Project description:This project aims to identify novel RNA binding proteins in the baker's yeast, Saccharomyces cerevisiae. Since interactions between RNAs and proteins may be transient, yeast cells were crosslinked with UV light at 254 nm which promotes the covalent link between proteins and RNAs. After this, polyadenylated mRNAs were purified via oligo(dT) coupled to magentic beads under stringet conditions. Finally, samples were subjected to mass spectrometry analysis. To rule out the possibility of RNA-independent binding we also analysed other samples: i) samples digested with RNase one; ii) samples where we performed competition assays with polyadenylic acid.
Project description:Saccharomyces cerevisiae is an excellent microorganism for industrial succinic acid production, but high succinic acid concentration will inhibit the growth of Saccharomyces cerevisiae then reduce the production of succinic acid. Through analysis the transcriptomic data of Saccharomyces cerevisiae with different genetic backgrounds under different succinic acid stress, we hope to find the response mechanism of Saccharomyces cerevisiae to succinic acid.
Project description:We quantified the exact RNA binding sites of the Ssd1 protein in Saccharomyces cerevisiae, in exponential growth and heat shock conditions, using the CRAC protocol. We used a His-TEV-protein A tag (HTP) on the C-terminal of the genomic copy of Ssd1, with the 3'UTR replaced by the 3'UTR/terminator from the K. lactis Ssd1 homolog, followed by a KlURA3 selection marker.
Project description:Protein binding is essential to the transport, decay and regulation of almost all RNA molecules. However, the structural preference of protein binding on RNAs and their cellelar functions and dynamics upon changing environmental condictions are poorly understood. Here, we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins (RBPs) and structured RNAs in yeast at single-nucleotide resolution. We found that on average, in terms of percent total lengths, ~15% of mRNA untranslated regions (UTRs), ~37% of canonical ncRNAs and ~11% of long ncRNA (lncRNAs) are bound by proteins. The RBP binding sites, in general, tend to occur at single-stranded loops, with evolutionarily conserved signatures, and often facilitate a specific RNA structure conformation in vivo. We found that four nucleotide modifications of tRNA are significantly associated with RBP binding. We also identified various structural motifs bound by RBPs in the UTRs of mRNAs, associated with localization, degradation and stress responces. Moreover, we identified >200 novel lncRNAs bound by RBPs, and about half of them contain conserved secondary structures. We present the first ensemble pattern of RBP binding sites in the structured noncoding regions of a eukaryotic genome, emphasizing their structural context and cellular functions.
Project description:We employed CapitalBio Corporation to investigate the global transcriptional profiling of Saccharomyces cerevisiae treated with allicin.
Project description:This project aims to identify novel RNA binding proteins in the baker's yeast , Saccharomyces cerevisiae, involved in the oxidative stress,. Since interactions between RNAs and proteins may be transient, yeast cells, either untreated and growth in rich media and exposed to 0.5 mM hydrogen peroxide for 15 minutes were crosslinked with UV light at 254 nm which promotes the covalent link between proteins and RNAs. After this, polyadenylated mRNAs were purified via oligo(dT) coupled to magentic beads under stringent conditions. Finally, samples were subjected to mass spectrometry analysis. To rule out the possibility of RNA-independent binding we also analysed other samples where we performed competition assays with polyadenylic acid
Project description:We employed CapitalBio Corporation to investigate the global transcriptional profiling of Saccharomyces cerevisiae treated with dictamnine. Keywords: response to dictamnine
Project description:Protein binding is essential to the transport, decay and regulation of almost all RNA molecules. However, the structural preference of protein binding on RNAs and their cellelar functions and dynamics upon changing environmental condictions are poorly understood. Here, we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins (RBPs) and structured RNAs in yeast at single-nucleotide resolution. We found that on average, in terms of percent total lengths, ~15% of mRNA untranslated regions (UTRs), ~37% of canonical ncRNAs and ~11% of long ncRNA (lncRNAs) are bound by proteins. The RBP binding sites, in general, tend to occur at single-stranded loops, with evolutionarily conserved signatures, and often facilitate a specific RNA structure conformation in vivo. We found that four nucleotide modifications of tRNA are significantly associated with RBP binding. We also identified various structural motifs bound by RBPs in the UTRs of mRNAs, associated with localization, degradation and stress responces. Moreover, we identified >200 novel lncRNAs bound by RBPs, and about half of them contain conserved secondary structures. We present the first ensemble pattern of RBP binding sites in the structured noncoding regions of a eukaryotic genome, emphasizing their structural context and cellular functions. Duplicate gPAR-CLIP libraries were sequenced from yeast strains for each of three conditions: log-phase growth, growth after 2 hour glucose starvation, and growth after 2 hour nitrogen starvation. polyA RNAs were isolated for all conditions. Total RNA were isolated from log phase growth conditions. Sucrose gradient fractionation was performed: some RNAs were isolated from the "light" fraction (lighter than 40S ribosome) and some from the "heavy" fraction. gPAR-CLIP libraries were used to determine regions of RNA bound by proteins.