Figure 1.

Analysis of Ribo Reporter Induction

(A) Diagram of Ribo1, ILRibo1, 5′SSRibo1, and 3′SSRibo1 genes. Exons are represented by rectangles, and the intron by a line with sequences at the ends. The reporter genes are based on previously described ACT1-PGK1 constructs (Hilleren and Parker, 2003; Alexander et al., 2010) expressed under control of a tetO7-CYC1-UAS promoter (Bellí et al., 1998). The lines above indicate amplicons analyzed in RT-qPCR or ChIP analyses: 1, 2, 3, 4, and 5 correspond to the promoter, exon 1/5′SS, 3′SS, 5′ end of exon 2 and 3′ end of exon 2, respectively. Amplicon 2′ corresponds to spliced Ribo1 mRNA or the 5′ end of the intronless ILRibo1. Lariat introns (6) were assayed with an oligo that hybridizes across the 2′-5′phosphodiester bond at the branch site (Vogel et al., 1997). See the Supplemental Information for full details of strains, reporter genes, and primer sequences.

(B) RT-qPCR analysis of accumulation of pre-mRNA, mRNA, or lariat RNA species (amplicon indicated in parentheses) showing the increase compared to time of doxycyclin addition (T0). Error bars indicate standard error for RT performed in triplicate and qPCR also performed in triplicate.

(C) ChIP analysis (presented as percentage of input relative to uninduced level at T0) to detect RNAPII at the promoter (amplicon 1) using anti-Rpb3 antibodies (Neoclone) with the same cultures as above.

(D) ChIP analysis to detect RNAPII at the 5′ end of exon 2 (amplicon 4), otherwise as above.

(E) 3D representation of the RNAPII ChIP data, showing RNAPII occupancy at all positions tested (x axis) at times indicated on the z axis. In ChIP assays, error bars indicate standard error for qPCR performed in triplicate. The kinetic resolution of the ChIP assay was tested by RT-qPCR measurement of Ribo1 transcription during the formaldehyde treatment, showing that transcript accumulation ceases immediately, and indicating that RNAPII activity is halted very rapidly (data not shown).

See also Figure S1.