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Naf1p, an essential nucleoplasmic factor specifically required for accumulation of box H/ACA small nucleolar RNPs - PubMed

Naf1p, an essential nucleoplasmic factor specifically required for accumulation of box H/ACA small nucleolar RNPs

Christophe Dez et al. Mol Cell Biol. 2002 Oct.

Abstract

Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. The ways in which these particles are assembled and correctly localized in the dense fibrillar component of the nucleolus remain largely unknown. Recently, the essential Saccharomyces cerevisiae Naf1p protein (encoded by the YNL124W open reading frame) was found to interact in a two-hybrid assay with two core protein components of mature H/ACA snoRNPs, Cbf5p and Nhp2p (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569-4574, 2001). Here we show that several H/ACA snoRNP components are weakly but specifically immunoprecipitated with epitope-tagged Naf1p, suggesting that the latter protein is involved in H/ACA snoRNP biogenesis, trafficking, and/or function. Consistent with this, we find that depletion of Naf1p leads to a defect in 18S rRNA accumulation. Naf1p is unlikely to directly assist H/ACA snoRNPs during pre-rRNA processing in the dense fibrillar component of the nucleolus for two reasons. Firstly, Naf1p accumulates predominantly in the nucleoplasm. Secondly, Naf1p sediments in a sucrose gradient chiefly as a free protein or associated in a complex of the size of free snoRNPs, whereas extremely little Naf1p is found in fractions containing preribosomes. These results are more consistent with a role for Naf1p in H/ACA snoRNP biogenesis and/or intranuclear trafficking. Indeed, depletion of Naf1p leads to a specific and dramatic decrease in the steady-state accumulation of all box H/ACA snoRNAs tested and of Cbf5p, Gar1p, and Nop10p. Naf1p is unlikely to be directly required for the synthesis of H/ACA snoRNP components. Naf1p could participate in H/ACA snoRNP assembly and/or transport.

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Figures

FIG. 1.
FIG. 1.

Analysis of the interactions between Naf1p-ZZ and H/ACA snoRNP components. Total extracts were produced in native conditions from yeast cells expressing either free ZZ tag (lanes 1 to 3), ZZ-Nop1p (lanes 4 to 6), Cbf5p-ZZ (lanes 7 to 9), or Naf1p-ZZ (lanes 10 to 12). Immunoprecipitation experiments were carried out using IgG-Sepharose beads in a buffer containing either 150 mM (lanes 2, 5, 8, and 11) or 500 mM (lanes 3, 6, 9, and 12) KOAc. After precipitation and being washed, beads were separated in two equal fractions. (A) Western blot analysis. Beads from one set of fractions were resuspended in protein-denaturing buffer. Aliquots of the resulting supernatants (lanes 2, 3, 5, 6, 8, 9, 11, and 12) and 1/20 of the corresponding amount of total proteins from the input extract (T and lanes 1, 4, 7, and 10) were submitted to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to cellulose membranes. Tagged proteins were detected by use of rabbit PAP, Nsr1p by a monoclonal antibody, and Nop1p, Gar1p, Nhp2p, and Nop10p by polyclonal sera. Northern blot (B) and analysis (C) of [32P]pCp-labeled RNAs. RNAs retained on the beads of the second set of fractions were purified. Aliquots of precipitated RNAs (lanes 2, 3, 5, 6, 8, 9, 11, and 12) and 1/10 of the corresponding amount of total RNAs from the input extract (T and lanes 1, 4, 7, and 10) were submitted to denaturing 6% polyacrylamide gel electrophoresis. In panel B, separated RNAs were transferred to nylon membranes, and various H/ACA and C/D snoRNAs were detected by hybridization with specific oligonucleotide probes. Phosphorimager scans of the Northern blots were used to quantify snoRNA levels. The amounts of RNAs precipitated, expressed as percentages of input RNA levels, are indicated in the table on the right. In panel C, RNAs were 3′ end labeled with [32P]pCp and separated on a 6% sequencing gel. M, molecular weight markers (pBR322 digested with HaeIII and TaqI). Positions of RNAs inferred from their size are indicated.

FIG. 2.
FIG. 2.

Naf1p depletion affects 18S rRNA accumulation. GAL::zz-naf1 cells were grown in a medium containing galactose (gal), raffinose, and sucrose, were washed, and were shifted to a medium containing glucose (glu). Cell aliquots were collected from the culture grown in galactose, raffinose, and sucrose (lanes 1) and after 6 (lanes 2), 12 (lanes 3), 24 (lanes 4), 48 (lanes 5) and 72 (lanes 6) h of growth in glucose-containing medium. Total proteins and RNAs were extracted from these samples for Western (A) and Northern (B) blot analysis. (A) Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a cellulose membrane. ZZ-Naf1p was detected by use of rabbit PAP and Nsr1p by using a monoclonal antibody. The third panel corresponds to an overexposed film to show the residual production of ZZ-Naf1p even after prolonged growth on glucose-containing medium. (B) High-molecular-weight RNAs were separated on a formaldehyde-agarose gel, and low-molecular-weight RNAs were separated on a denaturing 6% polyacrylamide gel. Gels were transferred to nylon membranes. Mature rRNAs and pre-rRNA processing intermediates were detected by use of specific oligonucleotide probes. (C) Cartoon of the pre-rRNA processing pathway. In wild-type cells, the 35S pre-rRNA is first cleaved at site A0 within the 5′ external transcribed spacer (5′ ETS), producing intermediate 33S, which is very rapidly cleaved at site A1, the 5′ end of 18S rRNA, to produce intermediate 32S. 32S is then cleaved at site A2 within the internal transcribed spacer 1 (ITS1), releasing 20S, the immediate precursor to 18S rRNA, and 27SA2. 27SA2 is then processed via two alternative pathways. It is either cut at site A3 to produce 27SA3, which is then trimmed by 5′-to-3′ exonucleases up to site B1(S), producing 27SB(S). Alternatively, it can be processed into 27SB(L) by an as-yet-unknown mechanism. 27SB(S) and 27SB(L) are then processed in the same manner to produce 25S and 5.8S(S) or 5.8S(L), respectively. In cells depleted of Naf1p, a fraction of 35S is directly cut at site A3, producing 23S that is degraded by the exosome. For a detailed review of the pre-rRNA processing pathway in yeast, see reference .

FIG. 3.
FIG. 3.

Pulse-chase analysis of pre-rRNA processing in ZZ-Naf1p-depleted cells. GAL::zz-naf1 cells were grown in a medium containing galactose (GAL), raffinose, and sucrose (lanes 1 to 5), were washed, and shifted to a medium containing glucose (GLU) for 28 h (lanes 6 to 10) or 52 h (lanes 11 to 15). Cells were labeled for 3 min with [methyl-3H]methionine, chased with an excess of cold methionine, and grown for a further 1 (lanes 1, 6, and 11), 2 (lanes 2, 7, and 12), 5 (lanes 3, 8, and 13), 10 (lanes 4, 9, and 14), and 20 (lanes 5, 10, and 15) min before collection of cells and freezing in liquid nitrogen. Extracted RNAs were separated on a formaldehyde-agarose gel and transferred to a nylon membrane. Labeled RNAs were detected by autoradiography. Pre-rRNA intermediates and mature rRNAs are indicated on the left.

FIG. 4.
FIG. 4.

Naf1p-ZZ accumulates mainly within the nucleoplasm. Shown are electron micrographs of a whole cell (×27,600 magnification) (A), a nucleus (×41,400 magnification) (B), and a nucleolus (×46,000 magnification) (C). Naf1p-ZZ was detected by electron microscopy after treatment of the grids with anti-protein A antibodies followed by incubation with colloidal gold-conjugated protein A. No, nucleolus; Nu, nucleoplasm. In panel C, a section of the nucleolar dense fibrillar component is indicated by arrow heads. Bar = 300 nm.

FIG. 5.
FIG. 5.

Sedimentation profile of Naf1p-ZZ in a glycerol gradient. A native extract from cells expressing Naf1p-ZZ was loaded on a 10 to 30% glycerol gradient and centrifuged for 10 h at 25,000 rpm in an SW41 Ti rotor. Nineteen fractions were collected and divided in two equal aliquots from which proteins or RNAs were extracted. Proteins were separated by SDS-polyacrylamide gel electrophoresis, while RNAs were separated by denaturing polyacrylamide gel electrophoresis. Proteins or RNAs were then transferred to cellulose or nylon membranes, respectively. Naf1p-ZZ and Nhp2p were detected by use of a polyclonal anti-Nhp2p serum, ribosomal protein L3 by a monoclonal anti-L3 antibody, and ribosomal protein S8 by a polyclonal anti-S8 serum. H/ACA snR10 and snR36 snoRNAs were detected using specific oligonucleotide probes. Lane numbers correspond to fraction numbers. Fraction 1, top of the gradient; fraction 19, bottom of the gradient; and T, protein or RNA aliquot from the nonfractionated input extract.

FIG. 6.
FIG. 6.

Analysis of steady-state levels of H/ACA snoRNP components and of the snR44 host gene mRNA in Naf1p-depleted cells. GAL::zz-naf1 (lanes 1 to 6) or GAL::naf1/CBF5-TAP (lanes 7 to 12) cells were grown in a medium containing galactose (gal), raffinose, and sucrose, were washed, and shifted to a medium containing glucose (glu). Cell aliquots were collected from the cultures grown in galactose, raffinose, and sucrose (lanes 1 and 7) and after 6 (lanes 2 and 8), 12 (lanes 3 and 9), 24 (lanes 4 and 10), 48 (lanes 5 and 11), and 72 (lanes 6 and 12) h of growth in glucose-containing medium. Total RNAs and proteins were extracted from these samples for Northern (A) and Western (B) blot analysis. A large set of RNAs was detected by use of complementary oligonucleotide probes. Phosphorimager scans of the Northern blots were used to quantify RNA levels. Values indicated are percentages of the figures obtained for RNAs originating from cells grown on galactose, raffinose, and sucrose. Cbf5p-TAP was detected using rabbit PAP. The remaining proteins were detected as described in previous figure legends.

FIG. 6.
FIG. 6.

Analysis of steady-state levels of H/ACA snoRNP components and of the snR44 host gene mRNA in Naf1p-depleted cells. GAL::zz-naf1 (lanes 1 to 6) or GAL::naf1/CBF5-TAP (lanes 7 to 12) cells were grown in a medium containing galactose (gal), raffinose, and sucrose, were washed, and shifted to a medium containing glucose (glu). Cell aliquots were collected from the cultures grown in galactose, raffinose, and sucrose (lanes 1 and 7) and after 6 (lanes 2 and 8), 12 (lanes 3 and 9), 24 (lanes 4 and 10), 48 (lanes 5 and 11), and 72 (lanes 6 and 12) h of growth in glucose-containing medium. Total RNAs and proteins were extracted from these samples for Northern (A) and Western (B) blot analysis. A large set of RNAs was detected by use of complementary oligonucleotide probes. Phosphorimager scans of the Northern blots were used to quantify RNA levels. Values indicated are percentages of the figures obtained for RNAs originating from cells grown on galactose, raffinose, and sucrose. Cbf5p-TAP was detected using rabbit PAP. The remaining proteins were detected as described in previous figure legends.

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