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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 impairs 27S pre-rRNA processing. (A) Wild-type control strain BY4741 (Wild-type) and strain RBY175 (GAL::RPL35) were transformed with YCplac33 and then grown at 30°C in SGal-Ura and shifted for 8 h to SD-Ura. Cells were pulse-labelled with [5,6-3H]uracil for 2 min followed by a chase with a large excess of unlabelled uracil for the times indicated. Total RNA was extracted and 20 000 cpm was loaded and separated on (A) a 1.2% agarose-6% formaldehyde gel or (B) a 7% polyacrylamide-8M urea, transferred to nylon membranes and visualized by fluorography. The positions of the different pre-rRNAs and mature rRNA are indicated.
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Figure 3: Depletion of L35 impairs 27S pre-rRNA processing. (A) Wild-type control strain BY4741 (Wild-type) and strain RBY175 (GAL::RPL35) were transformed with YCplac33 and then grown at 30°C in SGal-Ura and shifted for 8 h to SD-Ura. Cells were pulse-labelled with [5,6-3H]uracil for 2 min followed by a chase with a large excess of unlabelled uracil for the times indicated. Total RNA was extracted and 20 000 cpm was loaded and separated on (A) a 1.2% agarose-6% formaldehyde gel or (B) a 7% polyacrylamide-8M urea, transferred to nylon membranes and visualized by fluorography. The positions of the different pre-rRNAs and mature rRNA are indicated.

Mentions: To determine whether L35 is required for pre-rRNA processing, we analysed the effects of L35 depletion on the kinetics of rRNA production by [5,6-3H]-uracil pulse-chase labelling experiments. Both the wild-type and GAL::RPL35 strains were first transformed with an empty YCplac33 plasmid (CEN, URA3, see ‘Materials and Methods’ section) to make them prototrophic for uracil. Then, they were pre-grown in liquid SGal-Ura medium and finally shifted to liquid SD-Ura medium for 8 h. At this time point, the GAL::RPL35 strain was doubling every ca. 8–9 h compared with ca. 2 h for the wild-type strain. The cells were pulse-labelled for 2 min, then chased for 5, 15, 30 and 60 min with a large excess of non-radioactive uracil. Total RNA was extracted from each sample and analysed by agarose and acrylamide gel electrophoresis, followed by transfer to nylon membranes and fluorography. In wild-type cells, the 35S precursor was converted rapidly into 32S and then into 27S and 20S species, which were further processed into mature 25S and 18S rRNA, respectively. After 15 min of chase, almost all label was in the mature rRNAs. In contrast, in the L35-depleted strain, synthesis of 25S and 5.8S rRNAs was inhibited relative to synthesis of 18S and 5S rRNAs (Figure 3A and B). Processing of 35S pre-rRNA was delayed since the 35S pre-rRNA could be detected after the pulse and the early chase time points. Consistently, traces of the aberrant 23S species appeared (Figure 3A). Processing of 27SB pre-rRNAs was also slowed since these pre-rRNAs persisted even after the 60 min of chase; as a consequence no labelled 25S and 5.8S rRNA were detected (Figure 3A and B). Pulse-chase analyses were also performed with the Δrpl35A and Δrpl35B strains. These revealed that the Δrpl35A mutation led to similar, albeit milder, effects as those observed upon L35 depletion, while the Δrpl35B mutant only showed a slight delay of processing of both 35S and 27SB pre-rRNAs (data not shown). Thus, the deficit in 60S r-subunits upon mutation in or depletion of L35 is a consequence of impaired 27SB pre-rRNA processing leading to a reduced synthesis of both the mature 25S and 5.8S rRNAs.Figure 3.


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 impairs 27S pre-rRNA processing. (A) Wild-type control strain BY4741 (Wild-type) and strain RBY175 (GAL::RPL35) were transformed with YCplac33 and then grown at 30°C in SGal-Ura and shifted for 8 h to SD-Ura. Cells were pulse-labelled with [5,6-3H]uracil for 2 min followed by a chase with a large excess of unlabelled uracil for the times indicated. Total RNA was extracted and 20 000 cpm was loaded and separated on (A) a 1.2% agarose-6% formaldehyde gel or (B) a 7% polyacrylamide-8M urea, transferred to nylon membranes and visualized by fluorography. The positions of the different pre-rRNAs and mature rRNA are indicated.
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Related In: Results  -  Collection

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Figure 3: Depletion of L35 impairs 27S pre-rRNA processing. (A) Wild-type control strain BY4741 (Wild-type) and strain RBY175 (GAL::RPL35) were transformed with YCplac33 and then grown at 30°C in SGal-Ura and shifted for 8 h to SD-Ura. Cells were pulse-labelled with [5,6-3H]uracil for 2 min followed by a chase with a large excess of unlabelled uracil for the times indicated. Total RNA was extracted and 20 000 cpm was loaded and separated on (A) a 1.2% agarose-6% formaldehyde gel or (B) a 7% polyacrylamide-8M urea, transferred to nylon membranes and visualized by fluorography. The positions of the different pre-rRNAs and mature rRNA are indicated.
Mentions: To determine whether L35 is required for pre-rRNA processing, we analysed the effects of L35 depletion on the kinetics of rRNA production by [5,6-3H]-uracil pulse-chase labelling experiments. Both the wild-type and GAL::RPL35 strains were first transformed with an empty YCplac33 plasmid (CEN, URA3, see ‘Materials and Methods’ section) to make them prototrophic for uracil. Then, they were pre-grown in liquid SGal-Ura medium and finally shifted to liquid SD-Ura medium for 8 h. At this time point, the GAL::RPL35 strain was doubling every ca. 8–9 h compared with ca. 2 h for the wild-type strain. The cells were pulse-labelled for 2 min, then chased for 5, 15, 30 and 60 min with a large excess of non-radioactive uracil. Total RNA was extracted from each sample and analysed by agarose and acrylamide gel electrophoresis, followed by transfer to nylon membranes and fluorography. In wild-type cells, the 35S precursor was converted rapidly into 32S and then into 27S and 20S species, which were further processed into mature 25S and 18S rRNA, respectively. After 15 min of chase, almost all label was in the mature rRNAs. In contrast, in the L35-depleted strain, synthesis of 25S and 5.8S rRNAs was inhibited relative to synthesis of 18S and 5S rRNAs (Figure 3A and B). Processing of 35S pre-rRNA was delayed since the 35S pre-rRNA could be detected after the pulse and the early chase time points. Consistently, traces of the aberrant 23S species appeared (Figure 3A). Processing of 27SB pre-rRNAs was also slowed since these pre-rRNAs persisted even after the 60 min of chase; as a consequence no labelled 25S and 5.8S rRNA were detected (Figure 3A and B). Pulse-chase analyses were also performed with the Δrpl35A and Δrpl35B strains. These revealed that the Δrpl35A mutation led to similar, albeit milder, effects as those observed upon L35 depletion, while the Δrpl35B mutant only showed a slight delay of processing of both 35S and 27SB pre-rRNAs (data not shown). Thus, the deficit in 60S r-subunits upon mutation in or depletion of L35 is a consequence of impaired 27SB pre-rRNA processing leading to a reduced synthesis of both the mature 25S and 5.8S rRNAs.Figure 3.

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