Project description:We used CRISPR/Cas9 to delete the DBP1 ORF in SK1 budding yeast cells. We then used ribosome profiling and mRNA-seq to observe gene expression profiles of wild-type and dbp1∆ cells during meiosis.
Project description:Covalent nucleotide modifications in noncoding RNAs such as tRNAs affect a plethora of biological processes, with new functions continuing to be discovered for even well-known tRNA modifications. To systematically compare the functions of a large set of ncRNA modifications in gene regulation, we carried out ribosome profiling and RNA-Seq in budding yeast for 57 nonessential genes involved in tRNA modification.
Project description:Techniques for systematically monitoring protein translation have lagged far behind methods for measuring mRNA levels. Here we present a ribosome profiling strategy, based on deep sequencing of ribosome protected mRNA fragments, that enables genome-wide investigation of translation with sub-codon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control both for determining absolute protein abundance and for responding to environmental stress. We also observed distinct phases during translation involving a large decrease in ribosome density going from early to late peptide elongation as well as wide-spread, regulated initiation at non-AUG codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible. Examine replicates of ribosome footprints and mRNA abundance in biological replicates of log-phase growth and acute amino acid starvation
Project description:Cell growth underlies nearly all eukaryotic physiology, yet its quantitative principles remain unclear. Using single-molecule ribosome tracking, spike-in RNA sequencing, and quantitative proteomics across 15 nutrient-limited conditions in budding yeast, we define how growth is controlled in the budding yeast Saccharomyces cerevisiae. Ribosome concentration scales linearly with growth rate, while peptide elongation speed remains constant at ~9 amino acids s⁻¹, implying elongation is not a regulatory lever as in bacteria. Instead, total mRNA concentration increases proportionally with ribosomes to accelerate growth. A simple kinetic model of mRNA–ribosome binding accurately predicts the fraction of active ribosomes, growth rate, and responses to transcriptional or size perturbations. Consistent with this model, transient inhibition of mRNA degradation boosts growth by elevating mRNA concentration. These results reveal that eukaryotic cells accelerate proliferation primarily by proportionally scaling mRNA and ribosome abundance, establishing a quantitative framework for understanding eukaryotic biosynthesis.
Project description:Cell growth underlies nearly all eukaryotic physiology, yet its quantitative principles remain unclear. Using single-molecule ribosome tracking, spike-in RNA sequencing, and quantitative proteomics across 15 nutrient-limited conditions in budding yeast, we define how growth is controlled in the budding yeast Saccharomyces cerevisiae. Ribosome concentration scales linearly with growth rate, while peptide elongation speed remains constant at ~9 amino acids s⁻¹, implying elongation is not a regulatory lever as in bacteria. Instead, total mRNA concentration increases proportionally with ribosomes to accelerate growth. A simple kinetic model of mRNA–ribosome binding accurately predicts the fraction of active ribosomes, growth rate, and responses to transcriptional or size perturbations. Consistent with this model, transient inhibition of mRNA degradation boosts growth by elevating mRNA concentration. These results reveal that eukaryotic cells accelerate proliferation primarily by proportionally scaling mRNA and ribosome abundance, establishing a quantitative framework for understanding eukaryotic biosynthesis.