Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae

Abstract

A genetic screen for mutations synthetically lethal with temperature sensitive alleles of nop2 led to the identification of the nucleolar proteins Nop12p and Nop13p in Saccharomyces cerevisiae. NOP12 was identified by complementation of a synthetic lethal growth phenotype in strain YKW35, which contains a single nonsense mutation at codon 359 in an allele termed nop12-1. Database mining revealed that Nop12p was similar to a related protein, Nop13p. Nop12p and Nop13p are not essential for growth and each contains a single canonical RNA recognition motif (RRM). Both share sequence similarity with Nsr1p, a previously identified, non-essential, RRM-containing nucleolar protein. Likely orthologs of Nop12p were identified in Drosophila and Schizosaccharomyces pombe. Deletion of NOP12 resulted in a cold sensitive (cs) growth phenotype at 15 degrees C and slow growth at 20 and 25 degrees C. Growth of a nop12Delta strain at 15 and 20 degrees C resulted in impaired synthesis of 25S rRNA, but not 18S rRNA. A nop13 null strain did not produce an observable growth phenotype under the laboratory conditions examined. Epitope-tagged Nop12p, which complements the cs growth phenotype and restores normal 25S rRNA levels, was localized to the nucleolus by immunofluorescence microscopy. Epitope-tagged Nop13p was distributed primarily in the nucleolus, with a lesser portion localizing to the nucleoplasm. Thus, Nop12p is a novel nucleolar protein required for pre-25S rRNA processing and normal rates of cell growth at low temperatures.

Figure 1

nop12-1 is synthetically lethal with nop2-4. (A) Diagram of strain YJPA24 used for the sl screen. Retention of pJPA84 is selected on minimal medium minus tryptophan. Spontaneous loss of pPW67 at permissive temperature (25°C) results in white sectors in red colonies and confers resistance to 5-FOA. (B) Growth at 25°C on medium containing 5-FOA. YJPA22 was transformed with pRS314, or plasmids bearing NOP2 or different nop2 ts alleles. NOP2 supports loss of pPW67, whereas pRS314 does not. Temperature sensitive nop2 alleles support spontaneous loss of pPW67 and growth to different extents. (C) Allele specificity. The nop12-1 allele in YKW35 is synthetically lethal with nop2-4, nop2-9 and nop2-10, but not with nop2-3, nop2-5 or nop2-6. YKW35 transformants that carry nop2-4, nop2-9 or nop2-10 cannot spontaneously lose pPW67 and remain viable. (D) Complementation of synthetic lethality. Region of chromosome XV that complements the sl phenotype in YKW157. Open arrows denote endpoints of insert in pKW28. Filled arrows denote insert in pKW30. RPS15 is also known as RPS21. (E) NOP12 supports growth on medium containing 5-FOA. YKW35 bearing nop2-4 (on pJPA30) can afford to lose pPW67 and remain viable on medium containing 5-FOA if transformed with NOP2 (pBH49) or NOP12 (pKW39), but not with control plasmid pRS313, or plasmids bearing RPS15 (pKW36) or RPP2A (pKW38).

Figure 2

CLUSTAL alignment of Nop12p (encoded by YOL041c), Nop13p (encoded by YNL175c) and Nsr1p (11). Identical amino acids are boxed. RRM elements RNP-1 (underlined octapeptide) and RNP-2 (underlined hexapeptide) are indicated. RNP-1 and RNP-2 elements in Nsr1p that are not present in Nop12p are indicated (dashed underline). Amino acid residues within the conserved RRM that are present in either Nop12p or Nop13p and both Nsr1p and mammalian nucleolin are denoted with a filled circle. Lysine₃₅₉, which is mutated to a stop codon in nop12-1, is denoted with a black box.

Figure 3

Cold sensitivity in a nop12Δ strain. (A) Replica platings of serial dilutions of wild-type (WT), nop12Δ, nop13Δ and nsr1Δ strains grown for 2 or 13 days at the indicated temperatures on YPD medium. (B) Growth in liquid culture of wild-type (NOP12) and nop12Δ strains at 30°C and after shift to 20 and 15°C. Cultures were diluted during the time course to maintain OD₆₀₀ < 0.5.

Figure 4

Pulse–chase analysis of rRNA synthesis. Wild-type and nop12Δ strains were cultured at 30°C, shifted to 15°C for 5 h, pulse-labeled with [³H-methyl]methionine for 5 min at 15°C and chased for the indicated times (in min) at 15°C. RNAs were separated by gel electrophoresis, transferred to nylon membrane and visualized by fluorography. Positions of pre-rRNAs and mature rRNAs are indicated. Ratios of band intensities (25S/18S) as determined by NIHImage software are shown below each lane.

Figure 5

Northern analysis of steady-state levels of pre-rRNAs and rRNAs. (A) Northern blots of total RNA were prepared from the nop12Δ strain (Δ) or the isogenic NOP12 strain (12). Early log cultures at 25°C were continued at 25°C or shifted to either 20 or 15°C for 5 h prior to RNA extraction. The blot was hybridized sequentially with probes to ITS1 and ITS2 (which detect 35S, 27S and 20S pre-rRNAs); 25S and 18S rRNAs and ACT1 mRNA. (B–E) Quantification of hybridization results. Data for individual bands were acquired with a PhosphorImager, corrected for local background and used to calculate ratios (without any further computational manipulation). Thus, the absolute values on the ordinate axis are arbitrary and presented without units.

Figure 6

Nop12p–HA complements the nop12Δ cs phenotype. (A) Replica platings of serial dilutions were grown for 2 or 11 days at the indicated temperatures on YPD. The strains compared are wild-type (WT), nop12Δ and the nop12Δ strain transformed with a centromeric plasmid expressing HA-tagged Nop12p from its endogenous promoter (pKW42). (B) Ethidium bromide-stained rRNAs. Total RNAs were isolated from the strains described above grown at 15°C for 5 h and were separated on a formaldehyde agarose gel. Ratios of band intensities (25S/18S) as determined by NIHImage software are shown below each lane. (C) Western blot. Total cell protein extracts prepared from wild-type cells, or cells expressing HA epitope tagged Nop12p or Nop13p, were analyzed by western blotting with mAb 16B12, followed by chemiluminescent detection. Positions of molecular standards are indicated (in kDa).

Figure 7

Immunofluorescence localization. Yeast cells expressing epitope tagged Nop12p or Nop13p were prepared for microscopy and incubated with antibodies as described in Materials and Methods. Nop12p–HA (a) and Nop13p–HA (e) were localized simultaneously with Nop1p (b and f). Counterstaining with DAPI revealed the distribution of chromatin (c and g). Phase contrast images (d and h) of the same fields of cells are shown. Bar, 5 µm.

Figure 8

Characterization of the nop12-1 allele. (A) A nop12 null strain was transformed with the following plasmids: pKW42 (expresses Nop12p + C-terminal HA epitope tag); pRS313 (vector); pKW44 (carrying ‘nop12-1’, which encodes Nop12-1p); and pKW46 (carrying ‘nop12-1–HA’, which encodes Nop12-1p–HA). Replica platings of serial dilutions were grown for 2 or 13 days at the indicated temperatures on YPD. (B) Immunofluorescence localization in nop12Δ strains transformed with pKW46 or pKW42 was done as described in Figure 7. Nop12-1p–HA (a) and Nop12p–HA (e) were localized simultaneously with Nop1p (b and f). Counter-staining with DAPI (c and g) and phase contrast images (d and h) of the same fields of cells are shown. Bar, 5 µm.