Project description:Time course after the addition of the transcriptional inhibitor thiolutin at 3 and 10 µg/mL to an exponential growing culture of S. cerevisiae in YPD. Keywords: time course, mRNA stability analysis
Project description:Time course after the addition of the transcriptional inhibitor thiolutin at 3 and 10 µg/mL to an exponential growing culture of S. cerevisiae in YPD. Total RNA from BQS252 yeast strain (Mat a, ura3-52 derived from FY1679) growing in exponential phase in YPD was extracted at 30 minutes after thiolutin 3 and 10 µg/mL addition. All the samples were hybridized on the same nylon array. 2 replicates.
Project description:Time course after the addition of the transcriptional inhibitor thiolutin at 3µg/mL to an exponential growing culture of S. cerevisiae in YPD. Keywords: time course, mRNA stability analysis
Project description:Time course after the addition of the transcriptional inhibitor thiolutin at 3µg/mL to an exponential growing culture of S. cerevisiae in YPD. Total RNA from BQS252 yeast strain (Mat a, ura 3-52 derived from FY1679) growing in exponential phase in YPD was extracted at different times after thiolutin 3µg/mL addition. All the samples were hybridized on the same nylon array. In order to estimate mRNA stability the intensities where normalized using global poly(A) data from a RNA dot-blot and assuming a first order decaying. There is only 1 replicate.
Project description:The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3’ to 5’ degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5’ monophosphate ends on mRNAs in wild-type and dcp2∆ xrn1∆ yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low level endonucleotyic cleavage were observed suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5’ monophosphates on some mRNAs suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential new step to regulate gene expression.
