Figure 5.
Examination of mRNA turnover in polyploid and cell size mutant strains. (A) Global transcriptional study. Genomic Run-On (GRO) in the wild type haploid BY4741 and isogenic polyploid strains was performed to obtain a nascent transcription rate (nTRII). nTRII was divided by the median cell volume as a proxy of SRII. [mRNA] was calculated as described in Figure 3 legend. Essentially the same result was obtained by hybridization with radioactive oligo d(T) in a dot-blot protocol (not shown). The whole cell population mRNA half-life (HL) was determined by dividing [mRNA] by SRII. (B) RNA pol II protein regulation. Identical protein amounts were used to perform Western blot analyses for RNA pol II quantification by the antiSer2-phosphorylated antibody that measures elongating RNA pol II, and the Anti-N-terminal-Rpb1 antibody that recognizes all the RNA pol II molecules. The shown quantification corresponds to the averages and standard deviations (SD) of four independent biological replicates. Total protein concentration does not vary with cell volume (see Supplementary Figure S8). The levels of total and elongating RNA pol II were normalized against the internal G-6-PDH control. Examples of representative Western blots are shown in panel (C). The same study on aliquots with the same samples and with Anti-Rbp3 antibody is shown in Supplementary Figure S8. (C) The RT-qPCR analysis of the Rpb1 mRNA levels in the same cells normalized against ACT1 mRNA. (D) Regulation of the RNA pol II subunits that encode mRNAs. The qRT-PCR analysis of RPB1 mRNA (red dots) was done on the samples of the same yeast strains set. Data represent the average and SD of three biological repeats. The average SR and SEM of the three genes RPB1, RPB2 and RPB3, which encode the three largest RNA pol II subunits in the same strains, is also shown (blue triangles). These individual SR gene data correspond to the GRO data used for the total SRII used in panel (A).