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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 results in a deficit in 60S r-subunits. (A) Growth comparison of the strains BY4741 (Wild-type) and RBY175 (GAL::RPL35). The cells were grown in YPGal and streaked on YPGal (Gal) and YPD (Glc) plates, which were incubated at 30°C for 3 days. (B) The above strains were grown in YPGal at 30°C and shifted to YPD. Cells were harvested at the indicated times after the shift and cell extracts were prepared. Equivalent amounts of cell extracts were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8. (C) 10 A260 of cell extract from the GAL::RPL35 strain grown in YPGal (Gal) or shifted for 6 h (6 h Glc) or 12 h (12 h Glc) to YPD were subjected to polysome analysis in 7–50% sucrose gradients. 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 L35 results in a deficit in 60S r-subunits. (A) Growth comparison of the strains BY4741 (Wild-type) and RBY175 (GAL::RPL35). The cells were grown in YPGal and streaked on YPGal (Gal) and YPD (Glc) plates, which were incubated at 30°C for 3 days. (B) The above strains were grown in YPGal at 30°C and shifted to YPD. Cells were harvested at the indicated times after the shift and cell extracts were prepared. Equivalent amounts of cell extracts were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8. (C) 10 A260 of cell extract from the GAL::RPL35 strain grown in YPGal (Gal) or shifted for 6 h (6 h Glc) or 12 h (12 h Glc) to YPD were subjected to polysome analysis in 7–50% sucrose gradients. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows.

Mentions: To study the consequences of the L35 depletion we generated a conditional GAL::RPL35 strain (see ‘Materials and Methods’ section). Growth of this strain was identical to that of the isogenic wild-type strain on YPGal plates, showing that the production of N-terminally HA-tagged L35A derived from the GAL-RPL35A construct ensures wild-type growth (Figure 2A). As expected, only very residual growth of this strain was observed on YPD plates (Figure 2A). After shifting a mid-logarithmic culture of GAL::RPL35 from liquid YPGal medium to YPD medium, the growth rate rapidly decrease to a doubling time of >10 h after 12 h in YPD, as compared with the 1.5–2 h for the isogenic wild-type control strain (data not shown). Western blot analysis revealed a marked reduction of L35 in the GAL::RPL35 cells that coincided with the decrease in the growth rate in YPD medium. Interestingly, L1 is also reduced but to a lesser extent and S8 levels remained practically unaffected during the time-course of the shift (Figure 2B).Figure 2.


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 results in a deficit in 60S r-subunits. (A) Growth comparison of the strains BY4741 (Wild-type) and RBY175 (GAL::RPL35). The cells were grown in YPGal and streaked on YPGal (Gal) and YPD (Glc) plates, which were incubated at 30°C for 3 days. (B) The above strains were grown in YPGal at 30°C and shifted to YPD. Cells were harvested at the indicated times after the shift and cell extracts were prepared. Equivalent amounts of cell extracts were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8. (C) 10 A260 of cell extract from the GAL::RPL35 strain grown in YPGal (Gal) or shifted for 6 h (6 h Glc) or 12 h (12 h Glc) to YPD were subjected to polysome analysis in 7–50% sucrose gradients. 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 - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926614&req=5

Figure 2: Depletion of L35 results in a deficit in 60S r-subunits. (A) Growth comparison of the strains BY4741 (Wild-type) and RBY175 (GAL::RPL35). The cells were grown in YPGal and streaked on YPGal (Gal) and YPD (Glc) plates, which were incubated at 30°C for 3 days. (B) The above strains were grown in YPGal at 30°C and shifted to YPD. Cells were harvested at the indicated times after the shift and cell extracts were prepared. Equivalent amounts of cell extracts were subjected to western blot analysis with antibodies against the r-proteins L35, L1 and S8. (C) 10 A260 of cell extract from the GAL::RPL35 strain grown in YPGal (Gal) or shifted for 6 h (6 h Glc) or 12 h (12 h Glc) to YPD were subjected to polysome analysis in 7–50% sucrose gradients. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows.
Mentions: To study the consequences of the L35 depletion we generated a conditional GAL::RPL35 strain (see ‘Materials and Methods’ section). Growth of this strain was identical to that of the isogenic wild-type strain on YPGal plates, showing that the production of N-terminally HA-tagged L35A derived from the GAL-RPL35A construct ensures wild-type growth (Figure 2A). As expected, only very residual growth of this strain was observed on YPD plates (Figure 2A). After shifting a mid-logarithmic culture of GAL::RPL35 from liquid YPGal medium to YPD medium, the growth rate rapidly decrease to a doubling time of >10 h after 12 h in YPD, as compared with the 1.5–2 h for the isogenic wild-type control strain (data not shown). Western blot analysis revealed a marked reduction of L35 in the GAL::RPL35 cells that coincided with the decrease in the growth rate in YPD medium. Interestingly, L1 is also reduced but to a lesser extent and S8 levels remained practically unaffected during the time-course of the shift (Figure 2B).Figure 2.

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