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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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MCM3 interacts with XPO1 and MCM5 via an NES motif that is essential for inhibiting MCM chromatin binding and cell proliferation. (A) MCM3 interacts with XPO1 in an NES-dependent manner. Shown are Western blots probed for proteins co-IP’d from HEK cells ectopically expressing MYC tagged mMCM3 (M3) or a version with four Leucines mutated within the putative NES (M3-L4A; see ‘B’). The middle lane contains extracts that treated with calf intestinal alkaline phosphatase (CIAP) prior to IP. (B) Schematic of mutation made in the putative NES motif (top) and CDK phosphorylation sites (bottom) within MCM3. (C) Western blot analysis of the indicated chromatin-bound (‘Extracted’) and soluble proteins isolated from HeLa cells infected with control (LacZ vector) or MYC-tagged MCM3 lentiviral constructs (listed at top). (D) Quantification of Western blot data by densitometry. Intensities of bands in ‘A’ were normalized against GAPDH in soluble fractions, or fibrillarin in extracted fractions. These values relative to WT cells are plotted. Experiments were repeated twice. (E) Images of HeLA cell culture plates (crystal violet stained) infected with lentiviruses expressing control (LacZ) or the indicated MCM3 proteins variants. (F) MCM3 interacts with MCM5 via the NES motif. HeLa cells were transduced with vectors expressing MYC-tagged LacZ, MCM3WT, MCM3L4A or MCM3S5A, immunoprecipitated (right panel), and probed for MCM3, MCM5 or MCM7.
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gks176-F4: MCM3 interacts with XPO1 and MCM5 via an NES motif that is essential for inhibiting MCM chromatin binding and cell proliferation. (A) MCM3 interacts with XPO1 in an NES-dependent manner. Shown are Western blots probed for proteins co-IP’d from HEK cells ectopically expressing MYC tagged mMCM3 (M3) or a version with four Leucines mutated within the putative NES (M3-L4A; see ‘B’). The middle lane contains extracts that treated with calf intestinal alkaline phosphatase (CIAP) prior to IP. (B) Schematic of mutation made in the putative NES motif (top) and CDK phosphorylation sites (bottom) within MCM3. (C) Western blot analysis of the indicated chromatin-bound (‘Extracted’) and soluble proteins isolated from HeLa cells infected with control (LacZ vector) or MYC-tagged MCM3 lentiviral constructs (listed at top). (D) Quantification of Western blot data by densitometry. Intensities of bands in ‘A’ were normalized against GAPDH in soluble fractions, or fibrillarin in extracted fractions. These values relative to WT cells are plotted. Experiments were repeated twice. (E) Images of HeLA cell culture plates (crystal violet stained) infected with lentiviruses expressing control (LacZ) or the indicated MCM3 proteins variants. (F) MCM3 interacts with MCM5 via the NES motif. HeLa cells were transduced with vectors expressing MYC-tagged LacZ, MCM3WT, MCM3L4A or MCM3S5A, immunoprecipitated (right panel), and probed for MCM3, MCM5 or MCM7.

Mentions: To test these possibilities, we determined the ability of MCM4C3 to associate with other MCMs in HEK cells, which are efficient for transfection and expression of exogenous constructs. Whereas MCM7 could be co-immunoprecipitated with epitope-tagged mouse MCM4+ or MCM4C3, MCM6 was only pulled down with MCM4+ (Figure 3A). Little or no MCM2, 3 or 5 was co-IP’d with MCM4+ or MCM4C3 (not shown). Similar results were obtained with endogenous proteins in Mcm4C3/C3 MEFs (Figure 3B). Additionally, although anti-MCM6 IP’d MCM7 in WT cells, it did not in Chaos3 cells (Figure 3B). The disrupted MCM4/6 interaction likely causes MCM2–7 to break into subcomplexes (Figure 3C), as was also suggested by gel filtration studies (11), and our observations of relatively weaker interactions between MCM3 and MCM7 compared to MCM3 and MCM5 (Figure 4F). Since MCM4+ and MCM4C3co-IP’d each other, this suggests that the mutation does not abolish interactions between MCM2–7 hexamers.Figure 3.


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)

MCM3 interacts with XPO1 and MCM5 via an NES motif that is essential for inhibiting MCM chromatin binding and cell proliferation. (A) MCM3 interacts with XPO1 in an NES-dependent manner. Shown are Western blots probed for proteins co-IP’d from HEK cells ectopically expressing MYC tagged mMCM3 (M3) or a version with four Leucines mutated within the putative NES (M3-L4A; see ‘B’). The middle lane contains extracts that treated with calf intestinal alkaline phosphatase (CIAP) prior to IP. (B) Schematic of mutation made in the putative NES motif (top) and CDK phosphorylation sites (bottom) within MCM3. (C) Western blot analysis of the indicated chromatin-bound (‘Extracted’) and soluble proteins isolated from HeLa cells infected with control (LacZ vector) or MYC-tagged MCM3 lentiviral constructs (listed at top). (D) Quantification of Western blot data by densitometry. Intensities of bands in ‘A’ were normalized against GAPDH in soluble fractions, or fibrillarin in extracted fractions. These values relative to WT cells are plotted. Experiments were repeated twice. (E) Images of HeLA cell culture plates (crystal violet stained) infected with lentiviruses expressing control (LacZ) or the indicated MCM3 proteins variants. (F) MCM3 interacts with MCM5 via the NES motif. HeLa cells were transduced with vectors expressing MYC-tagged LacZ, MCM3WT, MCM3L4A or MCM3S5A, immunoprecipitated (right panel), and probed for MCM3, MCM5 or MCM7.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks176-F4: MCM3 interacts with XPO1 and MCM5 via an NES motif that is essential for inhibiting MCM chromatin binding and cell proliferation. (A) MCM3 interacts with XPO1 in an NES-dependent manner. Shown are Western blots probed for proteins co-IP’d from HEK cells ectopically expressing MYC tagged mMCM3 (M3) or a version with four Leucines mutated within the putative NES (M3-L4A; see ‘B’). The middle lane contains extracts that treated with calf intestinal alkaline phosphatase (CIAP) prior to IP. (B) Schematic of mutation made in the putative NES motif (top) and CDK phosphorylation sites (bottom) within MCM3. (C) Western blot analysis of the indicated chromatin-bound (‘Extracted’) and soluble proteins isolated from HeLa cells infected with control (LacZ vector) or MYC-tagged MCM3 lentiviral constructs (listed at top). (D) Quantification of Western blot data by densitometry. Intensities of bands in ‘A’ were normalized against GAPDH in soluble fractions, or fibrillarin in extracted fractions. These values relative to WT cells are plotted. Experiments were repeated twice. (E) Images of HeLA cell culture plates (crystal violet stained) infected with lentiviruses expressing control (LacZ) or the indicated MCM3 proteins variants. (F) MCM3 interacts with MCM5 via the NES motif. HeLa cells were transduced with vectors expressing MYC-tagged LacZ, MCM3WT, MCM3L4A or MCM3S5A, immunoprecipitated (right panel), and probed for MCM3, MCM5 or MCM7.
Mentions: To test these possibilities, we determined the ability of MCM4C3 to associate with other MCMs in HEK cells, which are efficient for transfection and expression of exogenous constructs. Whereas MCM7 could be co-immunoprecipitated with epitope-tagged mouse MCM4+ or MCM4C3, MCM6 was only pulled down with MCM4+ (Figure 3A). Little or no MCM2, 3 or 5 was co-IP’d with MCM4+ or MCM4C3 (not shown). Similar results were obtained with endogenous proteins in Mcm4C3/C3 MEFs (Figure 3B). Additionally, although anti-MCM6 IP’d MCM7 in WT cells, it did not in Chaos3 cells (Figure 3B). The disrupted MCM4/6 interaction likely causes MCM2–7 to break into subcomplexes (Figure 3C), as was also suggested by gel filtration studies (11), and our observations of relatively weaker interactions between MCM3 and MCM7 compared to MCM3 and MCM5 (Figure 4F). Since MCM4+ and MCM4C3co-IP’d each other, this suggests that the mutation does not abolish interactions between MCM2–7 hexamers.Figure 3.

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