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Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

Bonnart C, Gérus M, Hoareau-Aveilla C, Kiss T, Caizergues-Ferrer M, Henry Y, Henras AK - Nucleic Acids Res. (2012)

Bottom Line: Overexpression of a dominant negative version of HCA66, accumulating at the centrosome but absent from the nucleoli, alters centrosome function but has no effect on pre-rRNA processing, suggesting that HCA66 acts independently in each process.In yeast and HeLa cells, depletion of MTOC components does not impair ribosome synthesis.Hence our results suggest that both in yeast and human cells, assembly of a functional MTOC and ribosome synthesis are not closely connected processes.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France.

ABSTRACT
Ribosome production, one of the most energy-consuming biosynthetic activities in living cells, is adjusted to growth conditions and coordinated with the cell cycle. Connections between ribosome synthesis and cell cycle progression have been described, but the underlying mechanisms remain only partially understood. The human HCA66 protein was recently characterized as a component of the centrosome, the major microtubule-organizing center (MTOC) in mammalian cells, and was shown to be required for centriole duplication and assembly of the mitotic spindle. We show here that HCA66 is also required for nucleolar steps of the maturation of the 40S ribosomal subunit and therefore displays a dual function. Overexpression of a dominant negative version of HCA66, accumulating at the centrosome but absent from the nucleoli, alters centrosome function but has no effect on pre-rRNA processing, suggesting that HCA66 acts independently in each process. In yeast and HeLa cells, depletion of MTOC components does not impair ribosome synthesis. Hence our results suggest that both in yeast and human cells, assembly of a functional MTOC and ribosome synthesis are not closely connected processes.

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Defects in centrosome function induced by the expression of a dominant negative version of HCA66 do not impair ribosome synthesis in HeLa cells. (A) FACS analysis showing the cell cycle profiles of HeLa cell cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 48 h after transfection. Total cells (adherent and floating cells) were analyzed in each case. The arrowheads below each graph indicate the positions of the G1 and G2/M peaks. Arrows indicate the presence of a sub-G1 peak, prominent in cells expressing EGFP-HCA661–86. (B) Proportion of dead cells in cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 24, 48 or 72 h after electroporation. The histogram represents the percentage of trypan blue positive (dead) cells in each population, considering total cells. (C) Agarose gel electrophoresis of genomic DNAs extracted from HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. Genomic DNAs have been extracted from total cells. Molecular weight ladder: SmartLadder (Eurogentec). (D) Northern-blot analysis using probe a (Figure 2B and Supplementary Table S1) showing the accumulation levels of some pre-rRNAs in HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. RNAs were extracted from total cells.
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gks234-F7: Defects in centrosome function induced by the expression of a dominant negative version of HCA66 do not impair ribosome synthesis in HeLa cells. (A) FACS analysis showing the cell cycle profiles of HeLa cell cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 48 h after transfection. Total cells (adherent and floating cells) were analyzed in each case. The arrowheads below each graph indicate the positions of the G1 and G2/M peaks. Arrows indicate the presence of a sub-G1 peak, prominent in cells expressing EGFP-HCA661–86. (B) Proportion of dead cells in cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 24, 48 or 72 h after electroporation. The histogram represents the percentage of trypan blue positive (dead) cells in each population, considering total cells. (C) Agarose gel electrophoresis of genomic DNAs extracted from HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. Genomic DNAs have been extracted from total cells. Molecular weight ladder: SmartLadder (Eurogentec). (D) Northern-blot analysis using probe a (Figure 2B and Supplementary Table S1) showing the accumulation levels of some pre-rRNAs in HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. RNAs were extracted from total cells.

Mentions: For transfection experiments presented in Figures 1A, 3 and 7, 107 HeLa cells resuspended in 200 µl Opti-MEM (Gibco) were mixed with 10 µg of plasmid DNA or 1 nmol of siRNA duplex. The samples were transferred into 4 mm cuvettes (Eurogentec), incubated 5 min on ice and cells were electro-transformed with a Bio-Rad Gene Pulser apparatus (Exponential protocol, 250 V, 950 µF). Cells were immediately recovered, resuspended in 10 ml of supplemented DMEM (see above) and plated.Figure 3.


Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

Bonnart C, Gérus M, Hoareau-Aveilla C, Kiss T, Caizergues-Ferrer M, Henry Y, Henras AK - Nucleic Acids Res. (2012)

Defects in centrosome function induced by the expression of a dominant negative version of HCA66 do not impair ribosome synthesis in HeLa cells. (A) FACS analysis showing the cell cycle profiles of HeLa cell cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 48 h after transfection. Total cells (adherent and floating cells) were analyzed in each case. The arrowheads below each graph indicate the positions of the G1 and G2/M peaks. Arrows indicate the presence of a sub-G1 peak, prominent in cells expressing EGFP-HCA661–86. (B) Proportion of dead cells in cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 24, 48 or 72 h after electroporation. The histogram represents the percentage of trypan blue positive (dead) cells in each population, considering total cells. (C) Agarose gel electrophoresis of genomic DNAs extracted from HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. Genomic DNAs have been extracted from total cells. Molecular weight ladder: SmartLadder (Eurogentec). (D) Northern-blot analysis using probe a (Figure 2B and Supplementary Table S1) showing the accumulation levels of some pre-rRNAs in HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. RNAs were extracted from total cells.
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Related In: Results  -  Collection

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gks234-F7: Defects in centrosome function induced by the expression of a dominant negative version of HCA66 do not impair ribosome synthesis in HeLa cells. (A) FACS analysis showing the cell cycle profiles of HeLa cell cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 48 h after transfection. Total cells (adherent and floating cells) were analyzed in each case. The arrowheads below each graph indicate the positions of the G1 and G2/M peaks. Arrows indicate the presence of a sub-G1 peak, prominent in cells expressing EGFP-HCA661–86. (B) Proportion of dead cells in cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 24, 48 or 72 h after electroporation. The histogram represents the percentage of trypan blue positive (dead) cells in each population, considering total cells. (C) Agarose gel electrophoresis of genomic DNAs extracted from HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. Genomic DNAs have been extracted from total cells. Molecular weight ladder: SmartLadder (Eurogentec). (D) Northern-blot analysis using probe a (Figure 2B and Supplementary Table S1) showing the accumulation levels of some pre-rRNAs in HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. RNAs were extracted from total cells.
Mentions: For transfection experiments presented in Figures 1A, 3 and 7, 107 HeLa cells resuspended in 200 µl Opti-MEM (Gibco) were mixed with 10 µg of plasmid DNA or 1 nmol of siRNA duplex. The samples were transferred into 4 mm cuvettes (Eurogentec), incubated 5 min on ice and cells were electro-transformed with a Bio-Rad Gene Pulser apparatus (Exponential protocol, 250 V, 950 µF). Cells were immediately recovered, resuspended in 10 ml of supplemented DMEM (see above) and plated.Figure 3.

Bottom Line: Overexpression of a dominant negative version of HCA66, accumulating at the centrosome but absent from the nucleoli, alters centrosome function but has no effect on pre-rRNA processing, suggesting that HCA66 acts independently in each process.In yeast and HeLa cells, depletion of MTOC components does not impair ribosome synthesis.Hence our results suggest that both in yeast and human cells, assembly of a functional MTOC and ribosome synthesis are not closely connected processes.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France.

ABSTRACT
Ribosome production, one of the most energy-consuming biosynthetic activities in living cells, is adjusted to growth conditions and coordinated with the cell cycle. Connections between ribosome synthesis and cell cycle progression have been described, but the underlying mechanisms remain only partially understood. The human HCA66 protein was recently characterized as a component of the centrosome, the major microtubule-organizing center (MTOC) in mammalian cells, and was shown to be required for centriole duplication and assembly of the mitotic spindle. We show here that HCA66 is also required for nucleolar steps of the maturation of the 40S ribosomal subunit and therefore displays a dual function. Overexpression of a dominant negative version of HCA66, accumulating at the centrosome but absent from the nucleoli, alters centrosome function but has no effect on pre-rRNA processing, suggesting that HCA66 acts independently in each process. In yeast and HeLa cells, depletion of MTOC components does not impair ribosome synthesis. Hence our results suggest that both in yeast and human cells, assembly of a functional MTOC and ribosome synthesis are not closely connected processes.

Show MeSH
Related in: MedlinePlus