Requirement of fission yeast Cid14 in polyadenylation of rRNAs

Abstract

Polyadenylation in eukaryotes is conventionally associated with increased nuclear export, translation, and stability of mRNAs. In contrast, recent studies suggest that the Trf4 and Trf5 proteins, members of a widespread family of noncanonical poly(A) polymerases, share an essential function in Saccharomyces cerevisiae that involves polyadenylation of nuclear RNAs as part of a pathway of exosome-mediated RNA turnover. Substrates for this pathway include aberrantly modified tRNAs and precursors of snoRNAs and rRNAs. Here we show that Cid14 is a Trf4/5 functional homolog in the distantly related fission yeast Schizosaccharomyces pombe. Unlike trf4 trf5 double mutants, cells lacking Cid14 are viable, though they suffer an increased frequency of chromosome missegregation. The Cid14 protein is constitutively nucleolar and is required for normal nucleolar structure. A minor population of polyadenylated rRNAs was identified. These RNAs accumulated in an exosome mutant, and their presence was largely dependent on Cid14, in line with a role for Cid14 in rRNA degradation. Surprisingly, both fully processed 25S rRNA and rRNA processing intermediates appear to be channeled into this pathway. Our data suggest that additional substrates may include the mRNAs of genes involved in meiotic regulation. Polyadenylation-assisted nuclear RNA turnover is therefore likely to be a common eukaryotic mechanism affecting diverse biological processes.

FIG. 1.

S. pombe cid14 is a functional homologue of S. cerevisiae TRF4 and is required for normal cell growth. (A) trf4ts top1Δ mutants transformed with plasmids carrying full-length cid1, cid14, and TRF4, as indicated, under the control of the GAL1 promoter or “empty” vector were streaked onto synthetic defined plates containing galactose. Plates were photographed after 3 days of incubation at 25°C or the nonpermissive temperature of 36°C. (B) Tenfold serial dilutions of the indicated strains spanning the range from 10⁶ to 10² cells, as indicated, were spotted onto YE-agar plates and photographed after 2 days of incubation at 30°C. (C) Micrographs of Hoechst 33342-stained living wild-type and cid14Δ cells. Bar: 10 μm.

FIG. 2.

Deletion of cid14 leads to an increased chromosome segregation failure rate but not to precocious sister chromatid separation during metaphase arrest. (A) Merged images of fluorescence micrographs showing GFP-Swi6 and DNA (Hoechst 33342) localization in living cells. Bar: 10 μm. Percentages of cells with lagging chromosomes are indicated. (B) Visualization of unequal segregation of GFP-Swi6 signal in cid14Δ cells. Individual GFP-swi6 (cid14⁺) and GFP-swi6 cid14Δ cells were observed as for panel A, over a 4-min period, with images collected every minute. (C) Six tetrads derived from diploid strains h⁺/h⁻ mad2::ura4⁺/mad2⁺ cid14::LEU2/cid14⁺ (left) and bub1::ura4⁺/bub1⁺ cid14::LEU2/cid14⁺ (right) were microdissected onto YE-agar, and the resulting colonies were photographed after 5 days of growth at 30°C. The genotypes of the segregants were determined by replica plating. Boxes indicate the position of cid14 double mutants with mad2 and bub1, respectively. (D) Merged images of fluorescence micrographs showing Ndc80-GFP localization in nda3-KM311, cid14Δ nda3-KM311, and cid12Δ nda3-KM311 cells in EMM medium after incubation at 20°C for 8 h. Fluorescence micrographs of living cells were acquired after staining with Hoechst 33342, revealing green fluorescence (Ndc80-GFP) and DNA (Hoechst). (E) Percentages of cells with more than three Ndc80-GFP spots, indicative of precocious sister chromatid separation, in each strain are shown (n = 200).

FIG. 3.

S. pombe Cid14 localizes to the nucleolus throughout the cell cycle. (A) Whole-cell protein extracts from wild-type (control) and cid14-HA strains were prepared by trichloroacetic acid precipitation following glass bead disruption. The extracts were separated by SDS-PAGE and subjected to immunoblotting using anti-HA antibodies. (B) Cells expressing Cid14-HA were synchronously released from the G₂ block imposed by a temperature-sensitive cdc25-22 mutation. Samples taken at the times indicated were used to score percentages of septated cells and subjected to immunoblotting using anti-HA (Cid14) or anti-Cdc2 (loading control) antibodies as indicated. (C) The size of soluble Cid14-HA was estimated by gel filtration chromatography. Samples of each 0.5-ml Superose-6 column fraction were processed for immunoblotting with an anti-HA antibody. The elution volume (ml) of each fraction is shown above the blots. The positions at which dextran blue (∼2,000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), and bovine serum albumin (66 kDa) migrated are indicated. (D and E) Merged images of fluorescence micrographs showing Cid14-GFP and DNA (Hoechst 33342) localization in (D) living wild-type cells grown at 30°C and (E) nda3-KM311 mutants after incubation at 20°C for 8 h. (F) Merged images of fluorescence micrographs showing Cid14-GFP and nucleolar localization (Et Br, ethidium bromide) in living cells grown at 30°C. Bar: 10 μm.

FIG. 4.

Aberrant nucleolar structures in the cid14Δ mutants. (A) Merged images of fluorescence micrographs showing Gar2-GFP and DNA (Hoechst 33342) localization in living cells. Arrowheads indicate cells with aberrant nucleolar structures. (B) Visualization of unequal segregation of Gar2-GFP signal in cid14Δ cells. Individual gar2-GFP (cid14⁺) and gar2-GFP cid14Δ cells were observed as in panel A, over a 10-min period, with images collected every 2 min. Bar: 10 μm.

FIG. 5.

Cid14-dependent polyadenylated 25S rRNAs are accumulated in a dis3-54 mutant. (A) Schematic diagram of the primers for RT-PCR. Alternative processing cleavage sites (P1 and P2) and transcription termination sites (T1-3) are indicated. (B) The 3′-terminal sequences of cloned reverse transcription products. (C) Total RNA from cultures of indicated strains grown at 30°C or the nonpermissive temperature of 20°C for dis3-54 mutants was reverse transcribed using the anchored oligo(dT) primer AT and then PCR amplified with AT and the F1 primer specific for 25S rRNA. RT-PCR products were separated on a 1% agarose gel, and the relative signals were quantified as indicated. A control using total RNA and a non-reverse-transcribed negative control were included. (D) Total RNA from cultures of cid14Δ strains containing the indicated plasmids (with or without mutation in the nucleotide transferase motif: GS X₁₀ DXD of Cid14) and grown in the presence of 15 μΜ thiamine was subjected to RACE-PAT assay as for panel C. RT-PCR products were separated on a 1% agarose gel. A non-reverse-transcribed negative control was included. (E) Reverse transcripts were made with the R1 or AT primer and RNA from wild-type and dis3-54 strains grown at 30°C. Serial dilutions of R1 transcripts (for total 25S rRNA) were PCR amplified with F2 and R1 and compared with AT transcripts (polyadenylated 25S rRNA) amplified with primer pair F2 and AT or F2 and R1.

FIG. 6.

Analysis of the polyadenylation status of 5.8S and 5S rRNA in different genetic backgrounds. (A and D) Schematic diagram of the primers for RT-PCR. (B and E) Total RNA from cultures of indicated strains grown at 30°C or the nonpermissive temperature of 20°C for dis3-54 mutants was reverse-transcribed with the oligo(dT) anchor primer AT and then PCR amplified with AT and forward primers F1 specific for 5.8S and 5S rRNA, respectively. For detection of mature rRNA, reverse transcripts were made with the R1 primer specific for 5.8S and 5S rRNA and then PCR amplified with pair F1 and R1. RT-PCR products were separated on a 1.5% agarose gel, and the relative signals were quantified as indicated. (C and F) Genomic sequences of 5.8S and 5S genes. Arrowheads indicate polyadenylation sites identified. (G) Tenfold serial dilutions of wild-type and cid14Δ strains spanning the range from 10⁶ to 10² cells, as indicated, were spotted onto YE-agar containing 2 μg/ml 5-FU or no drug (control). Plates were photographed after 3 days of incubation at 30°C.

FIG. 7.

Deletion of cid14 leads to stimulation of meiosis in pat1-114 cells. (A) Iodine staining of colonies derived from spores of diploid h⁺/h⁻ cid14::ura4⁺/cid14⁺ pat1-114/pat1⁺ and h⁺/h⁻ dis3-54/dis3⁺ pat1-114/pat1⁺ strains after 5 days of growth on YE-agar at 25°C. Iodine stains cells that have undergone meiosis and sporulation dark brown. (B) Micrographs of the strains indicated from panel A. Arrowheads indicate cells that have undergone meiosis and sporulation. Bar: 10 μm. (C) Total RNA from cultures of the indicated strains grown at 30°C was reverse transcribed using an oligo(dT) primer. The cDNA was amplified using quantitative PCR and SYBR green in triplicate with primers specific for the indicated genes. RNA amounts normalized to cdc2 mRNA were expressed relative to the wild type.