAi pathways control MCM mRNA levels, siRNA-mediated depletions of Dicer1 and Drosha were performed in Chaos3 and control MEFs. Dicer1 knockdown (68%) in Chaos3 MEFs was accompanied by increase in Mcm2, 3, 4, 5 and 7 by up to 1.6-fold (Figure 2A). Drosha knockdown (72%) caused 1.3- to 1.7-fold increases of Mcm RNAs. No changes were induced by Dicer or Drosha knockdown in WT cells. These data are consistent with the possibility that endogenous small RNA-mediated degradation mechanisms are responsible for Mcm mRNA depletion in Chaos3 cells. Interestingly, Dicer mRNA itself also increased upon Drosha knockdown (Figure 2A), suggesting a regulatory relationship as observed by others (29).Figure 2.

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