Nob1p is required for cleavage of the 3' end of 18S rRNA

Abstract

We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits. Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates. 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm. Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm. This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export. Pre-40S export is not, however, inhibited by depletion of Nob1p.

FIG. 1.

Pre-rRNA processing pathway. In wild-type cells, the 35S pre-rRNA is cleaved at site A₀, producing the 33S pre-rRNA. This molecule is rapidly cleaved at site A₁ to produce the 32S pre-rRNA, which is cleaved at site A₂ releasing the 20S and 27SA₂ pre-rRNAs. The 20S pre-rRNA is exported to the cytoplasm, where it is dimethylated by Dim1p and then cleaved at site D, by an unidentified enzyme, to generate the mature 18S rRNA. 27SA₂ is processed via two alternative pathways. It is either cut at site A₃ to generate 27SA₃, which is then trimmed to site B_1S, producing 27SB_S. Alternatively, it can be processed to 27SB_L by an as yet unknown mechanism. 27SB_S and 27SB_L are matured to the 5.8S and 25S following identical pathways. Cleavage at site C₂ generates the 7S and 26S pre-rRNAs. The 7S pre-rRNA is digested 3′ to 5′ to 6S pre-rRNA and then to the mature 5.8S rRNA. The 26S pre-rRNA is digested 5′ to 3′ to the 25S pre-rRNA and then to the mature 25S rRNA. For a review on pre-rRNA processing and the known processing enzymes, see reference (40).

FIG. 2.

Multiple sequence alignment of Nob1p and related proteins. The full sequences of Nob1p and its putative eukaryotic orthologues were aligned using T-Coffee and corrected manually where required. Sequences are identified by the SwissProt number and species abbreviation. Numbers within the sequences indicate residues omitted due to unreliable alignments in these regions. Letters and symbols on the consensus line are as follows: s, small residues; t, tiny; b, big; h, hydrophobic; a, aromatic; l, aliphatic; p, polar; c, charged; −, negatively charged; +, positively charged. Individual residues with more than 80% identity in the entire alignment are shown as capital letters on the consensus line. Conserved acidic (Asp/Glu) residues in the PIN domain are denoted by red asterisks above the alignment. Conserved cysteines marked by blue asterisks are part of the zinc ribbon motif. The figure was prepared using CHROMA (10).

FIG. 3.

Nob1p is associated with pre-40S ribosomal particles. (A) The upper panel shows sedimentation of TAP-tagged Nob1p on a 10 to 50% sucrose gradient. The levels of the Nob1-TAP protein were determined by immunoblot analysis. The lower panel shows Northern analysis of the levels of the 20S pre-rRNA. Positions of 40S and 60S ribosomal subunits and 80S ribosomes are indicated, as determined by ethidium staining of the RNA recovered from each fraction (data not shown). (B and C) Northern analysis (B) and primer extension analyses (C) of rRNAs and pre-rRNAs coprecipitated with Nob1-TAP. RNA was extracted from whole cells (Tot.) and affinity-purified fractions from tagged (+ lane) and nontagged isogenic wild-type strain (− lane). The Northern blot membrane was consecutively hybridized with the probes indicated (see Fig. 6A for the locations of the probes used). The primer extension stop detected with oligonucleotide 004 is due to the cytoplasmic dimethylation of the 20S pre-rRNA at two consecutive A residues near the 3′ end of the 18S rRNA.

FIG. 4.

Depletion of Nob1p inhibits growth. (A) Growth rates of the GAL::HA-nob1 (squares) and wild-type (diamonds) strains following a shift from galactose to glucose medium. (B) Western blot analysis of Nob1p depletion. Whole-cell extracts were prepared from samples harvested at the indicated times. Equal amount of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide), and the HA tag on HA-Nob1p was detected by Western blotting.

FIG. 5.

Depletion of Nob1p inhibits 18S rRNA synthesis. GAL::HA-Nob1 and the isogenic wild-type (WT) strain were grown at 30°C in SDGal-URA medium and then transferred to SDGlu-URA for 8 h. The cells were pulse-labeled with [5,6-³H]uracil for 1 min and then chased with an excess of cold uracil. Total RNA was extracted from cell samples harvested at the indicated time points and resolved on 1.2% agarose-formaldehyde (A) and 6% acrylamide-urea (B) gels. The positions of mature rRNAs, pre-rRNAs and tRNA species are indicated.

FIG. 6.

Depletion of Nob1p impairs pre-rRNA processing. (A) Structure and processing sites of the 35S pre-rRNA. This precursor contains the sequences for the mature 18S, 5.8S, and 25S rRNAs, which are separated by the two internal transcribed spacers ITS1 and ITS2 and flanked by the two external transcribed spacers 5′ETS and 3′ETS. The positions of the oligonucleotides probes are indicated. (B to E) Northern analyses of pre-rRNA processing. The GAL::HA-nob1 and wild-type (WT) strains were growth at 30°C in YPGal and then shifted to YPD. The cells were harvested at the times indicated, and total RNA was extracted. Equal amounts of RNA (5 μg) were resolved on a 1.2% agarose-formaldehyde gel (B to D) or 6% acrylamide-urea gel (E) and transferred to a nylon membrane. (B) Ethidium bromide staining of the gel. (C) Probe complementary to NOB1 mRNA. (D and E) Probes specific for the mature rRNAs and pre-rRNAs as indicated.

FIG. 7.

Accumulation of pre-rRNA fragments on Nob1p depletion. Northern hybridization of RNA extracted from the GAL::HA-nob1 and wild-type (WT) strains following glucose shift is shown. The membranes were consecutively hybridized with the probes indicated (see Fig. 6A for probe locations). Molecular weight markers (MspI-digested pBR322) are indicated on the right in thousands.

FIG. 8.

Nob1p is localized to the nucleus and cytoplasm. Indirect immunofluorescence was performed on cells expressing TAP-tagged Nob1p and DsRed-tagged Nop1p. (A) Localization of DsRed-Nop1p. (C) Indirect immunofluorescence of Nob1-TAP with rabbit anti-protein A. (E) DNA stained with DAPI. (B) Superimposition of the signals from DsRed-Nop1p and Nob1-TAP. (D) Superimposition of the signals from Nob1-TAP and DAPI staining. (F) Superimposition of the signals from Nop1p, Nob1-TAP, and DAPI staining.

FIG. 9.

Nob1p is not required for nuclear export of the small ribosomal subunit. (A to C) The ITS1-5′ probe preferentially deccrates the nuclei of wild-type cells. (D to F) ITS1-5′ decorates both the nuclei and cytoplasm of GAL::HA-nob1 cells growth in galactose. (G to I) ITS1-5′ strongly decorates the cytoplasm of GAL::HA-nob1 cells after 6 h of depletion in glucose. DNA was labeled with DAPI to visualize the nucleus.