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Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae.

Babiano R, de la Cruz J - Nucleic Acids Res. (2010)

Bottom Line: In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes.Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle.We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C(2).

View Article: PubMed Central - PubMed

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

ABSTRACT
Ribosome synthesis involves the concomitance of pre-rRNA processing and ribosomal protein assembly. In eukaryotes, this is a complex process that requires the participation of specific sequences and structures within the pre-rRNAs, at least 200 trans-acting factors and the ribosomal proteins. There is little information on the function of individual 60S ribosomal proteins in ribosome synthesis. Herein, we have analysed the contribution of ribosomal protein L35 in ribosome biogenesis. In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase, northern hybridization and primer extension analyses show that processing of the 27SB to 7S pre-rRNAs is strongly delayed upon L35 depletion. Most likely as a consequence of this, release of pre-60S ribosomal particles from the nucleolus to the nucleoplasm is also blocked. Deletion of RPL35A leads to similar although less pronounced phenotypes. Moreover, we show that L35 assembles in the nucleolus and binds to early pre-60S ribosomal particles. Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle. We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C(2).

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Depletion of L35 leads to a mild delay of the cell cycle at the G1 phase. (A) FACS analysis of unsynchronized cells from the BY4741 (Wild-type) or the RBY175 (GAL::RPL35) strains. Cells were grown in YPGal (Gal) or shifted for up to 12 h to YPD (Glc) at 30°C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively. Numbers refers to the 1C/2C area peak ratios (B) Cell morphology of the wild-type and GAL::RPL35 cells. Cells were stained with DAPI for localization of nuclei and then visualized by fluorescence and visible phase contrast microscopy. Only merged images are shown. Numbers refer to mean percentages of unbudded cells after three independent experiments.
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Figure 8: Depletion of L35 leads to a mild delay of the cell cycle at the G1 phase. (A) FACS analysis of unsynchronized cells from the BY4741 (Wild-type) or the RBY175 (GAL::RPL35) strains. Cells were grown in YPGal (Gal) or shifted for up to 12 h to YPD (Glc) at 30°C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively. Numbers refers to the 1C/2C area peak ratios (B) Cell morphology of the wild-type and GAL::RPL35 cells. Cells were stained with DAPI for localization of nuclei and then visualized by fluorescence and visible phase contrast microscopy. Only merged images are shown. Numbers refer to mean percentages of unbudded cells after three independent experiments.

Mentions: Recent studies indicate that the cell cycle can be impaired at different stages upon mutational inactivation or depletion of individual yeast ribosome synthesis factors and r-proteins [e.g. see (27,41,79–81)]. To study the contribution of L35 to cell-cycle progression, we analysed the cell-cycle status and the cellular morphology of the GAL::RPL35 and a isogenic wild-type strain under both permissive and non-permissive conditions. First, asynchronous wild-type cells were grown in YPGal medium and shifted for up to 12 h to YPD medium and then the DNA content was analysed by FACS sorting. We detected two nearly equal peaks corresponding to cells with unreplicated (1C) and duplicated genomes (2C) (Figure 8A). A similar pattern was observed for the GAL::RPL35 cells grown in YPGal medium (Figure 8A). However, after a 3-h shift to YPD, the 1C peak of GAL::RPL35 cells was slightly higher than the one of 2C. Notably, the imbalance between the 1C and 2C peak was more pronounced after a 12-h shift to YPD, which led to an increase in the 1C/2C area peak from 0.7 in YPGal to 1.4 after the 12 h-shift to YPD (Figure 8A). Microscopy inspection of DAPI-stained cells from the wild-type grown either in YPGal or shifted to YPD revealed normal cellular morphology and a mixture of unbudded cells (∼43% from ∼120 examined cells) and cells with variably sized buds (Figure 8B). Similar results were obtained for the GAL::RPL35 cells grown in YPGal medium. However, when the GAL::RPL35 cells were transferred to YPD medium, a progressive increase in the percentage of unbudded cells was observed, and after 12 h in YPD, about 65% cells from ca. 200 examined cells were small and unbudded (Figure 8B). Similar FACS sorting and microscopy analyses were also performed with the Δrpl35A and Δrpl35B strains. These revealed similar although slighter phenotypes than those of L35 depleted cells for only the Δrpl35A mutant (data not shown).Figure 8.


Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae.

Babiano R, de la Cruz J - Nucleic Acids Res. (2010)

Depletion of L35 leads to a mild delay of the cell cycle at the G1 phase. (A) FACS analysis of unsynchronized cells from the BY4741 (Wild-type) or the RBY175 (GAL::RPL35) strains. Cells were grown in YPGal (Gal) or shifted for up to 12 h to YPD (Glc) at 30°C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively. Numbers refers to the 1C/2C area peak ratios (B) Cell morphology of the wild-type and GAL::RPL35 cells. Cells were stained with DAPI for localization of nuclei and then visualized by fluorescence and visible phase contrast microscopy. Only merged images are shown. Numbers refer to mean percentages of unbudded cells after three independent experiments.
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Figure 8: Depletion of L35 leads to a mild delay of the cell cycle at the G1 phase. (A) FACS analysis of unsynchronized cells from the BY4741 (Wild-type) or the RBY175 (GAL::RPL35) strains. Cells were grown in YPGal (Gal) or shifted for up to 12 h to YPD (Glc) at 30°C. 1C and 2C peaks correspond to cells with unreplicated and duplicated genomes, respectively. Numbers refers to the 1C/2C area peak ratios (B) Cell morphology of the wild-type and GAL::RPL35 cells. Cells were stained with DAPI for localization of nuclei and then visualized by fluorescence and visible phase contrast microscopy. Only merged images are shown. Numbers refer to mean percentages of unbudded cells after three independent experiments.
Mentions: Recent studies indicate that the cell cycle can be impaired at different stages upon mutational inactivation or depletion of individual yeast ribosome synthesis factors and r-proteins [e.g. see (27,41,79–81)]. To study the contribution of L35 to cell-cycle progression, we analysed the cell-cycle status and the cellular morphology of the GAL::RPL35 and a isogenic wild-type strain under both permissive and non-permissive conditions. First, asynchronous wild-type cells were grown in YPGal medium and shifted for up to 12 h to YPD medium and then the DNA content was analysed by FACS sorting. We detected two nearly equal peaks corresponding to cells with unreplicated (1C) and duplicated genomes (2C) (Figure 8A). A similar pattern was observed for the GAL::RPL35 cells grown in YPGal medium (Figure 8A). However, after a 3-h shift to YPD, the 1C peak of GAL::RPL35 cells was slightly higher than the one of 2C. Notably, the imbalance between the 1C and 2C peak was more pronounced after a 12-h shift to YPD, which led to an increase in the 1C/2C area peak from 0.7 in YPGal to 1.4 after the 12 h-shift to YPD (Figure 8A). Microscopy inspection of DAPI-stained cells from the wild-type grown either in YPGal or shifted to YPD revealed normal cellular morphology and a mixture of unbudded cells (∼43% from ∼120 examined cells) and cells with variably sized buds (Figure 8B). Similar results were obtained for the GAL::RPL35 cells grown in YPGal medium. However, when the GAL::RPL35 cells were transferred to YPD medium, a progressive increase in the percentage of unbudded cells was observed, and after 12 h in YPD, about 65% cells from ca. 200 examined cells were small and unbudded (Figure 8B). Similar FACS sorting and microscopy analyses were also performed with the Δrpl35A and Δrpl35B strains. These revealed similar although slighter phenotypes than those of L35 depleted cells for only the Δrpl35A mutant (data not shown).Figure 8.

Bottom Line: In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes.Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle.We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C(2).

View Article: PubMed Central - PubMed

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

ABSTRACT
Ribosome synthesis involves the concomitance of pre-rRNA processing and ribosomal protein assembly. In eukaryotes, this is a complex process that requires the participation of specific sequences and structures within the pre-rRNAs, at least 200 trans-acting factors and the ribosomal proteins. There is little information on the function of individual 60S ribosomal proteins in ribosome synthesis. Herein, we have analysed the contribution of ribosomal protein L35 in ribosome biogenesis. In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase, northern hybridization and primer extension analyses show that processing of the 27SB to 7S pre-rRNAs is strongly delayed upon L35 depletion. Most likely as a consequence of this, release of pre-60S ribosomal particles from the nucleolus to the nucleoplasm is also blocked. Deletion of RPL35A leads to similar although less pronounced phenotypes. Moreover, we show that L35 assembles in the nucleolus and binds to early pre-60S ribosomal particles. Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle. We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C(2).

Show MeSH
Related in: MedlinePlus