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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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Some molecular mechanisms underlying the function of HCA66/Utp6p in ribosome synthesis are conserved in yeast and mammals. (A) Schematic representation of Utp6p (440 amino acids), HCA66 (597 amino acids) and the different modified versions of HCA66 bearing single (A99E, H137A, E153A, R170A) or double (K102A + D106A) amino acid substitutions within the HAT repeats. The conserved N-terminal domain is colored in green, the HAT motifs in red. (B) Northern-blot analysis showing the accumulation levels of some pre-rRNAs (indicated on the left) in HeLa cells transfected with siRNAs targeting the 3′-UTR of HCA66 mRNA, either alone or in combination with vectors expressing EGFP only or wild-type and modified versions of EGFP-HCA66. The 18S-E/30S ratios, averaged from three experiments, are reported on the histogram presented on the right. (C) Immunoprecipitation experiment using anti-FLAG antibodies and HeLa cells co-expressing FLAG-WDR36 (+) or FLAG alone (−) and wild-type (WT) or altered version (A99E or K102A + D106A) of EGFP-HCA66. A fraction of the total extracts used for the experiments (T) and of the immunoprecipitated material (IP) was analyzed by western blot.
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gks234-F6: Some molecular mechanisms underlying the function of HCA66/Utp6p in ribosome synthesis are conserved in yeast and mammals. (A) Schematic representation of Utp6p (440 amino acids), HCA66 (597 amino acids) and the different modified versions of HCA66 bearing single (A99E, H137A, E153A, R170A) or double (K102A + D106A) amino acid substitutions within the HAT repeats. The conserved N-terminal domain is colored in green, the HAT motifs in red. (B) Northern-blot analysis showing the accumulation levels of some pre-rRNAs (indicated on the left) in HeLa cells transfected with siRNAs targeting the 3′-UTR of HCA66 mRNA, either alone or in combination with vectors expressing EGFP only or wild-type and modified versions of EGFP-HCA66. The 18S-E/30S ratios, averaged from three experiments, are reported on the histogram presented on the right. (C) Immunoprecipitation experiment using anti-FLAG antibodies and HeLa cells co-expressing FLAG-WDR36 (+) or FLAG alone (−) and wild-type (WT) or altered version (A99E or K102A + D106A) of EGFP-HCA66. A fraction of the total extracts used for the experiments (T) and of the immunoprecipitated material (IP) was analyzed by western blot.

Mentions: For co-transfection experiments presented in Figure 6, 107 HeLa cells resuspended in 200 µl Opti-MEM (Gibco) were electro-transformed with 10 µg of plasmid DNA and 1 nmol of siRNA duplex as described earlier.


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)

Some molecular mechanisms underlying the function of HCA66/Utp6p in ribosome synthesis are conserved in yeast and mammals. (A) Schematic representation of Utp6p (440 amino acids), HCA66 (597 amino acids) and the different modified versions of HCA66 bearing single (A99E, H137A, E153A, R170A) or double (K102A + D106A) amino acid substitutions within the HAT repeats. The conserved N-terminal domain is colored in green, the HAT motifs in red. (B) Northern-blot analysis showing the accumulation levels of some pre-rRNAs (indicated on the left) in HeLa cells transfected with siRNAs targeting the 3′-UTR of HCA66 mRNA, either alone or in combination with vectors expressing EGFP only or wild-type and modified versions of EGFP-HCA66. The 18S-E/30S ratios, averaged from three experiments, are reported on the histogram presented on the right. (C) Immunoprecipitation experiment using anti-FLAG antibodies and HeLa cells co-expressing FLAG-WDR36 (+) or FLAG alone (−) and wild-type (WT) or altered version (A99E or K102A + D106A) of EGFP-HCA66. A fraction of the total extracts used for the experiments (T) and of the immunoprecipitated material (IP) was analyzed by western blot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks234-F6: Some molecular mechanisms underlying the function of HCA66/Utp6p in ribosome synthesis are conserved in yeast and mammals. (A) Schematic representation of Utp6p (440 amino acids), HCA66 (597 amino acids) and the different modified versions of HCA66 bearing single (A99E, H137A, E153A, R170A) or double (K102A + D106A) amino acid substitutions within the HAT repeats. The conserved N-terminal domain is colored in green, the HAT motifs in red. (B) Northern-blot analysis showing the accumulation levels of some pre-rRNAs (indicated on the left) in HeLa cells transfected with siRNAs targeting the 3′-UTR of HCA66 mRNA, either alone or in combination with vectors expressing EGFP only or wild-type and modified versions of EGFP-HCA66. The 18S-E/30S ratios, averaged from three experiments, are reported on the histogram presented on the right. (C) Immunoprecipitation experiment using anti-FLAG antibodies and HeLa cells co-expressing FLAG-WDR36 (+) or FLAG alone (−) and wild-type (WT) or altered version (A99E or K102A + D106A) of EGFP-HCA66. A fraction of the total extracts used for the experiments (T) and of the immunoprecipitated material (IP) was analyzed by western blot.
Mentions: For co-transfection experiments presented in Figure 6, 107 HeLa cells resuspended in 200 µl Opti-MEM (Gibco) were electro-transformed with 10 µg of plasmid DNA and 1 nmol of siRNA duplex as described earlier.

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