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A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.

Wild T, Horvath P, Wyler E, Widmann B, Badertscher L, Zemp I, Kozak K, Csucs G, Lund E, Kutay U - PLoS Biol. (2010)

Bottom Line: We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner.Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1.Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.

ABSTRACT
The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

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Requirement of small ribosomal proteins at different stages of 40S biogenesis.(A) Representative images from Rps2-YFP and Enp1 IF screening analysis after depletion of RPS by RNAi. Positive control: Crm1; negative controls: Allstars (Qiagen) and Rps4Y1 (Y-chromosome encoded). Rps3 depletion is shown as an example of differences detected with the Rps2-YFP (nuclear accumulation) and Enp1 IF (cytoplasmic accumulation) readouts. (B) Rio2 localization after depletion of RPS by RNAi (10 nM siRNAs, 72 h) in the absence or presence of 10 nM leptomycin B (LMB) for 2 h prior to fixation. Endogenous Rio2 was detected by immunofluorescence. Representative images are given for control, Crm1, Rps3, and Rps3A depletion. (C) List of RPS proteins that caused defects in nuclear accumulation of Rio2 upon LMB treatment when downregulated by RNAi in (B). An RPS was defined as a hit if more than 20% of cells were classified as “cytoplasmic” Rio2 after LMB treatment. The average of the two highest scoring siRNAs is given. *For RspA, only 1 siRNA gave a phenotype. (D) RPS were sorted according to the phenotypes detected in the Rps2-YFP, Enp1 IF, and Rio2 IF readouts and grouped with respect to their nuclear or cytoplasmic requirement for human 40S biogenesis. “Nuclear” classified RPS were either detected with Rps2-YFP only (red) or with both the Rps2-YFP and Enp1 IF readouts (orange). Classification of “cytoplasmic” RPS is based on Enp1 IF (yellow), Rio2 IF (+LMB) (brown), or both (green; depletion of these RPS also caused nuclear Rps2-YFP accumulation). FAU was scored as a nuclear Rps2-YFP hit and a cytoplasmic Rio2 IF (+LMB) hit (blue). RPS marked by an asterisk scored positive for one siRNA by at least 7 times the hit rate of negative controls. The yeast RPS classification into four groups (I–IV) is based on [48] and described in the text. For E. coli RPS, primary (1), secondary (2), and tertiary (3) binders of the Nomura assembly map [1],[51] are listed. The names of yeast and bacterial homologs are given in brackets. FAU and GNBL2L1 are the human ribosomal proteins Rps30 and RACK1, respectively.
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pbio-1000522-g004: Requirement of small ribosomal proteins at different stages of 40S biogenesis.(A) Representative images from Rps2-YFP and Enp1 IF screening analysis after depletion of RPS by RNAi. Positive control: Crm1; negative controls: Allstars (Qiagen) and Rps4Y1 (Y-chromosome encoded). Rps3 depletion is shown as an example of differences detected with the Rps2-YFP (nuclear accumulation) and Enp1 IF (cytoplasmic accumulation) readouts. (B) Rio2 localization after depletion of RPS by RNAi (10 nM siRNAs, 72 h) in the absence or presence of 10 nM leptomycin B (LMB) for 2 h prior to fixation. Endogenous Rio2 was detected by immunofluorescence. Representative images are given for control, Crm1, Rps3, and Rps3A depletion. (C) List of RPS proteins that caused defects in nuclear accumulation of Rio2 upon LMB treatment when downregulated by RNAi in (B). An RPS was defined as a hit if more than 20% of cells were classified as “cytoplasmic” Rio2 after LMB treatment. The average of the two highest scoring siRNAs is given. *For RspA, only 1 siRNA gave a phenotype. (D) RPS were sorted according to the phenotypes detected in the Rps2-YFP, Enp1 IF, and Rio2 IF readouts and grouped with respect to their nuclear or cytoplasmic requirement for human 40S biogenesis. “Nuclear” classified RPS were either detected with Rps2-YFP only (red) or with both the Rps2-YFP and Enp1 IF readouts (orange). Classification of “cytoplasmic” RPS is based on Enp1 IF (yellow), Rio2 IF (+LMB) (brown), or both (green; depletion of these RPS also caused nuclear Rps2-YFP accumulation). FAU was scored as a nuclear Rps2-YFP hit and a cytoplasmic Rio2 IF (+LMB) hit (blue). RPS marked by an asterisk scored positive for one siRNA by at least 7 times the hit rate of negative controls. The yeast RPS classification into four groups (I–IV) is based on [48] and described in the text. For E. coli RPS, primary (1), secondary (2), and tertiary (3) binders of the Nomura assembly map [1],[51] are listed. The names of yeast and bacterial homologs are given in brackets. FAU and GNBL2L1 are the human ribosomal proteins Rps30 and RACK1, respectively.

Mentions: The datasets relying on the Rps2-YFP and Enp1 reporters allowed us to define at which stage the absence of a specific RPS interfered with 40S biogenesis in human cells. In this analysis, 18 RPS clearly caused a nuclear 40S biogenesis defect (Figure 4A, D).


A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.

Wild T, Horvath P, Wyler E, Widmann B, Badertscher L, Zemp I, Kozak K, Csucs G, Lund E, Kutay U - PLoS Biol. (2010)

Requirement of small ribosomal proteins at different stages of 40S biogenesis.(A) Representative images from Rps2-YFP and Enp1 IF screening analysis after depletion of RPS by RNAi. Positive control: Crm1; negative controls: Allstars (Qiagen) and Rps4Y1 (Y-chromosome encoded). Rps3 depletion is shown as an example of differences detected with the Rps2-YFP (nuclear accumulation) and Enp1 IF (cytoplasmic accumulation) readouts. (B) Rio2 localization after depletion of RPS by RNAi (10 nM siRNAs, 72 h) in the absence or presence of 10 nM leptomycin B (LMB) for 2 h prior to fixation. Endogenous Rio2 was detected by immunofluorescence. Representative images are given for control, Crm1, Rps3, and Rps3A depletion. (C) List of RPS proteins that caused defects in nuclear accumulation of Rio2 upon LMB treatment when downregulated by RNAi in (B). An RPS was defined as a hit if more than 20% of cells were classified as “cytoplasmic” Rio2 after LMB treatment. The average of the two highest scoring siRNAs is given. *For RspA, only 1 siRNA gave a phenotype. (D) RPS were sorted according to the phenotypes detected in the Rps2-YFP, Enp1 IF, and Rio2 IF readouts and grouped with respect to their nuclear or cytoplasmic requirement for human 40S biogenesis. “Nuclear” classified RPS were either detected with Rps2-YFP only (red) or with both the Rps2-YFP and Enp1 IF readouts (orange). Classification of “cytoplasmic” RPS is based on Enp1 IF (yellow), Rio2 IF (+LMB) (brown), or both (green; depletion of these RPS also caused nuclear Rps2-YFP accumulation). FAU was scored as a nuclear Rps2-YFP hit and a cytoplasmic Rio2 IF (+LMB) hit (blue). RPS marked by an asterisk scored positive for one siRNA by at least 7 times the hit rate of negative controls. The yeast RPS classification into four groups (I–IV) is based on [48] and described in the text. For E. coli RPS, primary (1), secondary (2), and tertiary (3) binders of the Nomura assembly map [1],[51] are listed. The names of yeast and bacterial homologs are given in brackets. FAU and GNBL2L1 are the human ribosomal proteins Rps30 and RACK1, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2964341&req=5

pbio-1000522-g004: Requirement of small ribosomal proteins at different stages of 40S biogenesis.(A) Representative images from Rps2-YFP and Enp1 IF screening analysis after depletion of RPS by RNAi. Positive control: Crm1; negative controls: Allstars (Qiagen) and Rps4Y1 (Y-chromosome encoded). Rps3 depletion is shown as an example of differences detected with the Rps2-YFP (nuclear accumulation) and Enp1 IF (cytoplasmic accumulation) readouts. (B) Rio2 localization after depletion of RPS by RNAi (10 nM siRNAs, 72 h) in the absence or presence of 10 nM leptomycin B (LMB) for 2 h prior to fixation. Endogenous Rio2 was detected by immunofluorescence. Representative images are given for control, Crm1, Rps3, and Rps3A depletion. (C) List of RPS proteins that caused defects in nuclear accumulation of Rio2 upon LMB treatment when downregulated by RNAi in (B). An RPS was defined as a hit if more than 20% of cells were classified as “cytoplasmic” Rio2 after LMB treatment. The average of the two highest scoring siRNAs is given. *For RspA, only 1 siRNA gave a phenotype. (D) RPS were sorted according to the phenotypes detected in the Rps2-YFP, Enp1 IF, and Rio2 IF readouts and grouped with respect to their nuclear or cytoplasmic requirement for human 40S biogenesis. “Nuclear” classified RPS were either detected with Rps2-YFP only (red) or with both the Rps2-YFP and Enp1 IF readouts (orange). Classification of “cytoplasmic” RPS is based on Enp1 IF (yellow), Rio2 IF (+LMB) (brown), or both (green; depletion of these RPS also caused nuclear Rps2-YFP accumulation). FAU was scored as a nuclear Rps2-YFP hit and a cytoplasmic Rio2 IF (+LMB) hit (blue). RPS marked by an asterisk scored positive for one siRNA by at least 7 times the hit rate of negative controls. The yeast RPS classification into four groups (I–IV) is based on [48] and described in the text. For E. coli RPS, primary (1), secondary (2), and tertiary (3) binders of the Nomura assembly map [1],[51] are listed. The names of yeast and bacterial homologs are given in brackets. FAU and GNBL2L1 are the human ribosomal proteins Rps30 and RACK1, respectively.
Mentions: The datasets relying on the Rps2-YFP and Enp1 reporters allowed us to define at which stage the absence of a specific RPS interfered with 40S biogenesis in human cells. In this analysis, 18 RPS clearly caused a nuclear 40S biogenesis defect (Figure 4A, D).

Bottom Line: We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner.Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1.Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.

ABSTRACT
The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

Show MeSH
Related in: MedlinePlus