Project description:Gene expression levels are determined by the balance between rates of mRNA transcription and decay, and genetic variation in either of these processes can result in heritable differences in transcript abundance. Although the genetics of gene expression has been the subject of intense interest, the contribution of heritable variation in mRNA decay rates to gene expression variation has received far less attention. To this end, we developed a novel statistical framework and measured allele-specific differences in mRNA decay rates in a diploid yeast hybrid created by mating two genetically diverse parental strains. In total, we estimate that 31% of genes exhibit allelic differences in mRNA decay rate, of which 350 can be identified at a false discovery rate of 10%. Genes with significant allele-specific differences in mRNA decay rate have higher levels of polymorphism compared to other genes, with all gene regions contributing to allelic differences in mRNA decay rate. Strikingly, we find widespread evidence for compensatory evolution, such that variants influencing transcriptional initiation and decay having opposite effects, suggesting steady-state gene expression levels are subject to pervasive stabilizing selection. Our results demonstrate that heritable differences in mRNA decay rates are widespread, and are an important target for natural selection to maintain or fine-tune steady-state gene expression levels.
Project description:Gene expression levels are determined by the balance between rates of mRNA transcription and decay, and genetic variation in either of these processes can result in heritable differences in transcript abundance. Although the genetics of gene expression has been the subject of intense interest, the contribution of heritable variation in mRNA decay rates to gene expression variation has received far less attention. To this end, we developed a novel statistical framework and measured allele-specific differences in mRNA decay rates in a diploid yeast hybrid created by mating two genetically diverse parental strains. In total, we estimate that 31% of genes exhibit allelic differences in mRNA decay rate, of which 350 can be identified at a false discovery rate of 10%. Genes with significant allele-specific differences in mRNA decay rate have higher levels of polymorphism compared to other genes, with all gene regions contributing to allelic differences in mRNA decay rate. Strikingly, we find widespread evidence for compensatory evolution, such that variants influencing transcriptional initiation and decay having opposite effects, suggesting steady-state gene expression levels are subject to pervasive stabilizing selection. Our results demonstrate that heritable differences in mRNA decay rates are widespread, and are an important target for natural selection to maintain or fine-tune steady-state gene expression levels. We measured rates of allele-specific mRNA decay (ASD) in a diploid yeast produced by mating two genetically diverse haploid Saccharomyces cerevisiae strains: the laboratory strain BY4716 (BY), which is isogenic to the reference sequence strain S288C, and the wild Californian vineyard strain RM11-1a (RM). Briefly, we introduced rpb1-1, a temperature sensitive mutation in an RNA polymerase II subunit, to each of the haploid yeast strains, mated the strains, and grew the resulting hybrid diploid to mid-log phase at 24 M-BM-0C, before rapidly shifting the culture to 37 M-BM-0C to inhibit transcription. RNA-seq was performed on culture samples taken at 0, 6, 12, 18, 24, and 42 minutes subsequent to the temperature shift. To identify ASD, we used transcribed polymorphisms to distinguish between parental transcripts, and compared the relative levels of transcript abundance over the time course. Note, this experimental design internally controls for trans-acting regulatory variation as well as environmental factors. Under the null hypothesis of no ASD, the proportion of reads from the BY transcript (p_BY = N_BY / (N_BY + N_RM)) observed over the time course remains unchanged. However, genes with ASD will exhibit an increasing or decreasing proportion of BY reads as a function of time. In total, we measured ASD from three independent biological replicates.
Project description:We subjected yeast to two stresses, oxidative stress, which under current settings induces a fast and transient response in mRNA abundance, and DNA damage, which triggers a slow enduring response. Using microarrays, we performed a transcriptional arrest experiment to measure genome-wide mRNA decay profiles under each condition. Genome-wide decay kinetics in each condition were compared to decay experiments that were performed in a reference condition (only transcription inhibition without an additional stress) to quantify changes in mRNA stability in each condition. We found condition-specific changes in mRNA decay rates and coordination between mRNA production and degradation. In the transient response, most induced genes were surprisingly destabilized, while repressed genes were somewhat stabilized, exhibiting counteraction between production and degradation. This strategy can reconcile high steady-state level with short response time among induced genes. In contrast, the stress that induces the slow response displays the more expected behavior, whereby most induced genes are stabilized, and repressed genes destabilized. Our results show genome-wide interplay between mRNA production and degradation, and that alternative modes of such interplay determine the kinetics of the transcriptome in response to stress. Keywords: Four separate time courses
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:Analysis of the extent to which inter-individual variation in mRNA decay contributes to inter-individual variation in gene expression levels in humans. The study examines properties of genome-wide decay rates and the relationship between mRNA decay and gene expression across genes, across individuals, and finally across genotype classes.