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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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Readouts, phenotypes, and targets used for the systematic analysis of human ribosome biogenesis by RNAi.(A) Fluorescent images representing the different phenotypic categories used for detecting defects in 40S biogenesis (Rps2-YFP, Enp1 IF) and 60S biogenesis (Rpl29-GFP) after 3 d of RNAi in HeLa cells with the indicated siRNAs (10 nM). (B) Schematic representation of the siRNA target distribution to different protein categories. The list of all targets is given in Table S1.
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pbio-1000522-g001: Readouts, phenotypes, and targets used for the systematic analysis of human ribosome biogenesis by RNAi.(A) Fluorescent images representing the different phenotypic categories used for detecting defects in 40S biogenesis (Rps2-YFP, Enp1 IF) and 60S biogenesis (Rpl29-GFP) after 3 d of RNAi in HeLa cells with the indicated siRNAs (10 nM). (B) Schematic representation of the siRNA target distribution to different protein categories. The list of all targets is given in Table S1.

Mentions: For the 40S subunit, we applied two different readouts [45], namely an inducible, fluorescent ribosomal reporter protein, Rps2-YFP, and immunofluorescence (IF) analysis of the trans-acting factor Enp1(BYSL) [24]. Tetracycline-induced expression of Rps2-YFP allows for selective visualization of newly synthesized 40S subunits [45]. After 14 h of induction, Rps2-YFP-containing 40S have largely completed maturation, giving rise to a prominent cytoplasmic signal of the reporter (Figure 1A, control RNAi). RNAi against Crm1, the RanGTP-dependent nuclear export receptor required for both pre-40S and pre-60S export [34],[35], resulted in a nucleoplasmic accumulation of Rps2-YFP, reflecting the dependence of pre-40S export on Crm1. In contrast, inhibition of nucleolar steps of ribosome biogenesis, as exemplified by depletion of the box C/D snoRNPs-associated methyltransferase fibrillarin (Fbl) [10], led to accumulation of Rps2-YFP in nucleoli (Figure 1A). Thus, two phenotypes can be distinguished using the Rps2-YFP reporter, reflecting early (nucleolar) and late (nucleoplasmic) defects in nuclear pre-40S maturation.


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)

Readouts, phenotypes, and targets used for the systematic analysis of human ribosome biogenesis by RNAi.(A) Fluorescent images representing the different phenotypic categories used for detecting defects in 40S biogenesis (Rps2-YFP, Enp1 IF) and 60S biogenesis (Rpl29-GFP) after 3 d of RNAi in HeLa cells with the indicated siRNAs (10 nM). (B) Schematic representation of the siRNA target distribution to different protein categories. The list of all targets is given in Table S1.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1000522-g001: Readouts, phenotypes, and targets used for the systematic analysis of human ribosome biogenesis by RNAi.(A) Fluorescent images representing the different phenotypic categories used for detecting defects in 40S biogenesis (Rps2-YFP, Enp1 IF) and 60S biogenesis (Rpl29-GFP) after 3 d of RNAi in HeLa cells with the indicated siRNAs (10 nM). (B) Schematic representation of the siRNA target distribution to different protein categories. The list of all targets is given in Table S1.
Mentions: For the 40S subunit, we applied two different readouts [45], namely an inducible, fluorescent ribosomal reporter protein, Rps2-YFP, and immunofluorescence (IF) analysis of the trans-acting factor Enp1(BYSL) [24]. Tetracycline-induced expression of Rps2-YFP allows for selective visualization of newly synthesized 40S subunits [45]. After 14 h of induction, Rps2-YFP-containing 40S have largely completed maturation, giving rise to a prominent cytoplasmic signal of the reporter (Figure 1A, control RNAi). RNAi against Crm1, the RanGTP-dependent nuclear export receptor required for both pre-40S and pre-60S export [34],[35], resulted in a nucleoplasmic accumulation of Rps2-YFP, reflecting the dependence of pre-40S export on Crm1. In contrast, inhibition of nucleolar steps of ribosome biogenesis, as exemplified by depletion of the box C/D snoRNPs-associated methyltransferase fibrillarin (Fbl) [10], led to accumulation of Rps2-YFP in nucleoli (Figure 1A). Thus, two phenotypes can be distinguished using the Rps2-YFP reporter, reflecting early (nucleolar) and late (nucleoplasmic) defects in nuclear pre-40S maturation.

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