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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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Ranked high confidence hit list.Compilation of high confidence hits for the three different readouts, Rps2-YFP, Enp1 IF, and Rpl29-GFP. Rank calculation was as follows: [(hit rate siRNA-X1)−(hit rate negative control)]/[(hit rate positive control)−(hit rate negative control)], where (hit rate negative control) and (hit rate positive control) equals the average hit rate of the two negative or positive controls with the highest hit rate from the same 96-well plate as siRNA-X1. For each hit, the average of the two highest ranked siRNAs is shown. For the Rps2-YFP readout, overall nuclear accumulation (nucleoplasmic and nucleolar) of the reporter was used for hit definition. Note that for “cytoplasmic” Enp1, absolute hit rates are given (ranking could not be performed due to a lack of an appropriate positive control for cytoplasmic accumulation). We set a cutoff to >0.2 for Rps2-YFP, >0.3 for Enp1 IF, and >0.25 for Rpl29-GFP (see Text S1).
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pbio-1000522-g002: Ranked high confidence hit list.Compilation of high confidence hits for the three different readouts, Rps2-YFP, Enp1 IF, and Rpl29-GFP. Rank calculation was as follows: [(hit rate siRNA-X1)−(hit rate negative control)]/[(hit rate positive control)−(hit rate negative control)], where (hit rate negative control) and (hit rate positive control) equals the average hit rate of the two negative or positive controls with the highest hit rate from the same 96-well plate as siRNA-X1. For each hit, the average of the two highest ranked siRNAs is shown. For the Rps2-YFP readout, overall nuclear accumulation (nucleoplasmic and nucleolar) of the reporter was used for hit definition. Note that for “cytoplasmic” Enp1, absolute hit rates are given (ranking could not be performed due to a lack of an appropriate positive control for cytoplasmic accumulation). We set a cutoff to >0.2 for Rps2-YFP, >0.3 for Enp1 IF, and >0.25 for Rpl29-GFP (see Text S1).

Mentions: Based on the hit rates, we further selected all targets, which were represented by at least two siRNAs resulting in more than twice the hit rate of the negative controls. Subsequently, these targets were ranked relative to both the negative and positive controls of the respective 96-well plate (for details, see Text S1). Next, we defined a cutoff (see Text S1), generating a high confidence hit list of targets that are involved in ribosome biogenesis based on the phenotypic classification of the different readouts (Figure 2). Lists of all numerical data are given in Tables S2, S3, and S4. All primary images are stored in a newly developed database, accessible via a web browser interface at http://hcpb.ethz.ch. We note that this database has all the required features to provide a framework for integrating future high content RNAi screening results.


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)

Ranked high confidence hit list.Compilation of high confidence hits for the three different readouts, Rps2-YFP, Enp1 IF, and Rpl29-GFP. Rank calculation was as follows: [(hit rate siRNA-X1)−(hit rate negative control)]/[(hit rate positive control)−(hit rate negative control)], where (hit rate negative control) and (hit rate positive control) equals the average hit rate of the two negative or positive controls with the highest hit rate from the same 96-well plate as siRNA-X1. For each hit, the average of the two highest ranked siRNAs is shown. For the Rps2-YFP readout, overall nuclear accumulation (nucleoplasmic and nucleolar) of the reporter was used for hit definition. Note that for “cytoplasmic” Enp1, absolute hit rates are given (ranking could not be performed due to a lack of an appropriate positive control for cytoplasmic accumulation). We set a cutoff to >0.2 for Rps2-YFP, >0.3 for Enp1 IF, and >0.25 for Rpl29-GFP (see Text S1).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2964341&req=5

pbio-1000522-g002: Ranked high confidence hit list.Compilation of high confidence hits for the three different readouts, Rps2-YFP, Enp1 IF, and Rpl29-GFP. Rank calculation was as follows: [(hit rate siRNA-X1)−(hit rate negative control)]/[(hit rate positive control)−(hit rate negative control)], where (hit rate negative control) and (hit rate positive control) equals the average hit rate of the two negative or positive controls with the highest hit rate from the same 96-well plate as siRNA-X1. For each hit, the average of the two highest ranked siRNAs is shown. For the Rps2-YFP readout, overall nuclear accumulation (nucleoplasmic and nucleolar) of the reporter was used for hit definition. Note that for “cytoplasmic” Enp1, absolute hit rates are given (ranking could not be performed due to a lack of an appropriate positive control for cytoplasmic accumulation). We set a cutoff to >0.2 for Rps2-YFP, >0.3 for Enp1 IF, and >0.25 for Rpl29-GFP (see Text S1).
Mentions: Based on the hit rates, we further selected all targets, which were represented by at least two siRNAs resulting in more than twice the hit rate of the negative controls. Subsequently, these targets were ranked relative to both the negative and positive controls of the respective 96-well plate (for details, see Text S1). Next, we defined a cutoff (see Text S1), generating a high confidence hit list of targets that are involved in ribosome biogenesis based on the phenotypic classification of the different readouts (Figure 2). Lists of all numerical data are given in Tables S2, S3, and S4. All primary images are stored in a newly developed database, accessible via a web browser interface at http://hcpb.ethz.ch. We note that this database has all the required features to provide a framework for integrating future high content RNAi screening results.

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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