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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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Exp5 binds specifically to pre-60S particles in a RanGTP-dependent manner.(A) Schematic representation of the experimental setup. Expression of a tagged bait protein in HEK293 cells was induced by tetracycline. Cells were lysed and the bait protein was affinity-purified by help of its tandem strep/HA-tag using a two-step protocol. Next, the bait protein and associated proteins, bound to anti-HA IgG beads, were incubated with HeLa extracts with or without addition of RanQ69L-GTP. Subsequently, bound proteins were analyzed by Western blotting. (B) Exp5 binds to pre-60S particles in the presence of RanGTP. Pre-60S particles were purified from HEK293 cells bearing an inducible copy of TAP-tagged MRTO4 after 24 h of tetracycline treatment (cell lysate: input MRTO4 TAP). The purified particles were incubated with HeLa cell extract (input HeLa extract) in the absence or presence of 10 µM RanQ69L-GTP. Anti-HA IgG beads were used as a control for non-specific binding of exportins in presence of RanQ69L-GTP. (C) Binding of Exp5 is specific for pre-60S particles. Experiment as in (B) including affinity purification of pre-40S subunits by Dim2 (PNO1) TAP from HEK293 cells bearing an inducible copy of TAP-tagged Dim2. Note that ribosomal particle components are not detected in the dilute input of HEK293 lysates but are highly enriched during purification. Load of input and bound fractions as in (B).
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pbio-1000522-g006: Exp5 binds specifically to pre-60S particles in a RanGTP-dependent manner.(A) Schematic representation of the experimental setup. Expression of a tagged bait protein in HEK293 cells was induced by tetracycline. Cells were lysed and the bait protein was affinity-purified by help of its tandem strep/HA-tag using a two-step protocol. Next, the bait protein and associated proteins, bound to anti-HA IgG beads, were incubated with HeLa extracts with or without addition of RanQ69L-GTP. Subsequently, bound proteins were analyzed by Western blotting. (B) Exp5 binds to pre-60S particles in the presence of RanGTP. Pre-60S particles were purified from HEK293 cells bearing an inducible copy of TAP-tagged MRTO4 after 24 h of tetracycline treatment (cell lysate: input MRTO4 TAP). The purified particles were incubated with HeLa cell extract (input HeLa extract) in the absence or presence of 10 µM RanQ69L-GTP. Anti-HA IgG beads were used as a control for non-specific binding of exportins in presence of RanQ69L-GTP. (C) Binding of Exp5 is specific for pre-60S particles. Experiment as in (B) including affinity purification of pre-40S subunits by Dim2 (PNO1) TAP from HEK293 cells bearing an inducible copy of TAP-tagged Dim2. Note that ribosomal particle components are not detected in the dilute input of HEK293 lysates but are highly enriched during purification. Load of input and bound fractions as in (B).

Mentions: Therefore, we next addressed whether Exp5 could have a direct function in 60S export. An exportin/cargo relationship between Exp5 and pre-60S particles would predict a physical, RanGTP-regulated interaction between Exp5 and pre-60S particles [62]. To test this, we first purified a pre-60S particle from HEK293 cells by tandem affinity purification (TAP) using the TAP-tagged trans-acting factor MRTO4 (homologous to yeast Mrt4) as bait (Figure 6 and Figure S8). Protein composition analysis of this particle showed the presence of 60S trans-acting factors like the export adaptor Nmd3, C15orf15 (Rlp24, ribosomal-like protein 24), and ribosomal proteins of the large subunit (Figure 6B and Figure S8A). These particles were then incubated with HeLa cell extract in the absence or presence of RanQ69L-GTP, a Ran mutant locked in the GTP-bound state, to analyze RanGTP-dependent binding of nuclear export receptors to pre-60S. Note that the HeLa cell extract had been pre-depleted for ribosomes and tRNAs. The depletion of ribosomes ensured that the TAP-purified particles were the sole ribosomal particles present. The presence of tRNA might hamper the detection of a potential Exp5/60S interaction because tRNA binds efficiently to Exp5 [63],[64].


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)

Exp5 binds specifically to pre-60S particles in a RanGTP-dependent manner.(A) Schematic representation of the experimental setup. Expression of a tagged bait protein in HEK293 cells was induced by tetracycline. Cells were lysed and the bait protein was affinity-purified by help of its tandem strep/HA-tag using a two-step protocol. Next, the bait protein and associated proteins, bound to anti-HA IgG beads, were incubated with HeLa extracts with or without addition of RanQ69L-GTP. Subsequently, bound proteins were analyzed by Western blotting. (B) Exp5 binds to pre-60S particles in the presence of RanGTP. Pre-60S particles were purified from HEK293 cells bearing an inducible copy of TAP-tagged MRTO4 after 24 h of tetracycline treatment (cell lysate: input MRTO4 TAP). The purified particles were incubated with HeLa cell extract (input HeLa extract) in the absence or presence of 10 µM RanQ69L-GTP. Anti-HA IgG beads were used as a control for non-specific binding of exportins in presence of RanQ69L-GTP. (C) Binding of Exp5 is specific for pre-60S particles. Experiment as in (B) including affinity purification of pre-40S subunits by Dim2 (PNO1) TAP from HEK293 cells bearing an inducible copy of TAP-tagged Dim2. Note that ribosomal particle components are not detected in the dilute input of HEK293 lysates but are highly enriched during purification. Load of input and bound fractions as in (B).
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pbio-1000522-g006: Exp5 binds specifically to pre-60S particles in a RanGTP-dependent manner.(A) Schematic representation of the experimental setup. Expression of a tagged bait protein in HEK293 cells was induced by tetracycline. Cells were lysed and the bait protein was affinity-purified by help of its tandem strep/HA-tag using a two-step protocol. Next, the bait protein and associated proteins, bound to anti-HA IgG beads, were incubated with HeLa extracts with or without addition of RanQ69L-GTP. Subsequently, bound proteins were analyzed by Western blotting. (B) Exp5 binds to pre-60S particles in the presence of RanGTP. Pre-60S particles were purified from HEK293 cells bearing an inducible copy of TAP-tagged MRTO4 after 24 h of tetracycline treatment (cell lysate: input MRTO4 TAP). The purified particles were incubated with HeLa cell extract (input HeLa extract) in the absence or presence of 10 µM RanQ69L-GTP. Anti-HA IgG beads were used as a control for non-specific binding of exportins in presence of RanQ69L-GTP. (C) Binding of Exp5 is specific for pre-60S particles. Experiment as in (B) including affinity purification of pre-40S subunits by Dim2 (PNO1) TAP from HEK293 cells bearing an inducible copy of TAP-tagged Dim2. Note that ribosomal particle components are not detected in the dilute input of HEK293 lysates but are highly enriched during purification. Load of input and bound fractions as in (B).
Mentions: Therefore, we next addressed whether Exp5 could have a direct function in 60S export. An exportin/cargo relationship between Exp5 and pre-60S particles would predict a physical, RanGTP-regulated interaction between Exp5 and pre-60S particles [62]. To test this, we first purified a pre-60S particle from HEK293 cells by tandem affinity purification (TAP) using the TAP-tagged trans-acting factor MRTO4 (homologous to yeast Mrt4) as bait (Figure 6 and Figure S8). Protein composition analysis of this particle showed the presence of 60S trans-acting factors like the export adaptor Nmd3, C15orf15 (Rlp24, ribosomal-like protein 24), and ribosomal proteins of the large subunit (Figure 6B and Figure S8A). These particles were then incubated with HeLa cell extract in the absence or presence of RanQ69L-GTP, a Ran mutant locked in the GTP-bound state, to analyze RanGTP-dependent binding of nuclear export receptors to pre-60S. Note that the HeLa cell extract had been pre-depleted for ribosomes and tRNAs. The depletion of ribosomes ensured that the TAP-purified particles were the sole ribosomal particles present. The presence of tRNA might hamper the detection of a potential Exp5/60S interaction because tRNA binds efficiently to Exp5 [63],[64].

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