Deletion of the nuclear exosome component RRP6 leads to continued accumulation of the histone mRNA HTB1 in S-phase of the cell cycle in Saccharomyces cerevisiae

Abstract

The nuclear exosome, a macromolecular complex of 3' to 5' exonucleases, is required for the post-transcriptional processing of a variety of RNAs including rRNAs and snoRNAs. Additionally, this complex forms part of a nuclear surveillance network where it acts to degrade any aberrantly processed mRNAs in the nucleus. The exosome complex has been implicated in the biogenesis pathway of general messenger RNAs through its interaction with the 3'-end processing machinery. During the cell cycle, yeast histone mRNAs accumulate in the S-phase and are rapidly degraded as cells enter the G2-phase. To determine if the exosome contributes to the cyclic turnover of yeast histone mRNAs, we examined the pattern of accumulation of 'HTB1' mRNA during the cell cycle in a deletion strain of 'RRP6', a component of the nuclear exosome. Our results show that cells lacking Rrp6p continue to accumulate HTB1 mRNA as the cell cycle proceeds. This continued accumulation appears to result from a delay in exit from S-phase in rrp6 cells. The accumulation of HTB1 mRNA in rrp6 cells is influenced by the interaction of the nuclear exosome with the 3'-end processing machinery although there is no evidence for differential regulation of histone mRNA 3'-end processing during the yeast cell cycle.

Figure 1.

(A) Diagram of the structure of the neo-HTB1 chimeric gene. The gene is under the control of the GAL1 promoter. The HTB1 gene sequences (from nt position +1005) include the last 17 amino acids of the open reading frame (+1005 to +1062) (grey-shaded box), the 3′-UTR, 3′-end cleavage sites (arrow) and downstream sequences and are fused downstream of the neomycin phosphotransferase open reading frame (unshaded rectangle). The location of the DDE is indicated by a black box (+1276 to +1321). The sequences within the DDE are shown below the diagram and the sequence changes in pSAC 15, 20 and 21 are indicated below. (B) CFIA mutants affect the steady-state levels of neo-HTB1 mRNAs. The temperature-sensitive mutants rna14-3, rna15-2 and pcf11-2 were transformed with a plasmid containing the neo-HTB1 gene. RNA was extracted following incubation of the mutants for 1 h at the non-permissive temperature 37°C. Following electrophoresis and gel transfer, the neo-HTB1 mRNAs were detected using a digoxigenin-labelled DNA probe prepared using the primers neo_(F) and neo_(R), Table 2. The upper panel shows the 18S and 28S rRNAs used as a loading control. Note lane 3 is overloaded compared to the other lanes. Lane 1, 7: WT S. cerevisiae strain S-150B, 2: rna14-3, 3: rna15-2, 4: rna14-3/Δrrp6, 5: rna15-2/Δrrp6, 6: Δrrp6, 8: pcf11-2. *denotes processed mature mRNAs in lanes 4 and 5.(C) Growth of mutant strains on G418 plates. Mutant strains containing the neo-HTB1 plasmid were plated on YEPGal plates containing 200 μg/ml G418. Plates were incubated at 30°C.

Figure 2.

DDE mutants are preferentially unstable in rna14-3 and stabilized in Δrrp6 cells. Strains rna14-3, rna14-3/Δrrp6 and Δrrp6 were transformed with plasmids containing the WT neo-HTB1 gene or the DDE mutant genes pSAC15, pSAC 20 or pSAC 21. Cells were incubated at the permissive temperature and prior to harvesting, were incubated at 22°C or 37°C for 1 h. RNA was extracted and probed for neo-HTB1 and actin mRNAs. Lanes 1, 5, 9: WT neo-HTB1, Lanes 2, 6, 10: pSAC 15 neo-HTB1, Lanes 3, 7, 11: pSAC 20. Lanes, 4, 8, 12: pSAC 21. Note lane 9 in the 22°C and 37°C panels is under loaded.

Figure 3.

Deletion of RRP6 decreases the turnover of HTB1 mRNA following the S-phase of the cell cycle. Yeast strains W303A (A) and Δrrp6 (B) were treated with α-factor as described in the Materials and Methods section. Samples were taken at 10-min intervals following removal of the α-factor. The extracted RNA was electrophoresed on a denaturing gel and transferred to a nylon membrane. The HTB1 and actin mRNAs were detected as described in the Materials and Methods section. (C) Quantification of HTB1 mRNA levels during the cell cycle. Normalized levels of HTB1 mRNA in strains W303A (black line) and Δrrp6 (grey line) following removal of α-factor. The data shown are the average levels obtained from real-time RT-PCR and northern blot analysis of four independent cell cycle experiments. The error bars represent the SE between experiments. The primers used for real time RT-PCR amplification of HTB1 and actin mRNAs are HTB1_Endo_F/HTB1_Endo_R and actin_F(2)/actin_R(2), Table 2. (D) Growth curve of W303 and Δrrp6 at 30°C. Error bars represent the average error of triplicate reading of optical density at 600 nm. Black line, W303, grey line Δrrp6. (E) Flow cytometry analysis of synchronized W303 and Δrrp6 cells. Cells were synchronized as described in the Materials and Methods section and samples taken every 15 min after α-factor removal. The cells were stained with propidium iodide and sorted as described in the Materials and Methods section. Flow analysis for every second sample is shown. Vertical lines show the position of 1n and 2n cells.

Figure 4.

Cell cycle accumulation pattern of HTB1 mRNAs in rna14-3/Δrrp6 cells. α-factor was added to strains W303B and rna14-3/Δrrp6 and the cells were incubated overnight at 22°C. The α-factor was removed and cells were then incubated for a further hour at either 22°C or 37°C. The cells were then returned to 22°C for the remainder of the experiment. Samples were collected at 1 h intervals. HTB1 mRNA levels were detected as outlined in the Materials and Methods section. (A) Lanes 1–9, W303B incubated at 22°C, lanes 10–18, rna14/Δrrp6 incubated at 22°C. (B) Lanes 1–9, W303B incubated at 37°C for 1 h after α-factor removal. Lane 1 sample was taken immediately after the 1 h incubation at 37°C. Lanes 10–18, rna14-3/Δrrp6 incubated at 37°C for 1 h after α-factor removal. Lane 10 sample was taken immediately after the 1 h incubation at 37°C. The numbers above the figure show the time points after α-factor removal.

Figure 5.

The 3′-ends of HTB1 mRNAs are not differentially processed during the cell cycle. (A) Diagram of the 3′-end of the HTB1. The location of the 3′-end cleavage sites (arrows) and the DDE are shown. The locations of the forward (F) and reverse (R) primers used for RT-PCR are shown. The expected sizes of the RT-PCR products using the common F primer and specific R primers are shown below each R primer. (B) RT-PCR products of HTB1 mRNA from rna14-3/Δrrp6 cells grown at 22°C and then incubated for 1 h at either 22°C or 37°C prior to RNA extraction using the forward and reverse primers shown in A. Lane 1; F and R1 but no reverse transcriptase added. 2; DNA 100 bp ladder molecular weight marker, 3; primers F and R1, 4; primers F and R2, 5; primers F and R3, 6; primers F and R4, 7; primers F and R5, 8; primers F and R6. (C) RT-PCR products of W303 RNA extracted at 1 and 6 h following alpha factor release using the forward and reverse primers shown in A. Lanes 1, 8; DNA 100 bp ladder molecular weight marker. 2, 9; F and R1 but no reverse transcriptase added. 3, 10; primers F and R1. 4, 11; primers F and R2. 5, 12; F and R3. 6; 13; F and R5. (D) As for C but with RNA extracted from rna14-3/Δrrp6 cells at 1 and 6 h following alpha factor release. Note: lane 12 is under loaded.