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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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Deletion of either RPL35A or RPL35B leads to a deficit in 60S r-subunits. (A) Growth test of the rpl35A and rpl35B  mutants compared to their isogenic wild-type control strain. Strains RBY138 (Δrpl35A), RBY139 (Δrpl35B) and BY4741 (Wild-type) were grown in YPD and diluted to an OD600 of 0.05. A 10-fold series of dilutions was performed for each strain and 5 µl drops were plated on YPD plates. Plates were incubated at 30°C for 4 days. (B) The above strains were grown in YPD at 30°C and 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. (C) Equivalent amounts of the cell extracts described above were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8.
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Figure 1: Deletion of either RPL35A or RPL35B leads to a deficit in 60S r-subunits. (A) Growth test of the rpl35A and rpl35B mutants compared to their isogenic wild-type control strain. Strains RBY138 (Δrpl35A), RBY139 (Δrpl35B) and BY4741 (Wild-type) were grown in YPD and diluted to an OD600 of 0.05. A 10-fold series of dilutions was performed for each strain and 5 µl drops were plated on YPD plates. Plates were incubated at 30°C for 4 days. (B) The above strains were grown in YPD at 30°C and 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. (C) Equivalent amounts of the cell extracts described above were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8.

Mentions: Early studies indicated that both RPL35 genes are required for growth and normal accumulation of 60S r-subunits (50) but no further information is available on the role of L35 in ribosome biogenesis. To characterize in detail the contribution of L35 in ribosome biogenesis, we first studied the phenotypic consequences of the deletion of either the RPL35A or the RPL35B gene. As shown in Figure 1A, the deletion of the RPL35A gene (Δrpl35A strain) results in a severe growth defect while the deletion of the RPL35B gene (Δrpl35B strain) has only a slight effect on the growth rate. In agreement, while the generation time in YPD for the wild-type strain grown at 30°C is 1.4 h, the Δrpl35A or Δrpl35B mutant strains have a doubling time of 3.6 h and 1.7 h, respectively. Since genome-wide expression data suggest that RPL35A might contribute to 56% while RPL35B to 44% of total RPL35 mRNA levels (71), the functional disparity between the two deletion strains could not be totally explained by differences in mRNA expression levels. To evaluate whether the growth rate of the two deletion strains has any correlation with the L35 protein levels, we examined the polysome profiles of the deletion strains and compared them with that from an isogenic wild-type counterpart. As shown in Figure 1B, both Δrpl35A and Δrpl35B strains showed a deficit of free 60S versus free 40S r-subunits, a mild decrease in 80S ribosomes and polysomes, and most importantly, an accumulation of half-mer polysomes. Clearly, the Δrpl35B strain led to less severe alterations of the profiles than the Δrpl35A one. This result suggests that the reduced growth rate of the Δrpl35A strain is due to a deficit of 60S r-subunits, which is much larger than the one observed for the Δrpl35B strain. To confirm this, we quantified total r-subunits by using run-off and low-Mg2+ sucrose gradients. Indeed, in the Δrpl35A strain we observed a 25% reduction in the overall quantity of 60S r-subunits relative to the wild-type strain whereas only a 7% reduction was detected in the Δrpl35B strain (data not shown). In agreement with this net deficit in 60S r-subunits, western blot analysis indicated that the L35 levels, as well as those of L1 were decreased in the Δrpl35A strain, whereas the levels of the small subunit r-protein S8 were not significantly affected. No evident changes were observed in the Δrpl35B strain (Figure 1C).Figure 1.


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

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

Deletion of either RPL35A or RPL35B leads to a deficit in 60S r-subunits. (A) Growth test of the rpl35A and rpl35B  mutants compared to their isogenic wild-type control strain. Strains RBY138 (Δrpl35A), RBY139 (Δrpl35B) and BY4741 (Wild-type) were grown in YPD and diluted to an OD600 of 0.05. A 10-fold series of dilutions was performed for each strain and 5 µl drops were plated on YPD plates. Plates were incubated at 30°C for 4 days. (B) The above strains were grown in YPD at 30°C and 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. (C) Equivalent amounts of the cell extracts described above were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8.
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Figure 1: Deletion of either RPL35A or RPL35B leads to a deficit in 60S r-subunits. (A) Growth test of the rpl35A and rpl35B mutants compared to their isogenic wild-type control strain. Strains RBY138 (Δrpl35A), RBY139 (Δrpl35B) and BY4741 (Wild-type) were grown in YPD and diluted to an OD600 of 0.05. A 10-fold series of dilutions was performed for each strain and 5 µl drops were plated on YPD plates. Plates were incubated at 30°C for 4 days. (B) The above strains were grown in YPD at 30°C and 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. (C) Equivalent amounts of the cell extracts described above were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8.
Mentions: Early studies indicated that both RPL35 genes are required for growth and normal accumulation of 60S r-subunits (50) but no further information is available on the role of L35 in ribosome biogenesis. To characterize in detail the contribution of L35 in ribosome biogenesis, we first studied the phenotypic consequences of the deletion of either the RPL35A or the RPL35B gene. As shown in Figure 1A, the deletion of the RPL35A gene (Δrpl35A strain) results in a severe growth defect while the deletion of the RPL35B gene (Δrpl35B strain) has only a slight effect on the growth rate. In agreement, while the generation time in YPD for the wild-type strain grown at 30°C is 1.4 h, the Δrpl35A or Δrpl35B mutant strains have a doubling time of 3.6 h and 1.7 h, respectively. Since genome-wide expression data suggest that RPL35A might contribute to 56% while RPL35B to 44% of total RPL35 mRNA levels (71), the functional disparity between the two deletion strains could not be totally explained by differences in mRNA expression levels. To evaluate whether the growth rate of the two deletion strains has any correlation with the L35 protein levels, we examined the polysome profiles of the deletion strains and compared them with that from an isogenic wild-type counterpart. As shown in Figure 1B, both Δrpl35A and Δrpl35B strains showed a deficit of free 60S versus free 40S r-subunits, a mild decrease in 80S ribosomes and polysomes, and most importantly, an accumulation of half-mer polysomes. Clearly, the Δrpl35B strain led to less severe alterations of the profiles than the Δrpl35A one. This result suggests that the reduced growth rate of the Δrpl35A strain is due to a deficit of 60S r-subunits, which is much larger than the one observed for the Δrpl35B strain. To confirm this, we quantified total r-subunits by using run-off and low-Mg2+ sucrose gradients. Indeed, in the Δrpl35A strain we observed a 25% reduction in the overall quantity of 60S r-subunits relative to the wild-type strain whereas only a 7% reduction was detected in the Δrpl35B strain (data not shown). In agreement with this net deficit in 60S r-subunits, western blot analysis indicated that the L35 levels, as well as those of L1 were decreased in the Δrpl35A strain, whereas the levels of the small subunit r-protein S8 were not significantly affected. No evident changes were observed in the Δrpl35B strain (Figure 1C).Figure 1.

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