Utp23p is required for dissociation of snR30 small nucleolar RNP from preribosomal particles

Abstract

Yeast snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) that promotes 18S rRNA processing through forming transient base-pairing interactions with the newly synthesized 35S pre-rRNA. By using a novel tandem RNA affinity selection approach, followed by coimmunoprecipitation and in vivo cross-linking experiments, we demonstrate that in addition to the four H/ACA core proteins, Cbf5p, Nhp2p, Nop10p and Gar1p, a fraction of snR30 specifically associates with the Utp23p and Kri1p nucleolar proteins. Depletion of Utp23p and Kri1p has no effect on the accumulation and recruitment of snR30 to the nascent pre-ribosomes. However, in the absence of Utp23p, the majority of snR30 accumulates in large pre-ribosomal particles. The retained snR30 is not base-paired with the 35S pre-rRNA, indicating that its aberrant tethering to nascent preribosomes is likely mediated by pre-ribosomal protein(s). Thus, Utp23p may promote conformational changes of the pre-ribosome, essential for snR30 release. Neither Utp23p nor Kri1p is required for recruitment of snR30 to the nascent pre-ribosome. On the contrary, depletion of snR30 prevents proper incorporation of both Utp23p and Kri1p into the 90S pre-ribosome containing the 35S pre-rRNA, indicating that snR30 plays a central role in the assembly of functionally active small subunit processome.

Figure 1.

The snR30/U17 snoRNA functions in the nucleolytic processing of 18S rRNA. (A) Processing of yeast 35S pre-rRNA in the presence and absence of snR30. The 18S, 5.8S and 25S rRNAs and the external (5′ETS and 3′ETS) and internal (ITS1 and ITS2) transcribed spacer sequences, the major processing sites (arrows A₀, A₁, A₂, A₃ and D) and the resulting pre-rRNA intermediates (35S, 33S, 23S, 20S, 27SA₂ and 27SA₃) are indicated. Interaction of 18S rRNA with snR30 is needed for cleavages at the A₀, A₁ and A₂ sites. (B) Schematic presentation of the evolutionarily conserved interaction of snR30 and 18S rRNA (23).

Figure 2.

Affinity purification of yeast snR30 snoRNP. (A) Schematic structure of the pTOB-MS2-R30 expression construct. The promoter (SNR5-P) and terminator (SNR5-T) of the SNR5 and the coding region of the SNR30 gene (open arrow) are indicated. Sequences and predicted structures of the tobramycin (TOB) and MS2 coat protein-binding motifs (italics) and the spacer regions (lower case letters) are shown. The 5′-terminal nucleotides of the predicted snR30 transcript represent authentic snR30 sequences (capitals). Restriction sites are indicated. (B) Growth properties of yeast GAL::SNR30 cells carrying the pTOB-MS2-R30 and pR30 expression plasmids or the pFL45/R5P empty vector on galactose- (GAL) and glucose-containing (GLU) solid medium. (C) Expression of wild type and TOB-MS2-tagged snR30 RNAs. About 5 μg of RNAs extracted from yeast GAL::SNR30 cells transformed by the indicated plasmids and grown in galactose- or glucose-containing liquid medium were fractionated on a 6% sequencing gel and analyzed by northern blotting with a snR30-specific oligonucleotide probe. Lane M, relevant size markers in nucleotides. (D) Purification of TOB-MS2-R30 RNP. Extracts (Ext) prepared from GAL::SNR30 cells expressing TOB-MS2-R30 or wild-type snR30 were used as starting materials for tandem RNA affinity-selection. RNAs isolated from cell extracts (5 μg) or from the pellets of affinity-selection reactions (0.1 μg) were analyzed by northern blotting.

Figure 3.

In vivo association of snR30 and Utp23p. (A) Yeast snR30 coprecipitates with TAP-tagged Utp23p. Extracts (Ext) prepared from control cells (WT) or from cells expressing Utp23p-TAP were cleared by centrifugation at 10 000 g for 10 min (low speed) or at 180 000 g for 45 min (high speed) before IP of Utp23-TAP with IgG-sepharose. RNAs from the extracts or from the pellets of IP reactions were analyzed by northern blotting with oligonucleotide probes specific for snR30, U3, snR10 and U14. (B) In vivo cross-linking of snR30 and Utp23p. Yeast cells expressing wild-type (no tag) or TAP-tagged Utp23p were treated with formaldehyde and after extract preparation, Utp23p-TAP was immunoprecipitated under stringent conditions. Coprecipitation of snR30 and U3 was monitored by northern blot analysis. Lane M, size markers. (C) Human Utp23p interacts with the U17 snoRNA. HeLa cells transfected or not transfected (NT) with the pCMV-Flag-UTP23 expression vector were used for preparation of high speed extracts. RNAs prepared from the extracts or from the pellets of anti-Flag IPs were analyzed by northern blotting with U17- and 7SK-specific probes.

Figure 4.

In vivo association of snR30 and Kri1p. (A) Kri1p interacts with snR30. Extracts were prepared by high speed centrifugation from control cells (WT) or from cells expressing protein A-tagged Kri1p (Kri1p-ZZ) and TAP-tagged Utp23p (Utp23p-TAP). The tagged proteins were immunoprecipitated with IgG-sepharose and the coprecipitated RNAs were analyzed by northern blotting with snR30- and U3-specific probes. Lane M, size markers. (B) In vivo cross-linking of snR30 and Kri1p. Yeast control cells (WT) and cells expressing Utp23p-TAP or Kri1p-ZZ were treated with formaldehyde. After extract preparation, Utp23p-TAP and Kri1p-ZZ were immunoprecipitated under stringent conditions and coprecipitation of snR30 and U3 was assayed by northern blot analysis. (C) Mutually exclusive association of Utp23p and Kri1p with snR30. Extracts were prepared from yeast cells expressing HA-tagged Utp23p (Utp23p-HA) together with Kri1p-ZZ or Ltv1p-TAP following the low speed (low sp) or high speed (high sp) centrifugation protocol. After IP of Kri1p-ZZ and Ltv1p-TAP (lower panel), co-precipitation of Utp23p-HA was tested by western blotting. Size markers are indicated in kDa.

Figure 5.

Depletion of Utp23p increases snR30 accumulation in large pre-ribosomal complexes. (A) Expression of Utp23p-TAP in yeast UTP23-TAP and GAL::UTP23-TAP cells grown in glucose medium. Western blot analysis was used to monitor the accumulation of Utp23p-TAP and Nhp2p. Size markers are shown. (B) Utp23p and Kri1p are not required for accumulation of snR30. GAL::UTP23-TAP, GAL::KRI1-TAP and control Y0341 (WT) cells were grown on glucose for 10 h. Accumulation of snR30 and U3 snoRNAs was measured by northern blotting. (C) Sedimentation behavior of yeast snR30 in the absence of Utp23p. Extracts prepared from yeast UTP23-TAP and GAL::UTP23-TAP cells grown on glucose for 10 h were fractionated on 5–45% linear sucrose gradients. Distribution of snR30, U3, snR10 and U14 was detected by northern blotting. Ribosomal sedimentation profiles were obtained by recording absorbance at OD₂₅₄. Lane M, size markers.

Figure 6.

Protein-mediated tethering of snR30 to pre-ribosomes in the absence of Utp23p. (A) Sedimentation behavior of Rok1p in the absence and presence of Utp23p. Extracts prepared from UTP23-TAP and GAL::UTP23-TAP cells expressing Rok1p-myc and grown on glucose were fractionated on 5–45% sucrose gradients. Distribution of Rok1p-myc and Utp23p-TAP was monitored by western blot analyses. The ribosomal sedimentation profiles are shown. (B) Rok1p binds to the 35S pre-rRNA independently of Utp23p. Rok1p-myc was expressed in control (WT), UTP23-HA and GAL::UTP23-HA cells. After 10 h of growth in glucose medium, extracts were prepared and Rok1p-myc was immunoprecipitated. Coprecipitation of the 35S pre-rRNA was assayed by primer extension analyses using a terminally labeled deoxyoligonucleotide primer. (C) Yeast snR30 is not base-paired with pre-rRNAs in Utp23p-depleted cells. Cell extracts prepared from GAL::UTP23-TAP and UTP23-TAP cells grown on glucose was treated with proteinase K before sucrose gradient fractionation. Distribution of snR30 and U3 was monitored by northern blotting.

Figure 7.

Association of Utp23p and Kri1p with preribosomes depends on snR30. (A) Utp23p comigrates with the 35S pre-rRNA in the absence of snR30. Yeast GAL::SNR30 cells were grown on glucose to deplete endogenous snR30. After extract preparation, the sedimentation behaviors of Utp23p-TAP, the U3 snoRNA and the 35S pre-rRNA were analyzed. For gradient migration of Utp23p-TAP in the presence of snR30, see Figure 6A. (B) Association of Utp23p and Kri1p with 35S pre-rRNA. Yeast GAL::SNR30/UTP23-TAP, GAL::SNR30/KRI1-ZZ, UTP23-TAP, KRI1-ZZ and control (WT) cells were grown on glucose before extract preparation and IP of with IgG Sepharose. Precipitation of Utp23p-TAP and Kri1p-ZZ and coprecipitation of 35S pre-rRNA were monitored by western and northern blot analyses. (C) Incorporation of Kri1p into large preribosomal particles depends on snR30 expression. Sucrose gradient sedimentation of ectopically expressed Kri1-ZZ and snR30, together with endogenous U3 and 35S RNAs was analyzed in extracts prepared from yeast GAL::SNR30 and SNR30 strains grown on glucose.