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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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Related in: MedlinePlus

Depletion of Utp6p does not arrest cells in mitosis but induces pre-rRNA processing defects that delay G1 phase progression. (A) Western-blot analysis showing the depletion of 3HA-Utp6p and 3HA-Spc97p in strains GAL::3HA::UTP6 and GAL::3HA::SPC97, respectively, shifted from a galactose- (GAL) to a glucose-based medium (GLU). (B) FACS analysis of cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion, or wild-type cells (BY4741) as a control, showing the distribution of cells in the different cell cycle stages (G1, S and G2/M). (C) Northern-blot analysis of the pre-rRNA processing pathway in wild-type cells (BY4741) or cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion using the indicated oligonucleotide probes (Supplementary Table S1).
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gks234-F5: Depletion of Utp6p does not arrest cells in mitosis but induces pre-rRNA processing defects that delay G1 phase progression. (A) Western-blot analysis showing the depletion of 3HA-Utp6p and 3HA-Spc97p in strains GAL::3HA::UTP6 and GAL::3HA::SPC97, respectively, shifted from a galactose- (GAL) to a glucose-based medium (GLU). (B) FACS analysis of cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion, or wild-type cells (BY4741) as a control, showing the distribution of cells in the different cell cycle stages (G1, S and G2/M). (C) Northern-blot analysis of the pre-rRNA processing pathway in wild-type cells (BY4741) or cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion using the indicated oligonucleotide probes (Supplementary Table S1).

Mentions: Extractions of total RNAs from yeast cells (Figure 5C) were performed as described (52). Equal amounts of total RNAs (4 µg) were analyzed by Northern blot (see below).Figure 5.


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)

Depletion of Utp6p does not arrest cells in mitosis but induces pre-rRNA processing defects that delay G1 phase progression. (A) Western-blot analysis showing the depletion of 3HA-Utp6p and 3HA-Spc97p in strains GAL::3HA::UTP6 and GAL::3HA::SPC97, respectively, shifted from a galactose- (GAL) to a glucose-based medium (GLU). (B) FACS analysis of cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion, or wild-type cells (BY4741) as a control, showing the distribution of cells in the different cell cycle stages (G1, S and G2/M). (C) Northern-blot analysis of the pre-rRNA processing pathway in wild-type cells (BY4741) or cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion using the indicated oligonucleotide probes (Supplementary Table S1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks234-F5: Depletion of Utp6p does not arrest cells in mitosis but induces pre-rRNA processing defects that delay G1 phase progression. (A) Western-blot analysis showing the depletion of 3HA-Utp6p and 3HA-Spc97p in strains GAL::3HA::UTP6 and GAL::3HA::SPC97, respectively, shifted from a galactose- (GAL) to a glucose-based medium (GLU). (B) FACS analysis of cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion, or wild-type cells (BY4741) as a control, showing the distribution of cells in the different cell cycle stages (G1, S and G2/M). (C) Northern-blot analysis of the pre-rRNA processing pathway in wild-type cells (BY4741) or cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion using the indicated oligonucleotide probes (Supplementary Table S1).
Mentions: Extractions of total RNAs from yeast cells (Figure 5C) were performed as described (52). Equal amounts of total RNAs (4 µg) were analyzed by Northern blot (see below).Figure 5.

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