The role of nuclear cap binding protein Cbc1p of yeast in mRNA termination and degradation

Abstract

The cyc1-512 mutation in Saccharomyces cerevisiae causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly long cyc1-512 mRNAs which differ in length at their 3' termini. cyc1-512 can be suppressed by deletion of either of the nonessential genes CBC1 and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex. The upf1-Delta deletion suppressed the cyc1-512 defect by diminishing degradation of the longer subset of cyc1-512 mRNAs, suggesting that downstream elements or structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of cyc1-512 defects by cbc1-Delta occurred by two different mechanisms. The levels of the shorter cyc1-512 transcripts were enhanced in the cbc1-Delta mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longer cyc1-512 transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore, cbc1-Delta greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.

FIG. 1

Comparison of the growth of CBC1⁺, cbc1-Δ, and cbc2-Δ strains. The three isogenic strains were grown at 30°C on YPD glucose medium for 2 days, YPG glycerol medium for 4 days, or YPR raffinose medium for 2 days. (A) B-9037 (CBC1⁺); (B) B-9038 (cbc1-Δ); (C) B-12463 (cbc2-Δ).

FIG. 2

Northern blot analyses of the eight cyc1-512 mRNAs listed in Table 3. Poly(A) RNA was extracted from four strains treated with 4 μg of thiolutin/ml for 0 to 30 min. (A and E) B-9037 (CBC1⁺ UPF1⁺); (B and F) B-9038 (cbc1-Δ UPF1⁺); (C and G) B-9848 (CBC1⁺ upf1-Δ); (D and H) B-11559 (cbc1-Δ upf1-Δ). (A to D) cyc1-512 mRNAs; (E to H) ACT1 mRNA. The values presented in Table 3 were estimated from these blots by normalizing them to ACT1 mRNA values, whose rates of degradation for these strains are known.

FIG. 3

Schematic representation of the cyc1-512 mRNAs, showing their lengths in nucleotides and the steady-state increases due to suppression by cbc1-Δ or upf1-Δ.

FIG. 4

Northern blot analysis of CYH2 mRNA and pre-mRNA (left) from strains B-9037 (CBC1⁺ UPF1⁺), B-9038 (cbc1-Δ UPF1⁺), B-9848 (CBC1⁺ upf1-Δ), and B-11559 (cbc1-Δ upf1-Δ) treated with 4 μg of thiolutin/ml for 0 to 40 min as indicated above the blots. 18S rRNA is shown on the right-hand side.

FIG. 5

Decay in thiolutin-treated cells of cyc1-512 630-nt mRNA (A), CYH2 pre-mRNA (B), CYH2 mRNA (C), and ACT1 mRNA (D) from the following strains: B-9037 (normal; cyc1-512) (●); B-9038 (cyc1-512 cbc1-Δ) (○); B-9848 (cyc1-512 upf1-Δ) (▾); and B-11559 (cyc1-512 cbc1-Δ upf1-Δ) (▿). The decay was determined by Northern blot analysis of RNA extracted from cells treated with 4 μg of thiolutin/ml for 0 to 40 min. The results are presented as the percentage of remaining RNA versus time of incubation in the presence of thiolutin.

FIG. 6

cbc1-Δ does not suppress the nonsense mutations. Three strains, B-11623 (UPF1⁺ CBC1⁺), B-9198 (UPF1⁺ cbc1-Δ), and B-9199 (upf1-Δ CBC1⁺), were grown on YPD plates for 2 days, replicated onto omission media lacking either leucine (−Leu), histidine (−His), or methionine (−Met) and subsequently incubated for 3 days to test suppression of leu2-1 (UAA), his4-166 (UGA), and met8-1 (UAG).

FIG. 7

Northern blot analysis of B-11623 (cyc1-72), B-9198 (cyc1-72 cbc1-Δ), and B-9199 (cyc1-72 upf1-Δ) showing the CYC1 steady-state mRNA levels compared to the level of 18S ribosomal DNA (rDNA).

FIG. 8

Decay of ACT1 and CYH2 mRNAs and pre-mRNA at 37°C in B-10603 (RAT7⁺), B-10095 (rat7-1), B-10096 (rat7-1 cbc1-Δ), and B-10097 (rat7-1 upf1-Δ). The strains were grown at 23°C to the mid-log phase, shifted to the restricted temperature of 37°C for 15 min, and subsequently treated with 4 μg of thiolutin/ml to inhibit transcription, and the decay was followed for 0 to 50 min. Northern blots were probed for CYH2 mRNA (bottom band in each blot) and pre-mRNA (top band in each blot) (A) and for ACT1 mRNA (B). The numbers at the right denote the half-lives (t_1/2) of the mRNAs in minutes.

FIG. 9

Growth of B-10603 (RAT7⁺), B-10095 (rat7-1), B-10096 (rat7-1 cbc1-Δ), and B-10097 (rat7-1 upf1-Δ) strains on YPD medium at 30 and 37°C for 3 days, showing the suppression of growth in rat7-1 strains by cbc1-Δ.