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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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HCA66 is detected both at the centrosome and in the nucleoli in HeLa cells and is required for normal accumulation of 40S ribosomal subunits. (A) Immunofluorescence microscopy using HeLa cells expressing EGFP-HCA66 and antibodies detecting γ-tubulin (upper panel) or fibrillarin (lower panel). Scale bar, 10 µm. (B) Western-blot analysis of endogenous HCA66 distribution in HeLa cell subcellular fractions. The different lanes correspond to the total extract (Tot.) or the cytoplasmic (Cyto.), nuclear (Nuclei), nucleoplasmic (Np.), soluble nucleolar (Sol. No.) or insoluble nucleolar (Insol. No.) fractions. HDAC2 and nucleolin are markers of the nucleoplasmic and nucleolar material, respectively. (C) Sucrose gradient analysis of ribosomal subunit levels in HeLa cells treated with HCA66 siRNAs. Cytoplasmic extracts prepared from HeLa cells treated with HCA66 or scrambled siRNAs were centrifuged trough 4.5–45% sucrose gradients. Fractions were collected and the absorbance at 254 nm was measured during collection.
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gks234-F1: HCA66 is detected both at the centrosome and in the nucleoli in HeLa cells and is required for normal accumulation of 40S ribosomal subunits. (A) Immunofluorescence microscopy using HeLa cells expressing EGFP-HCA66 and antibodies detecting γ-tubulin (upper panel) or fibrillarin (lower panel). Scale bar, 10 µm. (B) Western-blot analysis of endogenous HCA66 distribution in HeLa cell subcellular fractions. The different lanes correspond to the total extract (Tot.) or the cytoplasmic (Cyto.), nuclear (Nuclei), nucleoplasmic (Np.), soluble nucleolar (Sol. No.) or insoluble nucleolar (Insol. No.) fractions. HDAC2 and nucleolin are markers of the nucleoplasmic and nucleolar material, respectively. (C) Sucrose gradient analysis of ribosomal subunit levels in HeLa cells treated with HCA66 siRNAs. Cytoplasmic extracts prepared from HeLa cells treated with HCA66 or scrambled siRNAs were centrifuged trough 4.5–45% sucrose gradients. Fractions were collected and the absorbance at 254 nm was measured during collection.

Mentions: Transfections of siRNAs described in Figures 1C and 2 were performed as follows. HeLa cells were collected by trypsinization, rinsed with sterile ZAP buffer (10 mM Sodium Phosphate pH 7.25, 250 mM sucrose, 1 mM MgCl2) and resuspended at a density of 5  ×  107 cells/ml in ZAP buffer. Two hundred microliters of cell suspension (107 cells) were incubated 5 min on ice, mixed with 1 nmol of siRNA duplex, transferred into 4 mm cuvettes (Eurogentec) and cells were electro-transformed with a Bio-Rad Gene Pulser apparatus (square wave protocol, 10 pulses of 5 ms at 240 V separated by 1-s intervals). Transfected cells were immediately mixed with 200 µl of pure fetal bovine serum (Gibco), incubated 3 min at room temperature, resuspended in 10 ml of supplemented DMEM (see above) and plated.Figure 1.


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)

HCA66 is detected both at the centrosome and in the nucleoli in HeLa cells and is required for normal accumulation of 40S ribosomal subunits. (A) Immunofluorescence microscopy using HeLa cells expressing EGFP-HCA66 and antibodies detecting γ-tubulin (upper panel) or fibrillarin (lower panel). Scale bar, 10 µm. (B) Western-blot analysis of endogenous HCA66 distribution in HeLa cell subcellular fractions. The different lanes correspond to the total extract (Tot.) or the cytoplasmic (Cyto.), nuclear (Nuclei), nucleoplasmic (Np.), soluble nucleolar (Sol. No.) or insoluble nucleolar (Insol. No.) fractions. HDAC2 and nucleolin are markers of the nucleoplasmic and nucleolar material, respectively. (C) Sucrose gradient analysis of ribosomal subunit levels in HeLa cells treated with HCA66 siRNAs. Cytoplasmic extracts prepared from HeLa cells treated with HCA66 or scrambled siRNAs were centrifuged trough 4.5–45% sucrose gradients. Fractions were collected and the absorbance at 254 nm was measured during collection.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3401428&req=5

gks234-F1: HCA66 is detected both at the centrosome and in the nucleoli in HeLa cells and is required for normal accumulation of 40S ribosomal subunits. (A) Immunofluorescence microscopy using HeLa cells expressing EGFP-HCA66 and antibodies detecting γ-tubulin (upper panel) or fibrillarin (lower panel). Scale bar, 10 µm. (B) Western-blot analysis of endogenous HCA66 distribution in HeLa cell subcellular fractions. The different lanes correspond to the total extract (Tot.) or the cytoplasmic (Cyto.), nuclear (Nuclei), nucleoplasmic (Np.), soluble nucleolar (Sol. No.) or insoluble nucleolar (Insol. No.) fractions. HDAC2 and nucleolin are markers of the nucleoplasmic and nucleolar material, respectively. (C) Sucrose gradient analysis of ribosomal subunit levels in HeLa cells treated with HCA66 siRNAs. Cytoplasmic extracts prepared from HeLa cells treated with HCA66 or scrambled siRNAs were centrifuged trough 4.5–45% sucrose gradients. Fractions were collected and the absorbance at 254 nm was measured during collection.
Mentions: Transfections of siRNAs described in Figures 1C and 2 were performed as follows. HeLa cells were collected by trypsinization, rinsed with sterile ZAP buffer (10 mM Sodium Phosphate pH 7.25, 250 mM sucrose, 1 mM MgCl2) and resuspended at a density of 5  ×  107 cells/ml in ZAP buffer. Two hundred microliters of cell suspension (107 cells) were incubated 5 min on ice, mixed with 1 nmol of siRNA duplex, transferred into 4 mm cuvettes (Eurogentec) and cells were electro-transformed with a Bio-Rad Gene Pulser apparatus (square wave protocol, 10 pulses of 5 ms at 240 V separated by 1-s intervals). Transfected cells were immediately mixed with 200 µl of pure fetal bovine serum (Gibco), incubated 3 min at room temperature, resuspended in 10 ml of supplemented DMEM (see above) and plated.Figure 1.

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