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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 results in a deficit in free 60S r-subunits and in the accumulation of half-mer polysomes. Strain JDY511 [YCplac33-RPL3] (RPL3) was grown in YPGalS at 30°C (A) or shifted to YPDS for 24 h (B). Strain JDY511 [pZGA196] (GAL::RPL3) was grown in YPGalS at 30°C (C) or shifted to YPDS for 6 h (D). Cells were harvested at an OD600 of 0.8, cell extracts were prepared and 10 A260 of each extract were resolved in 7–50% sucrose gradients. The A254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows.
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Figure 2: Depletion of Rpl3p results in a deficit in free 60S r-subunits and in the accumulation of half-mer polysomes. Strain JDY511 [YCplac33-RPL3] (RPL3) was grown in YPGalS at 30°C (A) or shifted to YPDS for 24 h (B). Strain JDY511 [pZGA196] (GAL::RPL3) was grown in YPGalS at 30°C (C) or shifted to YPDS for 6 h (D). Cells were harvested at an OD600 of 0.8, cell extracts were prepared and 10 A260 of each extract were resolved in 7–50% sucrose gradients. The A254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows.

Mentions: Polysome extracts were prepared from cells harvested from the YPGalS cultures and at different times after the shift to YPDS. The GAL::RPL3 strain showed profiles very similar to those of the RPL3 strain when grown in YPGalS, although a slight deficit of free 60S versus 40S was reproducibly observed (Figure 2A and C). However, clear alterations in the profiles appeared after 6 h and became more pronounced at longer times in YPDS (Figure 2D and data not shown). The Rpl3p-depleted strain showed a strong decrease in the levels of free 60S r-subunits versus the levels of free 40S r-subunits and an overall decrease in the 80S peak and in polysomes (Figure 2D). In addition, there was an accumulation of half-mer polysomes (Figure 2D). Wild-type cells showed no alteration in the polysome profile when transferred to YPDS (Figure 2B). These results indicate that depletion of Rpl3p leads to a strong deficit in 60S r-subunits relative to 40S r-subunits.Figure 2.


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 results in a deficit in free 60S r-subunits and in the accumulation of half-mer polysomes. Strain JDY511 [YCplac33-RPL3] (RPL3) was grown in YPGalS at 30°C (A) or shifted to YPDS for 24 h (B). Strain JDY511 [pZGA196] (GAL::RPL3) was grown in YPGalS at 30°C (C) or shifted to YPDS for 6 h (D). Cells were harvested at an OD600 of 0.8, cell extracts were prepared and 10 A260 of each extract were resolved in 7–50% sucrose gradients. The A254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Depletion of Rpl3p results in a deficit in free 60S r-subunits and in the accumulation of half-mer polysomes. Strain JDY511 [YCplac33-RPL3] (RPL3) was grown in YPGalS at 30°C (A) or shifted to YPDS for 24 h (B). Strain JDY511 [pZGA196] (GAL::RPL3) was grown in YPGalS at 30°C (C) or shifted to YPDS for 6 h (D). Cells were harvested at an OD600 of 0.8, cell extracts were prepared and 10 A260 of each extract were resolved in 7–50% sucrose gradients. The A254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows.
Mentions: Polysome extracts were prepared from cells harvested from the YPGalS cultures and at different times after the shift to YPDS. The GAL::RPL3 strain showed profiles very similar to those of the RPL3 strain when grown in YPGalS, although a slight deficit of free 60S versus 40S was reproducibly observed (Figure 2A and C). However, clear alterations in the profiles appeared after 6 h and became more pronounced at longer times in YPDS (Figure 2D and data not shown). The Rpl3p-depleted strain showed a strong decrease in the levels of free 60S r-subunits versus the levels of free 40S r-subunits and an overall decrease in the 80S peak and in polysomes (Figure 2D). In addition, there was an accumulation of half-mer polysomes (Figure 2D). Wild-type cells showed no alteration in the polysome profile when transferred to YPDS (Figure 2B). These results indicate that depletion of Rpl3p leads to a strong deficit in 60S r-subunits relative to 40S r-subunits.Figure 2.

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