The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase

Abstract

Many or all of the sites of pseudouridine (Psi) formation in eukaryotic rRNA are selected by site-specific base-pairing with members of the box H + ACA class of small nucleolar RNAs (snoRNAs). Database searches previously identified strong homology between the rat nucleolar protein Nap57p, its yeast homolog Cbf5p, and the Escherichia coli Psi synthase truB/P35. We therefore tested whether Cbf5p is required for synthesis of Psi in the yeast rRNA. After genetic depletion of Cbf5p, formation of Psi in the pre-rRNA is dramatically inhibited, resulting in accumulation of the unmodified rRNA. Protein A-tagged Cbf5p coprecipitates all tested members of the box H + ACA snoRNAs but not box C + D snoRNAs or other RNA species. Genetic depletion of Cbf5p leads to depletion of all box H + ACA snoRNAs. These include snR30, which is required for pre-rRNA processing. Depletion of Cbf5p also results in a pre-rRNA processing defect similar to that seen on depletion of snR30. We conclude that Cbf5p is likely to be the rRNA Psi synthase and is an integral component of the box H + ACA class of snoRNPs, which function to target the enzyme to its site of action.

Figure 1

Structure of the yeast pre-rRNA and its processing pathway. (A) The 35S pre-rRNA. The sequences encoding the mature 18S, 5.8S, and 25S rRNAs (thick lines) are flanked by the 5′ and 3′ ETSs and separated by internal transcribed spacers 1 and 2 (ITS1 and ITS2). Sites of pre-rRNA processing are indicated with uppercase letters (A₀ to E) and the positions of hybridization of the oligonucleotides used are indicated with lowercase letters (a–h). (B) The pre-rRNA processing pathway. Processing of the primary 35S precursor starts at site A₀, yielding 33S pre-rRNA. This molecule is subsequently processed at sites A₁ and A₂, giving rise successively to the 32S pre-rRNA and to the 20S and 27SA₂ precursors. Cleavage at A₂ separates the pre-rRNAs destined for the small and large ribosomal subunit. The 20S precursor is then endonucleolytically cleaved at site D to yield the mature 18S rRNA. The 27SA₂ precursor is processed by two alternative pathways, forming the mature 5.8S and 25S rRNAs. The major pathway involves cleavage at a second site in ITS1, A₃, rapidly followed by exonucleolytic digestion to site B1_S, generating the 27SB_S precursor. Approximately 15% of the 27SA₂ molecules are processed by the second pathway at site B1_L, producing the 27SB_L pre-rRNA. At the same time as processing at B1 is completed, the 3′ end of mature 25S rRNA is generated by processing at site B₂. The subsequent processing of both 27SB species appears to follow a similar pathway. Cleavage at sites C₁ and C₂ releases the mature 25S rRNA and the 7S pre-rRNAs, which undergo rapid 3′ → 5′ exonuclease digestion to site E generating the mature 3′ end of 5.8S rRNA. Cbf5p is required for the early cleavages at sites A₁ and A₂; loss of these cleavages inhibits formation of the 20S and 27SA₂ pre-rRNA preventing synthesis of 18S rRNA. Cbf5p is also required for efficient processing at site A₀ and efficient processing of the 27SB and 7S pre-rRNAs in ITS2.

Figure 2

Genetic depletion of Cbf5p. (A) Schematic representation of the GAL::cbf5 allele. (B) Growth of the GAL::cbf5 (•) and CBF5 (○) strains following transfer to glucose medium. Cell density was measured at regular intervals, and the cultures were periodically diluted to be continuously kept in exponential growth. (C) Northern hybridization of the CBF5 mRNA from the CBF5 strain (lanes 1,2) and the GAL::cbf5 strain (lanes 3–10), following growth on rsg medium (0-hr lanes) and at intervals following transfer to glu medium (8 to 70-hr lanes). The positions of the CBF5 mRNA and 18S rRNA are indicated. As expected from the size of its ORF (1452 bp), the CBF5 mRNA is detected slightly under the 18S rRNA.

Figure 3

Northern analysis of rRNA and pre-rRNA synthesis in a GAL::cbf5 strain. (A) Probes against mature 25S and 18S rRNA (oligonucleotides a and h); (B) probe against the 5′ region of ITS1 (oligonucleotide b), (C); probe against ITS1 between sites A₂ and A₃ (oligonucleotide c); (D) probe against the 3′ region of ITS1 (oligonucleotide d); (E) probe against the 5′ region of ITS2 (probe f). (F) Shorter time course following transfer to glu medium, hybridized with oligonucleotide c. The oligonucleotides used are depicted in Fig. 1A. RNA was extracted from the CBF5 and GAL::cbf5 strains following growth on rsg medium (0-hr lanes) and at intervals following transfer to glu medium (8–70-hr lanes) and separated on a 1.2% agarose gel containing formaldehyde.

Figure 4

Northern analysis of rRNA and pre-rRNA synthesis in a GAL::cbf5 strain. (A) Probe against ITS2 (probe f); (B) probe against mature 5.8S rRNA (probe e). The oligonucleotides used are depicted in Fig. 1A. RNA was extracted from the CBF5 and GAL::cbf5 strains following growth on rsg medium (0-hr lanes) and at intervals following transfer to glu medium (8- to 70-hr lanes) and separated on an 8% polyacrylamide gel containing 8 m urea. (*) 5.8S species with intermediate length.

Figure 5

Primer-extension analysis of pre-rRNA processing in a GAL::cbf5 strain. (A) The 5′ end of the 35S primary transcript at site +1. (B) Site A₀. RNA was extracted from the CBF5 and GAL::cbf5 strains following growth on rsg medium (0-hr lanes) and at intervals following transfer to glu medium (8- to 70-hr lanes) and analyzed by primer extension with oligonucleotide a (Fig. 1A). A DNA sequence made with the same primer is shown as a size marker. Site +1 lies 730 nucleotides from the primer and the sequence is not useful.

Figure 6

Primer-extension analysis of pre-rRNA processing in a GAL::cbf5 strain. (A) Primer extension through sites B1_S, B1_L, A₃, and A₂. (B) Longer exposure of A showing the level of stop at site A₃. (C) Shorter exposure of A showing the level of stop at site B1. RNA was extracted from the CBF5 and GAL::cbf5 strains following growth on rsg medium (0-hr lanes) and at intervals following transfer to glu medium (8- to 70-hr lanes) and analyzed by primer extension using oligonucleotide g (Fig. 1A). A DNA sequence made with the same primer is shown as a size marker.

Figure 7

Θ formation in a GAL::cbf5 strain. Two-dimensional TLC analysis of ³²P-labeled 25S rRNA digested with RNAse T2. (A) RNA extracted from the CBF5 strain following growth in rsg medium. (B) RNA extracted from the CBF5 strain 24 hr after transfer to glu medium. (C) RNA extracted from the GAL::cbf5 strain following growth in rsg medium. (D) RNA extracted from the GAL::cbf5 strain 24 hr after transfer to glu medium. Spots corresponding to Ap, Cp, Gp, Up, and Θp are indicated. (E) Nucleotides separated by two-dimensional TLC were quantitated by PhosphorImager scanning. The ratio between incorporation into Θp and Ap in 35S pre-rRNA, 25S rRNA, and bulk tRNA is shown following growth in rsg medium, and 24 hr after transfer to glu medium. The Θp:Ap ratio in tRNA (right) is shown on a different scale from the 35S and 25S RNA samples (left) because of the greater representation of Θp in tRNA compared to rRNA. (Open bars) CBF5⁺/rsg; (light gray bars) CBF5⁺glu; (dark gray bars) GAL::cbf5 rsg; (solid bars) GAL::cbf5 glu.

Figure 7

Θ formation in a GAL::cbf5 strain. Two-dimensional TLC analysis of ³²P-labeled 25S rRNA digested with RNAse T2. (A) RNA extracted from the CBF5 strain following growth in rsg medium. (B) RNA extracted from the CBF5 strain 24 hr after transfer to glu medium. (C) RNA extracted from the GAL::cbf5 strain following growth in rsg medium. (D) RNA extracted from the GAL::cbf5 strain 24 hr after transfer to glu medium. Spots corresponding to Ap, Cp, Gp, Up, and Θp are indicated. (E) Nucleotides separated by two-dimensional TLC were quantitated by PhosphorImager scanning. The ratio between incorporation into Θp and Ap in 35S pre-rRNA, 25S rRNA, and bulk tRNA is shown following growth in rsg medium, and 24 hr after transfer to glu medium. The Θp:Ap ratio in tRNA (right) is shown on a different scale from the 35S and 25S RNA samples (left) because of the greater representation of Θp in tRNA compared to rRNA. (Open bars) CBF5⁺/rsg; (light gray bars) CBF5⁺glu; (dark gray bars) GAL::cbf5 rsg; (solid bars) GAL::cbf5 glu.

Figure 8

The box H + ACA snoRNAs are associated with Cbf5p–protein A (CBFS-Prot.A). Immunoprecipitations were performed at two salts concentration [150 and 500 mm Kacetate (KAc)] on two CBF5–Prot.A strains (YDL524-18 and YDL524-19) and at 150 mm Kacetate on the wild-type isogenic control (CBF5). RNA was extracted from equivalent amounts of total (T), supernatant (S), and pellet (P) fractions and separated on a 8% polyacrylamide gel containing 8 m urea. Probes used for the hybridizations are described in Materials and Methods.

Figure 9

Box H + ACA snoRNP components are codepleted in a GAL::cbf5 strain. H + ACA snoRNAs (A,B) and Gar1p (C) are codepleted with Cbf5p. Probes used for the hybridizations are described in Materials and Methods. RNA was extracted from the CBF5 and GAL::cbf5 strains following growth on rsg medium (0-hr lanes) and at intervals following transfer to glu medium (8- to 70-hr lanes) and separated on an 8% polyacrylamide gel containing 8 m urea. The anti-Gar1p antibody used was described by Girard et al. (1992) and cross-reacts weakly with Nop1p.

Figure 9

Box H + ACA snoRNP components are codepleted in a GAL::cbf5 strain. H + ACA snoRNAs (A,B) and Gar1p (C) are codepleted with Cbf5p. Probes used for the hybridizations are described in Materials and Methods. RNA was extracted from the CBF5 and GAL::cbf5 strains following growth on rsg medium (0-hr lanes) and at intervals following transfer to glu medium (8- to 70-hr lanes) and separated on an 8% polyacrylamide gel containing 8 m urea. The anti-Gar1p antibody used was described by Girard et al. (1992) and cross-reacts weakly with Nop1p.

Figure 9

Box H + ACA snoRNP components are codepleted in a GAL::cbf5 strain. H + ACA snoRNAs (A,B) and Gar1p (C) are codepleted with Cbf5p. Probes used for the hybridizations are described in Materials and Methods. RNA was extracted from the CBF5 and GAL::cbf5 strains following growth on rsg medium (0-hr lanes) and at intervals following transfer to glu medium (8- to 70-hr lanes) and separated on an 8% polyacrylamide gel containing 8 m urea. The anti-Gar1p antibody used was described by Girard et al. (1992) and cross-reacts weakly with Nop1p.