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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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Effects of Rpl3p depletion on steady-state levels of pre-rRNAs and mature rRNAs. Strains JDY511 [YCplac33-RPL3] (RPL3) and JDY511 [pZGA196] (GAL::RPL3) were grown in YPGalS medium and then shifted to YPD medium. Cells were harvested at the indicated times and total RNA was extracted. (A) RNA corresponding to equal amounts of OD600 units of cells were resolved on a 1.2% agarose–formaldehyde gel, transferred onto a nylon membrane and hybridized consecutively with different probes. (B) RNA corresponding to equal amounts of OD600 units of cells was resolved on a 7% polyacrylamide–urea gel, transferred onto a nylon membrane and hybridized consecutively with different probes. Probe names are indicated between parentheses (see Figure 1A for their location in the 35S pre-rRNA).
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Figure 3: Effects of Rpl3p depletion on steady-state levels of pre-rRNAs and mature rRNAs. Strains JDY511 [YCplac33-RPL3] (RPL3) and JDY511 [pZGA196] (GAL::RPL3) were grown in YPGalS medium and then shifted to YPD medium. Cells were harvested at the indicated times and total RNA was extracted. (A) RNA corresponding to equal amounts of OD600 units of cells were resolved on a 1.2% agarose–formaldehyde gel, transferred onto a nylon membrane and hybridized consecutively with different probes. (B) RNA corresponding to equal amounts of OD600 units of cells was resolved on a 7% polyacrylamide–urea gel, transferred onto a nylon membrane and hybridized consecutively with different probes. Probe names are indicated between parentheses (see Figure 1A for their location in the 35S pre-rRNA).

Mentions: To characterize the basis of the net deficit in 60S r-subunits of the GAL::RPL3 strain, we then analysed the effect of depletion of Rpl3p on pre-rRNA processing. Total RNA was isolated from RPL3 and GAL::RPL3 strains at various time points after transfer from liquid YPGalS to liquid YPDS, and steady-state levels of pre- and mature rRNA species were determined by northern blot and primer extension analyses. Different oligonucleotides hybridizing to defined regions of the 35S pre-RNA were used to monitor-specific processing intermediates (Figure 1A). As shown in Figure 3A, depletion of Rpl3p resulted in a marked decrease in 25S rRNA steady-state levels. This is likely due to an almost complete loss of the 27SB pre-rRNA species, which already becomes apparent at the shortest shift time point to YPDS. In addition, ongoing depletion of Rpl3p led to an accumulation of 35S pre-rRNA and aberrant 23S, 22S and 21S pre-rRNAs. These aberrant species extend from the 5′ end of the 35S pre-rRNA, site A0 and site A1 to site A3, respectively. Hybridizations also identified another fragment, which extends from the 5′ end of the 35S pre-rRNA to site D (Figure 3A). Depletion of Rpl3p also resulted in a mild reduction in the levels of the 20S pre-rRNA and slight reduction in the levels of 27SA2 compared to those from the GAL::RPL3 grown in YPGalS (Figure 3A).Figure 3.


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

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

Effects of Rpl3p depletion on steady-state levels of pre-rRNAs and mature rRNAs. Strains JDY511 [YCplac33-RPL3] (RPL3) and JDY511 [pZGA196] (GAL::RPL3) were grown in YPGalS medium and then shifted to YPD medium. Cells were harvested at the indicated times and total RNA was extracted. (A) RNA corresponding to equal amounts of OD600 units of cells were resolved on a 1.2% agarose–formaldehyde gel, transferred onto a nylon membrane and hybridized consecutively with different probes. (B) RNA corresponding to equal amounts of OD600 units of cells was resolved on a 7% polyacrylamide–urea gel, transferred onto a nylon membrane and hybridized consecutively with different probes. Probe names are indicated between parentheses (see Figure 1A for their location in the 35S pre-rRNA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Effects of Rpl3p depletion on steady-state levels of pre-rRNAs and mature rRNAs. Strains JDY511 [YCplac33-RPL3] (RPL3) and JDY511 [pZGA196] (GAL::RPL3) were grown in YPGalS medium and then shifted to YPD medium. Cells were harvested at the indicated times and total RNA was extracted. (A) RNA corresponding to equal amounts of OD600 units of cells were resolved on a 1.2% agarose–formaldehyde gel, transferred onto a nylon membrane and hybridized consecutively with different probes. (B) RNA corresponding to equal amounts of OD600 units of cells was resolved on a 7% polyacrylamide–urea gel, transferred onto a nylon membrane and hybridized consecutively with different probes. Probe names are indicated between parentheses (see Figure 1A for their location in the 35S pre-rRNA).
Mentions: To characterize the basis of the net deficit in 60S r-subunits of the GAL::RPL3 strain, we then analysed the effect of depletion of Rpl3p on pre-rRNA processing. Total RNA was isolated from RPL3 and GAL::RPL3 strains at various time points after transfer from liquid YPGalS to liquid YPDS, and steady-state levels of pre- and mature rRNA species were determined by northern blot and primer extension analyses. Different oligonucleotides hybridizing to defined regions of the 35S pre-RNA were used to monitor-specific processing intermediates (Figure 1A). As shown in Figure 3A, depletion of Rpl3p resulted in a marked decrease in 25S rRNA steady-state levels. This is likely due to an almost complete loss of the 27SB pre-rRNA species, which already becomes apparent at the shortest shift time point to YPDS. In addition, ongoing depletion of Rpl3p led to an accumulation of 35S pre-rRNA and aberrant 23S, 22S and 21S pre-rRNAs. These aberrant species extend from the 5′ end of the 35S pre-rRNA, site A0 and site A1 to site A3, respectively. Hybridizations also identified another fragment, which extends from the 5′ end of the 35S pre-rRNA to site D (Figure 3A). Depletion of Rpl3p also resulted in a mild reduction in the levels of the 20S pre-rRNA and slight reduction in the levels of 27SA2 compared to those from the GAL::RPL3 grown in YPGalS (Figure 3A).Figure 3.

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