Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae

Abstract

Using molecular genetic techniques, we have generated and characterized six temperature sensitive (ts) alleles of nop2. All failed to support growth at 37 degrees C and one was also formamide sensitive (fs) and failed to grow on media containing 3% formamide. Conditional lethality is not due to rapid turnover of mutant Nop2p proteins at 37 degrees C. Each allele contains between seven and 14 amino acid substitutions and one possesses a nonsense mutation near the C-terminus. Mapping experiments with one allele, nop2-4, revealed that a subset of the amino acid substitutions conferred the ts phenotype and that these mutations have an additive effect. All six mutants exhibited dramatic reductions in levels of 60S ribosome subunits under non-permissive conditions as well as some reduction at permissive temperature. Processing of 27S pre-rRNA to mature 25S rRNA was defective in all six mutants grown under non-permissive conditions. Levels of the 40S ribosomal subunit and 18S rRNA were not significantly affected. Amino acid substitutions in nop2 conditional alleles are discussed in the context of the hypothesis that Nop2p functions both as an RNA methyltransferase and a trans-acting factor in rRNA processing and large ribosomal subunit biogenesis.

Figure 1

Six ts nop2 alleles. (A) Replica platings of serial dilutions of L4717 (NOP2), YBH3 (nop2::LEU2, pJPA40) and six strains bearing plasmid-borne nop2 ts alleles. Strains were grown for 3 days at the indicated temperatures on YPD medium. (B) Rescue of conditional phenotypes with pJPA40 (NOP2). YBH23 (nop2-10) is sensitive to growth on YPD containing 3% formamide (+ FA) at 25°C, as well as to 37°C. YBH21 (nop2-9) and YBH23 transformed with pJPA40 grow as well as L4717 and YBH3 under non-permissive conditions.

Figure 2

(A) Growth of nop2 ts strains and strain YBH3 (NOP2) in YPD medium after shift from 25 to 37°C. (B) Growth of YBH23 (nop2-10), YBH15 (nop2-4) and YBH3 at 25°C after shift from YPD to YPD containing 3% formamide. Liquid cultures were diluted over the time course to maintain an OD₆₀₀ <0.5.

Figure 3

Western blot analysis. L4717, YBH3 and strains bearing nop2 ts alleles were shifted from 25 to 37°C and grown at 37°C for 6 h. Total cell protein extracts were prepared and analyzed by western blotting with an affinity-purified antipeptide polyclonal antibody directed against Nop2p (APpAb3). After stripping immunoglobulins from the membrane, it was reprobed with a monoclonal antibody (mAb D68). Nop2p (arrowhead) and non-specific protein bands (asterisks) are indicated.

Figure 4

mAb D68 recognizes Nop2p. (a–d) Immunofluorescence localization in strain L4717. mAb D68 co-localizes with anti-Nop2p affinity-purified antipeptide polyclonal antibody 3 (APpAb3; 8). Counterstaining of chromatin with DAPI and a phase contrast image of the same field are shown. (e–h) Detection of Nop2p with mAb D68 in strain YBH5 (GAL::NOP2; 14). YBH5 was shifted from YPG to YPD, grown for 1, 3, 6 or 12 h and analyzed by immunofluorescence localization with mAb D68. Panels are overexposed (by 2-fold compared with a) to reveal a low level of non-specific background staining in yeast cell cytoplasm. Bar, 5 µm.

Figure 5

Polysome analysis. Strains bearing nop2 ts alleles and YBH3 were grown at 25°C, shifted to 37°C or maintained at 25°C and grown for 2 h. YBH23 (nop2-10) was also shifted to medium containing 3% formamide (+ FA) and grown for 2 h at 25°C. Cell extracts were layered onto 15–50% sucrose gradients, centrifuged and analyzed using absorbance at 254 nm. See panel O for positions of ribosome subunits (40S and 60S), monosomes (80S) and polysomes (2–6). H, halfmer 43S preinitiation complexes are labeled.

Figure 6

Pulse–chase analysis of pre-rRNA processing. Strains bearing nop2 ts alleles and YBH31 (NOP2) were grown at 25°C and shifted to 37°C for 2 h. YBH23 (nop2-10) was shifted instead to medium containing 3% formamide (+ FA) and grown for 2 h at 25°C. A 2 min labeling pulse with [³H-methyl]-methionine was followed by chase times of 2, 4, 8 or 12 min. Labeled RNAs were separated on 1% glyoxal-agarose gels and visualized by fluorography. Precursor and mature rRNAs are indicated on the left. The 23S intermediate is indicated with an asterisk.

Figure 7

Amino acid substitutions in nop2 ts alleles. (A) Distribution of amino acid changes in six nop2 alleles. Nop2p is 618 amino acids in length. Amino acid substitutions are designated with a dot. A nonsense mutation in nop2-5 is denoted with an asterisk. Positions of changes are approximate. Silent mutations are not shown. (B) Allele numbers are listed above amino acid substitutions at the corresponding position in the Nop2p sequence, which is aligned with the sequence of human P120. Human P120 is 855 amino acids in length. Amino acids are numbered on the right. Four amino acids were mutated in two different alleles: E₁₀₈V in nop2-4 and E₁₀₈G in nop2-6; M₁₇₄T in nop2-9 and M₁₇₄I in nop2-4; P₃₁₅L in nop2-6 and P₃₁₅Q in nop2-10; and A₃₇₈T in both nop2-4 and nop2-5. The nonsense mutation in nop2-5 introduced a stop codon at K₆₀₅. Methyltransferase motifs N1, I-VI, ‘VIII’ and ‘X’ are shaded (31). The region of the alignment between E₂₅₇ and K₅₄₈ (endpoints highlighted in gray) is highly conserved among members of the Nop2p/P120 protein family. Approximate positions of BsgI and MfeI sites in the corresponding nucleotide sequence are indicated by arrowheads.

Figure 8

Mapping mutations in nop2-4. Restriction sites BsgI and MfeI were used to subclone regions of nop2-4 into a NOP2 plasmid to create alleles nop2-41 through nop2-46, which were introduced into a nop2::LEU2 strain (YBH3) by plasmid shuffling (Table 1). Serial dilutions of the resulting strains were replica plated onto YPD and grown at 25 or 37°C for 3 days. Asterisks represent approximate positions of amino acid substitutions in mutant alleles.

Figure 9

Site-directed mutagenesis of NOP2. Two point mutant alleles, nop2-441 (I₃₄₉T) and nop2-442 (A₃₇₈T), were constructed to contain the individual amino acid substitutions found in nop2-44 (Fig. 8). Mutant and control NOP2 strains were grown in YPD at 25°C and either maintained at 25°C or shifted to 37°C. Cell densities (OD₆₀₀) were measured at different times (in h) and liquid cultures were diluted during the time course to maintain an OD₆₀₀ <0.5.