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Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones.

Pausch P, Singh U, Ahmed YL, Pillet B, Murat G, Altegoer F, Stier G, Thoms M, Hurt E, Sinning I, Bange G, Kressler D - Nat Commun (2015)

Bottom Line: Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge.Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site.Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

View Article: PubMed Central - PubMed

Affiliation: LOEWE Center for Synthetic Microbiology (SYNMIKRO) and Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Straße, Marburg D-35043, Germany.

ABSTRACT
Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

No MeSH data available.


Related in: MedlinePlus

Chaperones are recruited to nascent ribosomal proteins.(a) The chaperone proteins were affinity purified (IgG-Sepharose pull-down) from extracts of cycloheximide-treated cells and the associated RNA was isolated from the TEV eluates. Each of the four chaperone purifications (NTAP-Rrb1, Syo1-FTpA, Sqt1-TAP, and Yar1-TAP) was assessed for their content of the four ribosomal protein (RP) mRNAs (RPL3, RPL5, RPL10 and RPS3) by real-time qRT–PCR. The data from one representative experiment are expressed as the relative enrichment of the specifically co-purified RP mRNA in each of the four chaperone purifications (see Methods section for details). For each cDNA, real-time qPCRs were performed in triplicates. A highly reproducible data set was obtained in an independent series of chaperone purifications. (b) The N-terminal residues of Rpl10 are sufficient to target Sqt1 to the nascent Rpl10(1–20)-yEGFP fusion protein. Sqt1-TAP, either expressed from the genomic locus (left panel) or from a multicopy plasmid (right panel) was affinity purified (IgG-Sepharose pull-down) from extracts of cells where expression of either the yEGFP (GFP) control protein or the Rpl10(1–20)-yEGFP [L10(1–20)-GFP] fusion protein has been induced from the CUP1 promoter for 10 min with 500 μM copper sulfate. The Sqt1-TAP purifications were assessed for their content of the RPL3, RPL10 and yEGFP (GFP) mRNAs by real-time qRT–PCR. The data from one representative experiment are expressed as the fold enrichment relative to the RPL3 mRNA. For each cDNA, real-time qPCRs were performed in triplicates. Note that the bar graphs of the left and right panel of this figure are at a different scale.
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f7: Chaperones are recruited to nascent ribosomal proteins.(a) The chaperone proteins were affinity purified (IgG-Sepharose pull-down) from extracts of cycloheximide-treated cells and the associated RNA was isolated from the TEV eluates. Each of the four chaperone purifications (NTAP-Rrb1, Syo1-FTpA, Sqt1-TAP, and Yar1-TAP) was assessed for their content of the four ribosomal protein (RP) mRNAs (RPL3, RPL5, RPL10 and RPS3) by real-time qRT–PCR. The data from one representative experiment are expressed as the relative enrichment of the specifically co-purified RP mRNA in each of the four chaperone purifications (see Methods section for details). For each cDNA, real-time qPCRs were performed in triplicates. A highly reproducible data set was obtained in an independent series of chaperone purifications. (b) The N-terminal residues of Rpl10 are sufficient to target Sqt1 to the nascent Rpl10(1–20)-yEGFP fusion protein. Sqt1-TAP, either expressed from the genomic locus (left panel) or from a multicopy plasmid (right panel) was affinity purified (IgG-Sepharose pull-down) from extracts of cells where expression of either the yEGFP (GFP) control protein or the Rpl10(1–20)-yEGFP [L10(1–20)-GFP] fusion protein has been induced from the CUP1 promoter for 10 min with 500 μM copper sulfate. The Sqt1-TAP purifications were assessed for their content of the RPL3, RPL10 and yEGFP (GFP) mRNAs by real-time qRT–PCR. The data from one representative experiment are expressed as the fold enrichment relative to the RPL3 mRNA. For each cDNA, real-time qPCRs were performed in triplicates. Note that the bar graphs of the left and right panel of this figure are at a different scale.

Mentions: Given that each of the distinct chaperones interacts with the N-terminal residues of the respective ribosomal protein client (Rpl3, amino acids 1–15; Rpl5, amino acids 2–20; Rpl10, amino acids 2–13; and Rps3, amino acids 14–29; Figs 1 and 2; refs 11, 34), we sought to explore the intuitive possibility that the chaperones are already recruited to nascent ribosomal proteins as these are translated from their mRNA. To this end, we purified each of the four different chaperones by immunoglobulin G (IgG)-sepharose pull-down from cell extracts of yeast cells that were, before harvesting, treated with cycloheximide, which blocks translation elongation, and thus preserves the translating ribosomes on the mRNAs (see Methods section). The purified chaperone and any associated molecules were then released from the IgG-sepharose beads by tobacco etch virus (TEV) protease cleavage and the RNA was subsequently isolated. To unambiguously reveal the specific presence of the corresponding ribosomal protein encoding mRNA, each of the four chaperone purifications was assessed for their content of the four ribosomal protein mRNAs (RPL3, RPL5, RPL10 and RPS3) by real-time quantitative reverse transcription PCR (real-time qRT–PCR). This analysis clearly showed that each of the four chaperones was specifically co-purifying the mRNA encoding its ribosomal protein client (Fig. 7a). While we observed a roughly 100-fold enrichment of the specific ribosomal protein mRNA in the case of Rrb1, Syo1 and Yar1, the enrichment of the RPL10 mRNA in the Sqt1 purification was clearly evident, albeit less pronounced (∼25-fold). Moreover, we could also detect co-purification of the specific ribosomal protein encoding mRNAs when cycloheximide was omitted (Supplementary Fig. 10a); thus, ruling out that the observed co-purification was simply due to an association with the nascent ribosomal proteins on elongation-blocked ribosomes during the 5-min period of the cycloheximide treatment. Finally, we expressed L10-N (amino acids 1–20) and L3-N (amino acids 1–23) yEGFP fusion constructs for 10 min from a copper-inducible promoter, followed by cycloheximide treatment, and assessed the content of the yEGFP mRNA in the Sqt1-TAP and NTAP-Rrb1 purification, respectively. While the co-translational association with the specific ribosomal protein encoding mRNA was reduced, the L10-N-yEGFP and L3-N-yEGFP were clearly enriched compared with the yEGFP control mRNA (Fig. 7b and Supplementary Fig. 10b). Notably, the selective co-purification of the L10-N-yEGFP mRNA was more evident when Sqt1-TAP was overexpressed from a multicopy plasmid (Fig. 7b), suggesting that genomically expressed Sqt1-TAP was efficiently titrated by the newly synthesized L10-N-yEGFP fusion protein (see also Supplementary Fig. 3b). We conclude that each of the four chaperones has the capacity to recognize its specific ribosomal protein substrate in a co-translational manner. The high affinity of the interaction between Sqt1 and L10-N (Kd ∼20 nM) suggests that chaperone recruitment to nascent ribosomal proteins may represent the default setting of this process in vivo.


Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones.

Pausch P, Singh U, Ahmed YL, Pillet B, Murat G, Altegoer F, Stier G, Thoms M, Hurt E, Sinning I, Bange G, Kressler D - Nat Commun (2015)

Chaperones are recruited to nascent ribosomal proteins.(a) The chaperone proteins were affinity purified (IgG-Sepharose pull-down) from extracts of cycloheximide-treated cells and the associated RNA was isolated from the TEV eluates. Each of the four chaperone purifications (NTAP-Rrb1, Syo1-FTpA, Sqt1-TAP, and Yar1-TAP) was assessed for their content of the four ribosomal protein (RP) mRNAs (RPL3, RPL5, RPL10 and RPS3) by real-time qRT–PCR. The data from one representative experiment are expressed as the relative enrichment of the specifically co-purified RP mRNA in each of the four chaperone purifications (see Methods section for details). For each cDNA, real-time qPCRs were performed in triplicates. A highly reproducible data set was obtained in an independent series of chaperone purifications. (b) The N-terminal residues of Rpl10 are sufficient to target Sqt1 to the nascent Rpl10(1–20)-yEGFP fusion protein. Sqt1-TAP, either expressed from the genomic locus (left panel) or from a multicopy plasmid (right panel) was affinity purified (IgG-Sepharose pull-down) from extracts of cells where expression of either the yEGFP (GFP) control protein or the Rpl10(1–20)-yEGFP [L10(1–20)-GFP] fusion protein has been induced from the CUP1 promoter for 10 min with 500 μM copper sulfate. The Sqt1-TAP purifications were assessed for their content of the RPL3, RPL10 and yEGFP (GFP) mRNAs by real-time qRT–PCR. The data from one representative experiment are expressed as the fold enrichment relative to the RPL3 mRNA. For each cDNA, real-time qPCRs were performed in triplicates. Note that the bar graphs of the left and right panel of this figure are at a different scale.
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Related In: Results  -  Collection

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f7: Chaperones are recruited to nascent ribosomal proteins.(a) The chaperone proteins were affinity purified (IgG-Sepharose pull-down) from extracts of cycloheximide-treated cells and the associated RNA was isolated from the TEV eluates. Each of the four chaperone purifications (NTAP-Rrb1, Syo1-FTpA, Sqt1-TAP, and Yar1-TAP) was assessed for their content of the four ribosomal protein (RP) mRNAs (RPL3, RPL5, RPL10 and RPS3) by real-time qRT–PCR. The data from one representative experiment are expressed as the relative enrichment of the specifically co-purified RP mRNA in each of the four chaperone purifications (see Methods section for details). For each cDNA, real-time qPCRs were performed in triplicates. A highly reproducible data set was obtained in an independent series of chaperone purifications. (b) The N-terminal residues of Rpl10 are sufficient to target Sqt1 to the nascent Rpl10(1–20)-yEGFP fusion protein. Sqt1-TAP, either expressed from the genomic locus (left panel) or from a multicopy plasmid (right panel) was affinity purified (IgG-Sepharose pull-down) from extracts of cells where expression of either the yEGFP (GFP) control protein or the Rpl10(1–20)-yEGFP [L10(1–20)-GFP] fusion protein has been induced from the CUP1 promoter for 10 min with 500 μM copper sulfate. The Sqt1-TAP purifications were assessed for their content of the RPL3, RPL10 and yEGFP (GFP) mRNAs by real-time qRT–PCR. The data from one representative experiment are expressed as the fold enrichment relative to the RPL3 mRNA. For each cDNA, real-time qPCRs were performed in triplicates. Note that the bar graphs of the left and right panel of this figure are at a different scale.
Mentions: Given that each of the distinct chaperones interacts with the N-terminal residues of the respective ribosomal protein client (Rpl3, amino acids 1–15; Rpl5, amino acids 2–20; Rpl10, amino acids 2–13; and Rps3, amino acids 14–29; Figs 1 and 2; refs 11, 34), we sought to explore the intuitive possibility that the chaperones are already recruited to nascent ribosomal proteins as these are translated from their mRNA. To this end, we purified each of the four different chaperones by immunoglobulin G (IgG)-sepharose pull-down from cell extracts of yeast cells that were, before harvesting, treated with cycloheximide, which blocks translation elongation, and thus preserves the translating ribosomes on the mRNAs (see Methods section). The purified chaperone and any associated molecules were then released from the IgG-sepharose beads by tobacco etch virus (TEV) protease cleavage and the RNA was subsequently isolated. To unambiguously reveal the specific presence of the corresponding ribosomal protein encoding mRNA, each of the four chaperone purifications was assessed for their content of the four ribosomal protein mRNAs (RPL3, RPL5, RPL10 and RPS3) by real-time quantitative reverse transcription PCR (real-time qRT–PCR). This analysis clearly showed that each of the four chaperones was specifically co-purifying the mRNA encoding its ribosomal protein client (Fig. 7a). While we observed a roughly 100-fold enrichment of the specific ribosomal protein mRNA in the case of Rrb1, Syo1 and Yar1, the enrichment of the RPL10 mRNA in the Sqt1 purification was clearly evident, albeit less pronounced (∼25-fold). Moreover, we could also detect co-purification of the specific ribosomal protein encoding mRNAs when cycloheximide was omitted (Supplementary Fig. 10a); thus, ruling out that the observed co-purification was simply due to an association with the nascent ribosomal proteins on elongation-blocked ribosomes during the 5-min period of the cycloheximide treatment. Finally, we expressed L10-N (amino acids 1–20) and L3-N (amino acids 1–23) yEGFP fusion constructs for 10 min from a copper-inducible promoter, followed by cycloheximide treatment, and assessed the content of the yEGFP mRNA in the Sqt1-TAP and NTAP-Rrb1 purification, respectively. While the co-translational association with the specific ribosomal protein encoding mRNA was reduced, the L10-N-yEGFP and L3-N-yEGFP were clearly enriched compared with the yEGFP control mRNA (Fig. 7b and Supplementary Fig. 10b). Notably, the selective co-purification of the L10-N-yEGFP mRNA was more evident when Sqt1-TAP was overexpressed from a multicopy plasmid (Fig. 7b), suggesting that genomically expressed Sqt1-TAP was efficiently titrated by the newly synthesized L10-N-yEGFP fusion protein (see also Supplementary Fig. 3b). We conclude that each of the four chaperones has the capacity to recognize its specific ribosomal protein substrate in a co-translational manner. The high affinity of the interaction between Sqt1 and L10-N (Kd ∼20 nM) suggests that chaperone recruitment to nascent ribosomal proteins may represent the default setting of this process in vivo.

Bottom Line: Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge.Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site.Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

View Article: PubMed Central - PubMed

Affiliation: LOEWE Center for Synthetic Microbiology (SYNMIKRO) and Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Straße, Marburg D-35043, Germany.

ABSTRACT
Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

No MeSH data available.


Related in: MedlinePlus