Project description:In budding yeast, damaged organelles and biomolecules are symmetrically segregated between mother and daughter cells, which can influence the replicating aging of mother cells. In some cases, older proteins are prone to be damaged and decline in their functions compared to newly synthesized proteins, likely to contribute the cellular aging. The purpose of this analysis is to uncover asymmetrically inherited older proteins, which are preferentially retained in mother cells during division into mother and rejuvenating daughter cells. To achieve this purpose, we conducted an originally developed strategy where mother and daughter cells are separated after just one cycle of synchronized culture, during which newly synthesized proteins are labeled with stable isotope amino acid. Synchronized culture was carried out using mating pheromone, which arrest the budding yeast in G1 phase. We successfully identified more than 20 proteins whose older forms are asymmetrically inherited by mother cells, among which are proteins involved in the intracellular homeostasis of the proton concentration and the response to the stress of misfolded proteins.
Project description:Sequencing of newly synthesized RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesized and pre-existing RNA in single cells. We validate the method on pre-alkylated RNA, and by demonstrating that more newly synthesized RNA was detected for genes with known high mRNA turnover. NASC-seq reveals rapidly up- and down-regulated genes during T-cell activation, and RNA arising from induced genes is essentially only newly synthesized. The newly synthesized and pre-existing transcriptomes after T-cell activation are distinct, confirming that we simultaneously measure gene expression at two time points in single cells. Altogether, NASC-seq is an accessible and powerful tool to investigate transcriptional dynamics that enables the precise monitoring of RNA synthesis at flexible time periods during homeostasis, perturbation responses and cellular differentiation.
Project description:In budding yeast, damaged organelles and biomolecules are symmetrically segregated between mother and daughter cells, which can influence the replicating aging of mother cells. In some cases, older proteins are prone to be damaged and decline in their functions compared to newly synthesized proteins, likely to contribute the cellular aging. The purpose of this analysis is to uncover asymmetrically inherited older proteins, which are preferentially retained in mother cells during division into mother and rejuvenating daughter cells. To achieve this purpose, we conducted an originally developed strategy where mother and daughter cells are separated after just one cycle of synchronized culture, during which newly synthesized proteins are labeled with stable isotope amino acid. Synchronized culture was carried out using virgin cells, which have never produce daughter cells and enriched in G1 phase. We successfully identified more than 20 proteins whose older forms are asymmetrically inherited by mother cells, among which are proteins involved in the intracellular homeostasis of the proton concentration and the response to the stress of misfolded proteins.
Project description:Variants of the essential genes in budding yeast S. cerevisae were cloned into a variomics library, and tested for their ability to confer resistance to three different drugs. Genes were tagged with molecular barcodes, and the relative change in abundance of the molecular barcodes are detected using a spotted Agilent synthesized microarray.
Project description:The SAGA co-activator has been implicated in the regulation of a smal subset of genes in budding yeast in transcriptomic analyses performed in steady-state levels of RNA. We used microarrays to analyse newly-synthesized RNA in several mutants for the SAGA complex to disclose and readdress the impact of this complex in RNA Polymerase II transcription.
Project description:Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into the role of differential mRNA stability in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed and pre-existing RNA pools in Karenia brevis. These isolated fractions were then used for analysis of global mRNA stability by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed/pre-existing RNA for 7086 array features using the online software HALO (Half-life Organizer). Overall, mRNA half-lives were substantially longer than reported in other organisms studied at the global level, ranging from 42 minutes to greater than 3 days, with a median of 33 hours. Thirteen percent of messages showed a half-life of 3 days, demonstrating their stability throughtout the course of the cell cycle and divison. Consistent with well-documented trends observed in other organisms, housekeeping processes, including energy metabolism and transport, were significantly enriched in the most highly stable messages. Shorter-lived transcripts included a higher proportion of transcriptional regulation, stress response, and other response/regulatory processes. Log phase cultures (n=6) were exposed to 0.2 mM 4-thiouracil for 2h to biosynthetically label newly transcribed RNA and total RNA was extracted. Following extraction, RNA was biotinylated to allow for purification of the thiolated newly synthesized RNA from the total RNA pool using streptavidin coated magnetic beads. From each replicate 3 pools of RNA, total RNA, pre-exisiting RNA, and newly synthesized RNA, were Cy3 labeled and hybridized to microarrys in a one color format. Based on the appearance of bioanlayzer profiles, total and pre-existing RNA were treated as total RNA, while newly synthesized RNA was treated as mRNA in the labeling protocol.
Project description:The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into the small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeast. The crystal structure reveals a homodimer that resembles bacterial RNase III but includes a novel N-terminal domain and newly identified catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses show that Dcr1 dimers bind cooperatively along the dsRNA substrate and cleave at precise intervals based on the distance between consecutive active sites. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, Dcr1 initiates processing in the interior and works outward. The distinct mechanism of budding-yeast Dicers establishes a novel paradigm for natural protein-based molecular rulers and imparts substrate preferences with ramifications for biological function.
Project description:The aim of the mass spectrometry analysis was to identify interaction partners of newly synthesized precursors of mitochondrial outer membrane proteins. To that aim, HA-tagged proteins were synthesized in vitro in extract of yeast cells and pull-down with anti-HA beads was performed.