Figure 3.

The mechanism that blocks crs1 processing in vegetative cells requires the nuclear exosome in conjunction with a factor implicated in turnover of meiotic transcripts in proliferating cells. a, (Top panel) Splicing of crs1 pre-mRNA was assayed by RT-PCR as in Fig. 2d. The relevant genotype of each strain assayed is indicated above the corresponding gel lane; complete genotypes are provided in Supplementary Table 3 (online). All strains were grown at 30°C in EMM2 medium with required supplements⁶⁰. (Bottom panel) Polyadenylation of crs1 pre-mRNA was assayed by RT-PCR as in Fig. 2a, using the same RNA preparations as in Panel a. b,The mmi1-ts6 mutation leads to processing of crs1 RNA in proliferating cells. Mutant cells (see Supplementary Table 3 online, for the complete genotype) were grown to early log phase at the permissive temperature (25°C) and then shifted to the non-permissive temperature (36°C) for the indicated times. (Left panel) RT-PCR assays of splicing (top) and polyadenylation (bottom) were performed as in Fig. 2a & d. (Right panel) Splicing and polyadenylation assays on an isogenic wild-type strain subjected to the same growth regimen as the mutant strain. In the polyadenylation assays shown in both Panels a and b, an asterisk designates a product unrelated to crs1 (see Fig. 2a legend). c, qPCR analysis of spliced and unspliced crs1 in the same RNA preparations analyzed in Panel b.