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Accumulation of H/ACA snoRNPs depends on the integrity of the conserved central domain of the RNA-binding protein Nhp2p - PubMed

  • ️Mon Jan 01 2001

Accumulation of H/ACA snoRNPs depends on the integrity of the conserved central domain of the RNA-binding protein Nhp2p

A Henras et al. Nucleic Acids Res. 2001.

Abstract

Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. How box H/ACA snoRNPs are assembled remains unknown. Here we show that yeast Nhp2p, a core component of these particles, directly binds RNA. In vitro, Nhp2p interacts with high affinity with RNAs containing irregular stem-loop structures but shows weak affinity for poly(A), poly(C) or for double-stranded RNAs. The central region of Nhp2p is believed to function as an RNA-binding domain, since it is related to motifs found in various RNA-binding proteins. Removal of two amino acids that shortens a putative beta-strand element within Nhp2p central domain impairs the ability of the protein to interact with H/ACA snoRNAs in cell extracts. In vivo, this deletion prevents cell viability and leads to a strong defect in the accumulation of H/ACA snoRNAs and Gar1p. These data suggest that proper direct binding of Nhp2p to H/ACA snoRNAs is required for the assembly of H/ACA snoRNPs and hence for the stability of some of their components. In addition, we show that converting a highly conserved glycine residue (G(59)) within Nhp2p central domain to glutamate significantly reduces cell growth at 30 and 37 degrees C. Remarkably, this modification affects the steady-state levels of H/ACA snoRNAs and the strength of Nhp2p association with these RNAs to varying degrees, depending on the nature of the H/ACA snoRNA. Finally, we show that the modified Nhp2p protein whose interaction with H/ACA snoRNAs is impaired cannot accumulate in the nucleolus, suggesting that only the assembled H/ACA snoRNP particles can be efficiently retained in the nucleolus.

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Figures

Figure 1
Figure 1

Purified proteins used in band retardation assays. Purified proteins were submitted to SDS–PAGE and revealed by Coomassie blue staining. Lane 1, Nhp2pZZ; lane 2, Nhp2G59EpZZ; lane 3, Nhp2ΔV76I77pZZ; lane 4, His-Nhp2p. Molecular weights (kDa) of markers (M) indicated on the left and right.

Figure 2
Figure 2

Nhp2p is an RNA-binding protein. Band retardation assays were performed with 10 fmol of labeled full-length snR36 (A), snR36 5′ stem–loop + insertion element + H box (B), snR36 5′ stem–loop + insertion element (C), snR36 3′ stem–loop + AUA box (D), snR36 3′ stem–loop (E). Nhp2pZZ purified from yeast or His-Nhp2p purified from E.coli were used at the concentrations indicated. Protein was omitted in reactions loaded in lanes 1 (A–E), lane 7 (D), lanes 9 (B, C and E) and lane 13 (D). One microliter of anti-Nhp2p polyclonal serum diluted 104-, 103-, 102- and 10-fold was added to binding reactions loaded in lanes 9–12 of (D), respectively. All reactions contained 100 ng/µl poly(A). Complexes were resolved by electrophoresis on native 10 or 12% polyacrylamide gels.

Figure 2
Figure 2

Nhp2p is an RNA-binding protein. Band retardation assays were performed with 10 fmol of labeled full-length snR36 (A), snR36 5′ stem–loop + insertion element + H box (B), snR36 5′ stem–loop + insertion element (C), snR36 3′ stem–loop + AUA box (D), snR36 3′ stem–loop (E). Nhp2pZZ purified from yeast or His-Nhp2p purified from E.coli were used at the concentrations indicated. Protein was omitted in reactions loaded in lanes 1 (A–E), lane 7 (D), lanes 9 (B, C and E) and lane 13 (D). One microliter of anti-Nhp2p polyclonal serum diluted 104-, 103-, 102- and 10-fold was added to binding reactions loaded in lanes 9–12 of (D), respectively. All reactions contained 100 ng/µl poly(A). Complexes were resolved by electrophoresis on native 10 or 12% polyacrylamide gels.

Figure 2
Figure 2

Nhp2p is an RNA-binding protein. Band retardation assays were performed with 10 fmol of labeled full-length snR36 (A), snR36 5′ stem–loop + insertion element + H box (B), snR36 5′ stem–loop + insertion element (C), snR36 3′ stem–loop + AUA box (D), snR36 3′ stem–loop (E). Nhp2pZZ purified from yeast or His-Nhp2p purified from E.coli were used at the concentrations indicated. Protein was omitted in reactions loaded in lanes 1 (A–E), lane 7 (D), lanes 9 (B, C and E) and lane 13 (D). One microliter of anti-Nhp2p polyclonal serum diluted 104-, 103-, 102- and 10-fold was added to binding reactions loaded in lanes 9–12 of (D), respectively. All reactions contained 100 ng/µl poly(A). Complexes were resolved by electrophoresis on native 10 or 12% polyacrylamide gels.

Figure 2
Figure 2

Nhp2p is an RNA-binding protein. Band retardation assays were performed with 10 fmol of labeled full-length snR36 (A), snR36 5′ stem–loop + insertion element + H box (B), snR36 5′ stem–loop + insertion element (C), snR36 3′ stem–loop + AUA box (D), snR36 3′ stem–loop (E). Nhp2pZZ purified from yeast or His-Nhp2p purified from E.coli were used at the concentrations indicated. Protein was omitted in reactions loaded in lanes 1 (A–E), lane 7 (D), lanes 9 (B, C and E) and lane 13 (D). One microliter of anti-Nhp2p polyclonal serum diluted 104-, 103-, 102- and 10-fold was added to binding reactions loaded in lanes 9–12 of (D), respectively. All reactions contained 100 ng/µl poly(A). Complexes were resolved by electrophoresis on native 10 or 12% polyacrylamide gels.

Figure 2
Figure 2

Nhp2p is an RNA-binding protein. Band retardation assays were performed with 10 fmol of labeled full-length snR36 (A), snR36 5′ stem–loop + insertion element + H box (B), snR36 5′ stem–loop + insertion element (C), snR36 3′ stem–loop + AUA box (D), snR36 3′ stem–loop (E). Nhp2pZZ purified from yeast or His-Nhp2p purified from E.coli were used at the concentrations indicated. Protein was omitted in reactions loaded in lanes 1 (A–E), lane 7 (D), lanes 9 (B, C and E) and lane 13 (D). One microliter of anti-Nhp2p polyclonal serum diluted 104-, 103-, 102- and 10-fold was added to binding reactions loaded in lanes 9–12 of (D), respectively. All reactions contained 100 ng/µl poly(A). Complexes were resolved by electrophoresis on native 10 or 12% polyacrylamide gels.

Figure 3
Figure 3

Competition experiments. Band retardation assays were performed with 10 fmol of labeled snR36 3′ stem–loop + AUA box and 100 ng/µl poly(A), except for the retardation assays of lanes 8–10 in (C), for which 100 ng/µl poly(C) were used. All reactions contained Nhp2pZZ at a concentration of 30 nM, except for those loaded in lanes 1 (A–C), and lane 8 (C), which lacked protein. Complexes were resolved by electrophoresis on native 12% polyacrylamide gels. (A) Nhp2pZZ interacts with C/D-type snoRNAs in vitro. The following competitor RNAs were added to binding reactions loaded in lanes 3–8: lane 3, snR36 3′ stem–loop + AUA box, 150× molar excess; lane 4, snR36 3′ stem–loop + AUA box, 400× molar excess; lane 5, U24, 150× molar excess; lane 6, U24, 500× molar excess; lane 7, snR78, 150× molar excess; lane 8, snR78, 500× molar excess. (B) Nhp2pZZ binds poorly to double-stranded RNAs. Double-stranded competitor RNAs, obtained by transcribing each strand of the pGEM1 polylinker followed by strand annealing, were added to the reactions loaded in lanes 3–6 at increasing concentrations: lane 3, 200× molar excess; lane 4, 300× molar excess; lane 5, 400× molar excess; lane 6, 500× molar excess. (C) Nhp2pZZ binds poorly to poly(A) and poly(C). The following competitor RNAs were added to binding reactions loaded in lanes 3–7 and 10: lane 3, poly(G), 300× molar excess of nucleotides; lane 4, poly(G), 600× molar excess of nucleotides; lane 5, poly(U), 700× molar excess of nucleotides; lane 6, poly(U), 1400× molar excess of nucleotides; lane 7, poly(C), 2800× molar excess of nucleotides; lane 10, poly(A), 2600× molar excess of nucleotides.

Figure 3
Figure 3

Competition experiments. Band retardation assays were performed with 10 fmol of labeled snR36 3′ stem–loop + AUA box and 100 ng/µl poly(A), except for the retardation assays of lanes 8–10 in (C), for which 100 ng/µl poly(C) were used. All reactions contained Nhp2pZZ at a concentration of 30 nM, except for those loaded in lanes 1 (A–C), and lane 8 (C), which lacked protein. Complexes were resolved by electrophoresis on native 12% polyacrylamide gels. (A) Nhp2pZZ interacts with C/D-type snoRNAs in vitro. The following competitor RNAs were added to binding reactions loaded in lanes 3–8: lane 3, snR36 3′ stem–loop + AUA box, 150× molar excess; lane 4, snR36 3′ stem–loop + AUA box, 400× molar excess; lane 5, U24, 150× molar excess; lane 6, U24, 500× molar excess; lane 7, snR78, 150× molar excess; lane 8, snR78, 500× molar excess. (B) Nhp2pZZ binds poorly to double-stranded RNAs. Double-stranded competitor RNAs, obtained by transcribing each strand of the pGEM1 polylinker followed by strand annealing, were added to the reactions loaded in lanes 3–6 at increasing concentrations: lane 3, 200× molar excess; lane 4, 300× molar excess; lane 5, 400× molar excess; lane 6, 500× molar excess. (C) Nhp2pZZ binds poorly to poly(A) and poly(C). The following competitor RNAs were added to binding reactions loaded in lanes 3–7 and 10: lane 3, poly(G), 300× molar excess of nucleotides; lane 4, poly(G), 600× molar excess of nucleotides; lane 5, poly(U), 700× molar excess of nucleotides; lane 6, poly(U), 1400× molar excess of nucleotides; lane 7, poly(C), 2800× molar excess of nucleotides; lane 10, poly(A), 2600× molar excess of nucleotides.

Figure 3
Figure 3

Competition experiments. Band retardation assays were performed with 10 fmol of labeled snR36 3′ stem–loop + AUA box and 100 ng/µl poly(A), except for the retardation assays of lanes 8–10 in (C), for which 100 ng/µl poly(C) were used. All reactions contained Nhp2pZZ at a concentration of 30 nM, except for those loaded in lanes 1 (A–C), and lane 8 (C), which lacked protein. Complexes were resolved by electrophoresis on native 12% polyacrylamide gels. (A) Nhp2pZZ interacts with C/D-type snoRNAs in vitro. The following competitor RNAs were added to binding reactions loaded in lanes 3–8: lane 3, snR36 3′ stem–loop + AUA box, 150× molar excess; lane 4, snR36 3′ stem–loop + AUA box, 400× molar excess; lane 5, U24, 150× molar excess; lane 6, U24, 500× molar excess; lane 7, snR78, 150× molar excess; lane 8, snR78, 500× molar excess. (B) Nhp2pZZ binds poorly to double-stranded RNAs. Double-stranded competitor RNAs, obtained by transcribing each strand of the pGEM1 polylinker followed by strand annealing, were added to the reactions loaded in lanes 3–6 at increasing concentrations: lane 3, 200× molar excess; lane 4, 300× molar excess; lane 5, 400× molar excess; lane 6, 500× molar excess. (C) Nhp2pZZ binds poorly to poly(A) and poly(C). The following competitor RNAs were added to binding reactions loaded in lanes 3–7 and 10: lane 3, poly(G), 300× molar excess of nucleotides; lane 4, poly(G), 600× molar excess of nucleotides; lane 5, poly(U), 700× molar excess of nucleotides; lane 6, poly(U), 1400× molar excess of nucleotides; lane 7, poly(C), 2800× molar excess of nucleotides; lane 10, poly(A), 2600× molar excess of nucleotides.

Figure 4
Figure 4

Inhibition of the accumulation of Gar1p and H/ACA snoRNAs due to the ΔV76I77 deletion within Nhp2p. Strains GAL::nhp2/pNHP2ZZ (Nhp2pZZ, lanes 1–5), GAL::nhp2/pnhp2ΔV76I77ZZ (Nhp2ΔV76I77pZZ, lanes 6–10) and GAL::nhp2/empty vector (empty vector, lanes 11–15) were grown in galactose-containing medium (lanes 1, 6 and 11), then were shifted to glucose-containing medium for 12 (lanes 2, 7 and 12), 24 (lanes 3, 8 and 13), 36 (lanes 4, 9 and 14) or 48 h (lanes 5, 10 and 15). Culture aliquots were collected and total proteins or total RNAs were extracted for western (A) or northern (B) analysis. (A) Proteins were separated by SDS–PAGE and transferred to cellulose membranes. Wild-type Nhp2p and ZZ-tagged proteins, Gar1p and Nsr1p proteins were detected by use of anti-Nhp2p, anti-Gar1p, anti-Nsr1p sera, respectively. (B) Extracted RNAs were separated on denaturing 6% polyacrylamide gels and transferred to nylon membranes. snoRNAs were detected by hybridization with specific oligonucleotide probes.

Figure 4
Figure 4

Inhibition of the accumulation of Gar1p and H/ACA snoRNAs due to the ΔV76I77 deletion within Nhp2p. Strains GAL::nhp2/pNHP2ZZ (Nhp2pZZ, lanes 1–5), GAL::nhp2/pnhp2ΔV76I77ZZ (Nhp2ΔV76I77pZZ, lanes 6–10) and GAL::nhp2/empty vector (empty vector, lanes 11–15) were grown in galactose-containing medium (lanes 1, 6 and 11), then were shifted to glucose-containing medium for 12 (lanes 2, 7 and 12), 24 (lanes 3, 8 and 13), 36 (lanes 4, 9 and 14) or 48 h (lanes 5, 10 and 15). Culture aliquots were collected and total proteins or total RNAs were extracted for western (A) or northern (B) analysis. (A) Proteins were separated by SDS–PAGE and transferred to cellulose membranes. Wild-type Nhp2p and ZZ-tagged proteins, Gar1p and Nsr1p proteins were detected by use of anti-Nhp2p, anti-Gar1p, anti-Nsr1p sera, respectively. (B) Extracted RNAs were separated on denaturing 6% polyacrylamide gels and transferred to nylon membranes. snoRNAs were detected by hybridization with specific oligonucleotide probes.

Figure 5
Figure 5

Effects of the ΔV76I77 deletion or the G59E substitution on the interaction of Nhp2p with H/ACA snoRNAs in cell extracts. Immunoprecipitation experiments were carried out using IgG–Sepharose and extracts from strains obtained by transforming JG540 with plasmids directing expression of either Nhp2pZZ (lanes 1 and 2), Nhp2ΔV76I77pZZ (lanes 3 and 4) or Nhp2G59EpZZ (lanes 5 and 6). RNAs extracted from one-fifteenth of the input extracts (I, lanes 1, 3 and 5) or from the pellets following immunoprecipitation (P, lanes 2, 4 and 6) were separated on a denaturing 6% polyacrylamide gel and transferred to a nylon membrane. Various H/ACA and C/D snoRNAs were detected by hybridization with specific oligonucleotide probes.

Figure 6
Figure 6

The ΔV76I77 deletion prevents the specific accumulation of Nhp2p within the nucleolus. Shown are immunolocalizations of Nhp2pZZ (WT) or Nhp2ΔV76I77pZZ (ΔV76I77) proteins expressed in wild-type strain JG540. ZZ-tagged proteins were detected by treatment with anti-protein A antibodies followed by incubation with colloidal gold-conjugated protein A. No, nucleolus; Nu, nucleoplasm.

Figure 7
Figure 7

Northern analysis of snoRNA levels in nhp2::LEU2 strains expressing Nhp2V56KpZZ, Nhp2G59EpZZ, Nhp2R68ApZZ or Nhp2D80ApZZ. Total RNAs were extracted from strains nhp2::LEU2/pNHP2ZZ (Nhp2pZZ, lane 1), nhp2::LEU2/pnhp2V56KZZ (Nhp2V56KpZZ, lane 2), nhp2::LEU2/pnhp2G59EZZ (Nhp2G59EpZZ, lane 3), nhp2::LEU2/pnhp2R68AZZ (Nhp2R68ApZZ, lane 4) and nhp2::LEU2/pnhp2D80AZZ (Nhp2D80ApZZ, lane 5), separated on a denaturing 6% polyacrylamide gel and transferred to a nylon membrane. Various H/ACA-type and C/D-type snoRNAs were detected by hybridization with specific oligonucleotide probes. Phosphorimager scans of the northern blot were used to quantify H/ACA snoRNA levels. These are given as percentage level in wild-type cells (to the right of the northern blot). Values were normalized using the levels of the C/D-type snR190 and U14 snoRNAs as internal standards. Values oscillate on average by ±5%.

Figure 8
Figure 8

The ΔV76I77 deletion or the G59E substitution within Nhp2p central domain alters the pattern of complexes obtained in band retardation assays. Band retardation experiments were performed using 10 fmol of labeled snR36 3′ stem + AUA box and Nhp2pZZ (lanes 2–6), Nhp2ΔV76I77pZZ (lanes 8–12) or Nhp2G59EpZZ (lanes 14–18) added at the indicated concentrations. In reactions loaded in lanes 1, 7 and 13, protein was omitted. Complexes were resolved by electrophoresis on native 12% polyacrylamide gels.

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