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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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L35B associates with all pre-60S ribosomal particles. L35B-eGFP was affinity purified from total cellular extracts of Δrpl35B cells expressing L35B-eGFP with GFP-Trap®_A beads as indicated in ‘Materials and Methods’ section. Wild-type cells, which express untagged L35B, serves as a negative control. RNA was extracted from the pellets obtained after purification (lanes IP) or from an amount of total extract corresponding to 1/100 of that used for purification (lanes T) and subjected to northern analysis of pre-rRNAs and mature rRNAs. Probes, between parentheses, are described in Figure S1A. Signal intensity was measured by phosphorimager scanning; values (below each IP lane) refer to as the percentage of each RNA recovered after purification.
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Figure 7: L35B associates with all pre-60S ribosomal particles. L35B-eGFP was affinity purified from total cellular extracts of Δrpl35B cells expressing L35B-eGFP with GFP-Trap®_A beads as indicated in ‘Materials and Methods’ section. Wild-type cells, which express untagged L35B, serves as a negative control. RNA was extracted from the pellets obtained after purification (lanes IP) or from an amount of total extract corresponding to 1/100 of that used for purification (lanes T) and subjected to northern analysis of pre-rRNAs and mature rRNAs. Probes, between parentheses, are described in Figure S1A. Signal intensity was measured by phosphorimager scanning; values (below each IP lane) refer to as the percentage of each RNA recovered after purification.

Mentions: To address the timing of the nucle(ol)ar L35 assembly, we affinity purified L35B-eGFP containing complexes with GFP-Trap beads (see ‘Materials and Methods’ section) and analysed which pre-rRNA species were present in these complexes by northern blot hybridization. As shown in Figure 7, and as expected for a 60S r-protein, there was a significant co-purification of mature 25S, 5.8S and 5S rRNAs with L35B-eGFP. We believe that the apparent lack of stochiometry observed for the purification efficiency of 25S versus 5.8S and 5S rRNAs is due to the use of different gel matrices for their resolution. Moreover, mature 18S rRNA was also efficiently co-purified with L35B-eGFP. This must reflect the common unspecific association of 40S with 60S r-subunits in 80S couples in Mg2+ containing buffers or ribosomes engaged in translation. Interestingly, purification of L35B-eGFP yielded all precursors (27S and 7S pre-rRNAs) of 25S and 5.8S (Figure 7). Significantly, quantification analysis indicated that all these pre-rRNAs are enriched to similar extents in the L35B-eGFP purified samples. In clear contrast, only background levels were detected for 35S and 20S pre-rRNAs. All these results are specific since no RNAs were detected upon affinity purification from extracts of the untagged strain (Figure 7).Figure 7.


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

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

L35B associates with all pre-60S ribosomal particles. L35B-eGFP was affinity purified from total cellular extracts of Δrpl35B cells expressing L35B-eGFP with GFP-Trap®_A beads as indicated in ‘Materials and Methods’ section. Wild-type cells, which express untagged L35B, serves as a negative control. RNA was extracted from the pellets obtained after purification (lanes IP) or from an amount of total extract corresponding to 1/100 of that used for purification (lanes T) and subjected to northern analysis of pre-rRNAs and mature rRNAs. Probes, between parentheses, are described in Figure S1A. Signal intensity was measured by phosphorimager scanning; values (below each IP lane) refer to as the percentage of each RNA recovered after purification.
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Related In: Results  -  Collection

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Figure 7: L35B associates with all pre-60S ribosomal particles. L35B-eGFP was affinity purified from total cellular extracts of Δrpl35B cells expressing L35B-eGFP with GFP-Trap®_A beads as indicated in ‘Materials and Methods’ section. Wild-type cells, which express untagged L35B, serves as a negative control. RNA was extracted from the pellets obtained after purification (lanes IP) or from an amount of total extract corresponding to 1/100 of that used for purification (lanes T) and subjected to northern analysis of pre-rRNAs and mature rRNAs. Probes, between parentheses, are described in Figure S1A. Signal intensity was measured by phosphorimager scanning; values (below each IP lane) refer to as the percentage of each RNA recovered after purification.
Mentions: To address the timing of the nucle(ol)ar L35 assembly, we affinity purified L35B-eGFP containing complexes with GFP-Trap beads (see ‘Materials and Methods’ section) and analysed which pre-rRNA species were present in these complexes by northern blot hybridization. As shown in Figure 7, and as expected for a 60S r-protein, there was a significant co-purification of mature 25S, 5.8S and 5S rRNAs with L35B-eGFP. We believe that the apparent lack of stochiometry observed for the purification efficiency of 25S versus 5.8S and 5S rRNAs is due to the use of different gel matrices for their resolution. Moreover, mature 18S rRNA was also efficiently co-purified with L35B-eGFP. This must reflect the common unspecific association of 40S with 60S r-subunits in 80S couples in Mg2+ containing buffers or ribosomes engaged in translation. Interestingly, purification of L35B-eGFP yielded all precursors (27S and 7S pre-rRNAs) of 25S and 5.8S (Figure 7). Significantly, quantification analysis indicated that all these pre-rRNAs are enriched to similar extents in the L35B-eGFP purified samples. In clear contrast, only background levels were detected for 35S and 20S pre-rRNAs. All these results are specific since no RNAs were detected upon affinity purification from extracts of the untagged strain (Figure 7).Figure 7.

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