The nucleolar protein Nop19p interacts preferentially with Utp25p and Dhr2p and is essential for the production of the 40S ribosomal subunit in Saccharomyces cerevisiae

Abstract

In eukaryotes, ribosome biogenesis is a process of major interest that requires more than 200 factors acting coordinately in time and space. Using genetic and proteomic studies, most of the components have now been identified. Based on its nucleolar localization, we characterized the protein encoded by the open reading frame YGR251W, we renamed Nop19p as playing an essential role in ribosome biogenesis. Depletion of the Nop19p in yeast impairs pre-rRNA processing at sites A₀, A₁ and A₂, leading to a strong decrease in 18S rRNA and 40S subunit levels. Nop19p is a component of 90S preribosomes which assembly is believed to result from stepwise incorporation of UTP modules. We show that Nop19p depletion does not impair the incorporation of UTP subcomplexes on preribosomes and conversely that depletion of UTP subcomplexes does not affect Nop19p recruitment on 90S preribosomes. TAP experiments under stringent conditions revealed that Nop19p interacts preferentially with the DEAH-box RNA helicase Dhr2p and Utp25p, both required for A 0, A 1 and A 2 cleavages. Nop19p appeared essential for the incorporation of Utp25p in preribosomes. In addition, our results suggest that in absence of Nop19p, Dhr2p remains trapped within aberrant preribosomes.

Figure 1

Scheme of the pre-rRNA processing pathway in S. cerevisiae. (A) Initial 35S pre-rRNA precursor with detailed cleavages sites. (B) Endonucleolytic and exonucleolytic cleavages leading to the production of mature 18S, 5.8S and 25S (reviewed in ref. 22).

Figure 2

Nop19p is a nucleolar protein associated with preribosomes. (A) Subcellular localization of Nop19p. Yeast strain expressing Nop19p-YFP and mCherry-Nop1p were grown exponentially and cell samples were used for fluorescence microscopy analysis. (B) Sedimentation profile of Nop19p on a sucrose gradient. A total extract prepared from NOP19::TAP cells growing exponentially was sedimented through a sucrose gradient and 17 fractions were collected. The corresponding A₂₅₄ profile is displayed with the characteristic annotated peaks. Each fraction was TCA precipitated and Nop19-TAP was detected by western blotting using PAP antibodies. (C) Nop19p co-immunoprecipitates with 35S and 23S pre-rRNAs. Northern-blot analysis of (pre-)rRNAs coprecipitated with TAP-tagged version of Nop19p (lanes 3–4 and 7–8) or from control experiments using extracts of cells lacking a tagged protein (lanes 1, 2, 5 and 6). Immunoprecipitation was performed on cell extracts using IgG-Sepharose. RNAs were extracted from the pellet after precipitation (lanes IP) or from total cell extract (lanes Tot) corresponding to 10% of the input used for the immunoprecipitation reactions. Following separation, RNAs were transferred to a nylon membrane and hybridized with anti-sense oligonucleotides corresponding to various (pre-) rRNAS and snoRNAs.

Figure 3

Nop19p depletion affects 40S ribosomal subunit accumulation in yeast cells. (A) Growth rate of wild-type and Gal::3HA::NOP19 strains following a transfer from permissive galactose medium to glucose medium for the times indicated. Cells were maintained in exponential growth throughout the time course by dilution into pre-warmed medium. (B) Western-blot analysis of 3HA-Nop19p depletion. Total proteins were extracted at the times indicated and analyzed by western blot. Accumulation of 3HA-Nop19p and Nop1p was respectively detected using anti-HA and Nop1p-specific antibodies. (C) Ribosome profiles in Nop19p-depleted cells. Gal::3HA::NOP19 and WT BY4741 strains were grown up to 0.6 (OD₆₀₀) on galactose medium and shifted to glucose for 6 h. Total cell extracts were prepared and centrifuges through 4.5% to 45% sucrose gradients and 17 fractions were collected. The A₂₅₄ absorbance profile is presented.

Figure 4

Nop19p depletion leads to a defect in A₀, A₁ and A₂ cleavages. (A) WT BY4741 and Gal::3HA::NOP19 strains were shifted from a galactose to a glucose medium. Samples were collected before and at different times after the nutritional shift. Total RNAs were extracted from these cell samples, and the accumulation of the different pre-rRNAs, rRNAs and sn(o)RNAs was analyzed by northern-blot. (B) Pulse-chase labeling of RNAs. WT BY4741 and Gal::3HA::NOP19 cells were grown in galactose containing medium and were next shifted in glucose for 3 h. Cells were then pulse labeled with [8-³H] adenine for 2 min. Samples were collected 0, 1, 2, 5, 10, 20 and 30 min after addition of an excess of cold adenine. Total RNAs were extracted from these samples, separated by gel electrophoresis and transferred to a nylon membrane.

Figure 5

Nop19p and U3 processome assembly. (A) Depletion of Nop19p in yeast does not affect the incorporation of components of the UTP-A, UTP-B or UTP-C modules within preribosomes. Strains expressing TAP-tagged versions of Utp17p, Pwp2p, Utp22p or Rrp5p that were otherwise WT or expressing 3HA-Nop19p under the control of the GAL1 promoter were transferred from galactose- to glucose-based medium and grown for 3 h. Total extracts prepared from these cells were sedimented through sucrose gradients. Western blot experiments were carried out using rabbit PAP to detect TAP-tagged proteins in the different fractions. (B) Sedimentation profile of Nop19-TAP in WT cells (upper left part) or in cells lacking Utp17p (upper right part), Pwp2p (lower left part) or Rrp5p (lower right part). The GAL1::UTP17, GAL1::PWP2 and GAL1::RRP5 strains expressing Nop19-TAP were shifted from galactose- to glucose-containing medium and grown for 14 h, to deplete the corresponding proteins. As a control, the Nop19::TAP strain grown in the presence of glucose was used. Total extracts prepared from these cell samples were sedimented through sucrose gradients. The proteins contained in each fraction were analyzed by western blotting using PAP antibodies to detect Nop19-TAP.

Figure 6

TAP purification of Nop19p-TAP under stringent conditions. (A) Nop19p-TAP was affinity purified under native conditions (see Materials and Methods). Nop19p-TAP-containing complexes bound to IgG were extensively washed with buffers containing 200 mM, 1 M or 2 M salt (KCl) concentration before Tev elution. Final purified samples were separated by SDS-polyacrylamide gel electrophoresis and observed after silver staining coloration. Samples were next subjected to mass spectrometry analysis. (B) Proteins specifically associated with Nop19p. Interactions identified by mass spectrometry analysis were individually verified. 3HA-tag versions of proteins of interest (Dhr2p, Utp25p, Dhr1p, Utp17p, Pwp2p, Utp22p, Rrp5p) were expressed in a NOP19::TAP background strain. Total extracts were immobilized on IgG and submitted to extensive washes with buffers containing 200 mM, 1 M or 2 M salt (KCl) before elution. Eluted proteins were analyzed by western blotting using PAP and HA antibodies.

Figure 7

Depletion of Nop19p affects the sedimentation profile of Dhr2p and Utp25p. Strains expressing TAP-tagged versions of Dhr2p (A) and Utp25p (B) that were otherwise WT or expressing 3HA-Nop19p under the control of the GAL1 promoter were transferred from galactose- to glucose-based medium and grown for 3 h. Total extracts prepared from these cells were sedimented through sucrose gradients. Western blot experiments were carried out using rabbit PAP to detect TAP-tagged proteins in the different fractions.