The transcriptional inhibitor thiolutin blocks mRNA degradation in yeast

Abstract

Thiolutin is commonly used as a general inhibitor of transcription in yeast. It has been used to calculate mRNA decay rates by stopping the transcription and then determining the relative abundance of individual mRNAs at different times after inhibition. We report here that thiolutin is also an inhibitor of mRNA degradation, and thus its use can lead to miscalculations of mRNA half-lives. The inhibition of mRNA decay seems to affect the mRNA degradation pathway without impeding poly(A) shortening, given that the decay rate of total poly(A) amount is not reduced by thiolutin. Moreover, the thiolutin-dependent inhibition of mRNA degradation has variable effects on different functional groups of genes, suggesting that they use various degradation pathways for their mRNAs.

The Rtr1p CTD phosphatase autoregulates its mRNA through a degradation pathway involving the REX exonucleases

“…This could be due to differences in the kinetics of transcriptional shutoff between the GAL promoter and the Thiolutin treatment. In addition, Thiolutin could have indirect effects that could impact RNA half-lives (Pelechano and Pérez-Ortín 2008); additionally, differences in promoters may also impact RNA half-life (Trcek et al 2011). However, the effects of the different mutations are the same regardless of the system used to measure RTR1 decay: Inactivation of Rtr1p and deletion of the binding site element stabilize RTR1, and inactivation of both does not result in an increase in stability.…”

“…Transcription was inhibited by the transcription inhibitor, Thiolutin (Enzo Life Sciences), at a final concentration of 3 μg/mL as described previously (Pelechano and Pérez-Ortín 2008). We tested 3, 6, 10, and 18 μg/mL Thiolutin and saw little difference on the RTR1 halflives.…”

Next-generation analysis of gene expression regulation – comparing the roles of synthesis and degradation

“…Second, these approaches severely disrupt cellular homeostasis, and therefore provide poor estimates of the actual degradation rates. For example, thiolutin has been shown to inhibit both mRNA synthesis and degradation in yeast 26, 27 . Even the well-regarded rpb1-1 mutation system in yeast appears to decrease transcription only 3-fold at the non-permissive temperature, with rates recovering after about one hour 28 .…”

Imaging gene expression in real-time using aptamers

“…The calculated half-life of IMAGEtags was 5.5 min, which is significantly shorter than half-life of the galactokinase mRNA (43 min) and the half-life of the ACT1 mRNA (29 min) ( Figure 5). Previously reported values for the half-life of ACT1 are 50 min measured by Northern blot (Pelechano and Perez-Ortin 2008), 46 min from overall decay and 11 min from poly A decay measured in a temperature sensitive rpb1-1 mutant (Wang, Liu et al 2002). The reported value for the half-life of galactokinase mRNA are 31 min from overall decay and 18 min from poly A decay measured in the temperature sensitive rpb1-1 mutant (Wang, Liu et al 2002).…”

“…Thiolutin from Streptomyces luteoreticuli was used to detect the mRNA half-life because it inhibits all yeast RNA polymerases (RNAPI, RNAPII and RNAPIII), especially at the level of initiation (Pelechano and Perez-Ortin 2008). Therefore to measure the half-life of IMAGEtag RNA, the amount of IMAGEtag RNA (under the control of the GAL1 promoter) was determined in cells that had been preincubated in galactose-containing medium then treated with 3 μg/ml thiolutin.…”