FIG. 5.

Spf30 functions in the exosome-mediated silencing pathway. (A) Spf30 deficiency alleviates silencing of a subtelomeric tel1L::ura4⁺ marker gene. Cells of indicated genotypes were serially diluted and spotted on nonselective (N/S), selective (−Ura), and counterselective (FOA) synthetic medium. 5-Fluoroorotic acid (5-FOA) is toxic to Ura⁺ cells. Plates were incubated at 32°C. (B) spf30-38 affects silencing at the mating-type locus. A colony color assay for ade6⁺ silencing when it is inserted within the mating-type locus (mat3::ade6⁺) was performed. The indicated strains were grown at 32°C on YES containing a low amount of adenine. (C) Spf30 deficiency does not overtly affect the production of siRNAs. Centromeric siRNAs were detected by Northern blotting, and blots were reprobed with snR58 for a loading control. (D) Polyadenylated cen-dh transcripts accumulate when Spf30 is deficient. The scheme of the polyadenylation assay is shown. Total RNA extracted from indicated strains grown at 30.5°C was reverse transcribed with the poly(dT) anchor primer and PCR amplified by using cen-dh strand-specific and anchor primers. (E) Dis3 overexpression partly suppresses spf30-38 silencing defect at 32°C. Strains transformed with the pCD174 episomal multicopy plasmid (pDis3) (26) or with an empty vector (pLEU2) were serially diluted and spotted onto selective media. (F) The steady-state amount of Dis3 protein is not altered in the spf30-38 mutant. Soluble protein extracts prepared from wild-type (wt) and spf30-38 cells expressing Dis3-HA and grown at 25 or 32°C or shifted at 37°C for 6 h were subjected to Western blotting with monoclonal 12CA5 anti-HA antibodies. Equal loading was verified by Ponceau staining. (G) Cryptic unstable transcripts accumulate in spf30-38 cells. Total RNA from indicated strains was reverse transcribed by using random hexamers, and cDNAs were quantified by real-time PCR.