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.
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. 5' monophosphorylated ends of poly(A) RNA from wild-type and dcp2D xrn1D strains were identified in duplicates and triplicates, respectively.
Project description:The production of Tobacco Acid Pyrophosphatase (TAP), an enzyme commonly used for the removal of the 5’cap of eukaryotic mRNAs, has been recently discontinued. Here we performed a comparison of current alternatives for the mapping of 5’cap mRNAs and the associated transcription start sites in Sacharomyces cerevisiae. Specifically we compared TAP with Cap-clip and a Decapping Pyrophosphohydrolase. Our results suggest that Cap-clip is a good alternative for TAP.
Project description:The production of Tobacco Acid Pyrophosphatase (TAP), an enzyme commonly used for the removal of the 5â??cap of eukaryotic mRNAs, has been recently discontinued. Here we performed a comparison of current alternatives for the mapping of 5â??cap mRNAs and the associated transcription start sites in Sacharomyces cerevisiae. Specifically we compared TAP with Cap-clip and a Decapping Pyrophosphohydrolase. Our results suggest that Cap-clip is a good alternative for TAP. We used two biological replicates of S. cerevisiae that was grown to exponential phase (OD600 ~1) in rich media (YPAD). Samples where processed until the dephosphorylation step (CIP treatment). After that each sample was split in 4 aliquots: TAP treatment, Cap-Clip treatment, Decapping Pyrophosphohydrolase treatment or no treatment (negative control). From that step all samples are processed in parallel.
Project description:Metabolic fluxes may be regulated "hierarchically," e.g., by changes of gene expression that adjust enzyme capacities (V(max)) and/or "metabolically" by interactions of enzymes with substrates, products, or allosteric effectors. In the present study, a method is developed to dissect the hierarchical regulation into contributions by transcription, translation, protein degradation, and posttranslational modification. The method was applied to the regulation of fluxes through individual glycolytic enzymes when the yeast Saccharomyces cerevisiae was confronted with the absence of oxygen and the presence of benzoic acid depleting its ATP. Metabolic regulation largely contributed to the approximately 10-fold change in flux through the glycolytic enzymes. This contribution varied from 50 to 80%, depending on the glycolytic step and the cultivation condition tested. Within the 50-20% hierarchical regulation of fluxes, transcription played a minor role, whereas regulation of protein synthesis or degradation was the most important. These also contributed to 75-100% of the regulation of protein levels. Keywords: Condition comparison
Project description:In eukaryotes, mRNA decay is thought to occur in an ordered manner, with an initial deadenylation step that triggers decapping and RNA degradation. We performed SLAM-seq experiments in yeast strains depleted either for the decapping or for the major deadenylase enzymes to evaluate changes in the half-life of mRNA. Our results suggest that deadenylation and decapping of mRNA are not necessarily linked for mRNA degradation, since the effects of inhibiting the enzymes on RNA stability were not correlated.