Mrd1p is required for release of base-paired U3 snoRNA within the preribosomal complex

Abstract

In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A(2) cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A(0) to A(2). Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.

FIG. 1.

The RBD mutations in Mrd1p and their effect on growth. (A) Schematic structure of Mrd1p. The RBD deletion mutations, Δ1 to Δ5, are indicated at the top. The positions of the RNP1 and RNP2 motifs are shown for RBD5. The consensus sequence of the RNP1 motif is shown below the protein structure. The wt amino acid sequences of RNP1 in RBD1 to -5 of Mrd1p and substitutions to leucines at positions 3 and 5 (bold) are shown for each RBD (mutRBD). (B) Western blot analysis of wt and mutated Mrd1p. Equal amounts of total protein were analyzed for each strain. (C) Growth of wt and Δ1 to Δ5 strains on 5-FAA plates. The wt MRD1 gene contained in a TRP1 plasmid (pRS424/MRD1) was lost under these conditions. (D) Growth of the Δ1, Δ2, and Δ4 strains, lacking a wt MRD1 gene, on YPD at 30°C and 37°C. (E) Growth of the Δ3, Δ5, and mutRBD5 strains compared to that after depletion of Mrd1p. The cells were grown on YPD plates to shut off synthesis of a GAL1 promoter-driven wt MRD1 gene.

FIG. 2.

Pre-rRNA processing in the Mrd1p mutant strains. (A) Schematic representation of 35S pre-rRNA, showing processing sites (A₀ to E) and positions of oligonucleotide probes (1 to 3) used for Northern blot hybridizations. (B) Effect of the Δ1 to Δ5 mutations on mature rRNAs and on pre-rRNAs. The lethal Δ3, Δ5, and mutRBD5 strains were depleted for the wt Mrd1p in YPD for 6 h. For comparison, RNA from Mrd1p-depleted cells (GAL::3HA-Mrd1) and wt cells were included. Top, methylene blue (MB)-stained Northern blot membrane. The filter was hybridized with probes specific for 25S and 18S rRNAs, and the ratios between the signals for 18S and 25S rRNA are shown at the bottom (18S/25S). The same membrane was hybridized with probes 1, 2, and 3 as indicated. Positions of 25S rRNA, 18S rRNA, and pre-rRNAs are indicated.

FIG. 3.

EM analyses of cotranscriptional pre-rRNA processing. (A) Bar graphs show a semiquantitative analysis (see Materials and Methods) of the fraction of rRNA genes in the various strains that displayed the following normal characteristics: a robust level of transcription (as in genes in panels B, D, and F), the presence of 5′ETS particles on at least some nascent transcripts (as in genes in panels B to I, thin arrows), the presence of SSU processome particles on at least some nascent transcripts (as in genes in panels B and F to I, larger arrows), and a normal level of cotranscriptional cleavage at the A₂ site (as in gene in panel B, bracket). (B to I) Examples of individual rRNA genes from the wt and seven mutant strains, as labeled in each panel. Chromatin spreads from the Δ3, Δ5, and mutRBD5 strains were obtained after 17 to 20 h under depletion conditions to turn off the wt MRD1 gene.

FIG. 4.

Cellular locations of the Δ1 to Δ5 and mutRBD5 proteins and their association with pre-rRNP and pre-rRNA. (A) The locations of the Myc-tagged wt Mrd1p and the Δ1 to Δ5 mutant proteins are shown by immunofluorescence (green). DAPI staining (blue) shows the intranuclear location of the chromosomal DNA. FITC, fluorescein isothiocyanate. (B) The location of the mutRBD5 protein is shown by immunofluorescence. DAPI staining is as in panel A. (C) Sucrose gradient analyses of Mrd1p in extracts from wt and mutant strains. All Mrd1p versions were tagged with a 13Myc tag, and the location was determined by Western blot analyses of the individual fractions. Fraction numbers are shown at the bottom. The positions of 40S, 60S, and 80S rRNAs, as determined from analyses of rRNA by agarose electrophoresis, are indicated at the top. The typical position of 35S pre-rRNA, determined by hybridization with probe 1, is also shown. (D) Coimmunoprecipitation of pre-rRNAs and U3 snoRNA with wt Mrd1p and with the lethal MRD1 mutants (Δ3, Δ5, and mutRBD5). Extracts from cells with untagged Mrd1p as a negative control and from cells with wt Mrd1p, Δ3, Δ5, and mutRBD5, all tagged with 13Myc, were immunoprecipitated with anti-Myc antibodies. wt Mrd1p was depleted in the mutant strains for 6 h in YPD. Immunoprecipitated RNA (IP) and RNA in 1/30 of the extracts (Input) were analyzed in parallel. The same membrane was hybridized with probe 1 and with a U3 snoRNA probe.

FIG. 5.

Association of U3, U14, and snR30 snoRNPs with pre-rRNP complexes and pre-rRNA in the presence of lethal MRD1 mutations. (A and C) Extracts from wt cells; cells depleted for Mrd1p (−Mrd1p); or cells depleted for wt Mrd1p for 6 h with Δ3, Δ5, mutRBD5, or Δ1 as indicated were fractionated in 10 to 50% sucrose gradients. The Δ1 cells were grown at 37°C for 3 h. At this temperature Δ1 Mrd1p is not functional. U3 snoRNA (U3) (A) and U14 snoRNA (U14) (C) were located by Northern blot hybridization. (B and D) Extracts from the same cells as described above were treated with proteinase K prior to centrifugation in 10 to 30% sucrose gradients. U3 and U14 snoRNAs were located as described above. (E and F) Position of snR30 in gradients from wt cells and from cells depleted for Mrd1p. (G and H) The positions of 5S rRNA, 5.8S rRNA, and 35S pre-rRNA in the gradients were determined by hybridization with specific probes (5S and 5.8S probes and probe 1 [Fig. 2A]; see Table S2 in the supplemental material).

FIG. 6.

Northern blot analyses of RNAs from wt, ΔRBD1, ΔRBD3/GAL::HA-Mrd1 (grown in YPD medium for 14 h), and ΔRBD1/ΔRBD3 strains. (A) 25S and 18S rRNAs shown by staining with methylene blue. (B) Pre-rRNAs after hybridization with probe 2. Positions of defined pre-rRNAs are indicated.