RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3′-end processing in Saccharomyces cerevisiae

Kinetics of repression and turnover of Ribo1 reporter transcripts using the RiboSys tetOFF strains. The tetOFF strains with intron-containing Ribo1 or intonless ILRibo1 (A,B), mutant 5′SSRibo1 (C), or mutant 3′SSRibo1 (D) reporters were grown in SD minimal medium with doxycycline (4 μg/ mL) added at time 0 to initiate repression of transcription. (A) Northern blot analysis of Ribo1 and ILRibo1 reporters showing relative mRNA levels following repression. Apparent half-lives of the reporter transcripts are shown below the blot. (B–D) Repression of RiboSys reporters showing relative abundance (log₂) of the Ribo1 and ILRibo1 mRNAs (B), 5′SSRibo1 pre-mRNA (C), and 3′SSRibo1 lariat-exon2 intermediate (D) assayed by RT-qPCR of the 3′SS region (3′SS) or using lariat-specific RT-qPCR (LAR). The apparent half-lives, calculated from the linear parts of the curves, are displayed.