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Functional analysis of Saccharomyces cerevisiae ribosomal protein Rpl3p in ribosome synthesis.

Rosado IV, Kressler D, de la Cruz J - Nucleic Acids Res. (2007)

Bottom Line: In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes.Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles.Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Universidad de Sevilla, Sevilla, Spain.

ABSTRACT
Ribosome synthesis in eukaryotes requires a multitude of trans-acting factors. These factors act at many steps as the pre-ribosomal particles travel from the nucleolus to the cytoplasm. In contrast to the well-studied trans-acting factors, little is known about the contribution of the ribosomal proteins to ribosome biogenesis. Herein, we have analysed the role of ribosomal protein Rpl3p in 60S ribosomal subunit biogenesis. In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability of early and intermediate pre-ribosomal particles, as evidenced by the low steady-state levels of 27SA(3), 27SB(S) and 7S(L/S) precursors. Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles. Interestingly, flow cytometry analysis indicates that Rpl3p-depleted cells arrest in the G1 phase. Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented.

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Depletion of Rpl3p leads to an arrest of the cell cycle at the G1 phase and an abnormal cell morphology. (A) Cell morphology of GAL::RPL3 cells grown in YPGalS (Gal+Sorbitol) or shifted for 12 h to YPDS (Glc+Sorbitol). Cells were stained with DAPI for localization of nuclei and then visualized by fluorescence and phase contrast microscopy. Merged images are shown. (B) FACS analysis of unsynchronized GAL::RPL3 cells grown in YPGalS or shifted for 12 h to YPDS at 30°C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively.
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Figure 6: Depletion of Rpl3p leads to an arrest of the cell cycle at the G1 phase and an abnormal cell morphology. (A) Cell morphology of GAL::RPL3 cells grown in YPGalS (Gal+Sorbitol) or shifted for 12 h to YPDS (Glc+Sorbitol). Cells were stained with DAPI for localization of nuclei and then visualized by fluorescence and phase contrast microscopy. Merged images are shown. (B) FACS analysis of unsynchronized GAL::RPL3 cells grown in YPGalS or shifted for 12 h to YPDS at 30°C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively.

Mentions: Our previous results (55) and unpublished observations (I.V.R., unpublished results) as well as results from Wozniak and co-workers (56) indicate that Rpl3p interacts functionally and physically with the WD-repeat Rrb1p protein, which has been suggested to act as the Rpl3p assembler onto pre-60S r-particles (55,56). Rrb1p has also been shown to be required for the metaphase/anaphase transition during the cell cycle and proper chromosome segregation (42). Rrb1p also functionally interacts with the origin recognition complex component Orc6p, involved in the initiation of DNA replication (42,57), and the yeast Pescadillo complex, which consists in yeast of Nop7p, Erb1p and Ytm1p and is required for both ribosome biogenesis and normal progression through the S phase of the cell cycle (42,58). Moreover, inactivation of human orthologues of Rrb1p, Nop7p, Erb1p/Bop1p, Orc6p and Rpl3p alters proper chromosome segregation (42). To study whether yeast Rpl3p is required for optimal progression through the cell cycle, we first examined cellular morphology of GAL::RPL3 cells by light microscopy. A normal morphology was observed for most cells when grown in galactose medium (Figure 6A). However, 6 h after transfer to glucose medium, GAL::RPL3 cells increased in size and a significant percentage (about 5%) showed an elongated shape and contained enlarged buds with pronounced apical growth. Apparently, these elongated cells contained duplicated, separated nuclei as shown by DAPI staining (Figure 6A).Figure 6.


Functional analysis of Saccharomyces cerevisiae ribosomal protein Rpl3p in ribosome synthesis.

Rosado IV, Kressler D, de la Cruz J - Nucleic Acids Res. (2007)

Depletion of Rpl3p leads to an arrest of the cell cycle at the G1 phase and an abnormal cell morphology. (A) Cell morphology of GAL::RPL3 cells grown in YPGalS (Gal+Sorbitol) or shifted for 12 h to YPDS (Glc+Sorbitol). Cells were stained with DAPI for localization of nuclei and then visualized by fluorescence and phase contrast microscopy. Merged images are shown. (B) FACS analysis of unsynchronized GAL::RPL3 cells grown in YPGalS or shifted for 12 h to YPDS at 30°C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919493&req=5

Figure 6: Depletion of Rpl3p leads to an arrest of the cell cycle at the G1 phase and an abnormal cell morphology. (A) Cell morphology of GAL::RPL3 cells grown in YPGalS (Gal+Sorbitol) or shifted for 12 h to YPDS (Glc+Sorbitol). Cells were stained with DAPI for localization of nuclei and then visualized by fluorescence and phase contrast microscopy. Merged images are shown. (B) FACS analysis of unsynchronized GAL::RPL3 cells grown in YPGalS or shifted for 12 h to YPDS at 30°C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively.
Mentions: Our previous results (55) and unpublished observations (I.V.R., unpublished results) as well as results from Wozniak and co-workers (56) indicate that Rpl3p interacts functionally and physically with the WD-repeat Rrb1p protein, which has been suggested to act as the Rpl3p assembler onto pre-60S r-particles (55,56). Rrb1p has also been shown to be required for the metaphase/anaphase transition during the cell cycle and proper chromosome segregation (42). Rrb1p also functionally interacts with the origin recognition complex component Orc6p, involved in the initiation of DNA replication (42,57), and the yeast Pescadillo complex, which consists in yeast of Nop7p, Erb1p and Ytm1p and is required for both ribosome biogenesis and normal progression through the S phase of the cell cycle (42,58). Moreover, inactivation of human orthologues of Rrb1p, Nop7p, Erb1p/Bop1p, Orc6p and Rpl3p alters proper chromosome segregation (42). To study whether yeast Rpl3p is required for optimal progression through the cell cycle, we first examined cellular morphology of GAL::RPL3 cells by light microscopy. A normal morphology was observed for most cells when grown in galactose medium (Figure 6A). However, 6 h after transfer to glucose medium, GAL::RPL3 cells increased in size and a significant percentage (about 5%) showed an elongated shape and contained enlarged buds with pronounced apical growth. Apparently, these elongated cells contained duplicated, separated nuclei as shown by DAPI staining (Figure 6A).Figure 6.

Bottom Line: In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes.Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles.Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Universidad de Sevilla, Sevilla, Spain.

ABSTRACT
Ribosome synthesis in eukaryotes requires a multitude of trans-acting factors. These factors act at many steps as the pre-ribosomal particles travel from the nucleolus to the cytoplasm. In contrast to the well-studied trans-acting factors, little is known about the contribution of the ribosomal proteins to ribosome biogenesis. Herein, we have analysed the role of ribosomal protein Rpl3p in 60S ribosomal subunit biogenesis. In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability of early and intermediate pre-ribosomal particles, as evidenced by the low steady-state levels of 27SA(3), 27SB(S) and 7S(L/S) precursors. Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles. Interestingly, flow cytometry analysis indicates that Rpl3p-depleted cells arrest in the G1 phase. Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented.

Show MeSH
Related in: MedlinePlus