Project description:We report the application of single-molecule-based sequencing technology for transcription profile analysis of S. cerevisiae strains with different genetic background. By combining the whole genome sequence of these strains, we sought to explore the effects of genome mutations on the transcription diversities. Comparsion of transcription profiles in S. cerevisiae strains
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 report the application of next-generation sequencing technology for transcription profile analysis of S. cerevisiae strains with different genetic background. By combining the whole genome sequence of these strains, we sought to explore the effects of genome mutations on the transcription diversities. Comparsion of transcription profiles in S. cerevisiae Chinese rice wine strain with laboratory strain
Project description:We report the application of single-molecule-based sequencing technology for transcription profile analysis of two S. cerevisiae strains. By combining the whole genome sequence of these strains,we sought to explore the effects of genomic structure variations on the transcription diversities. Comparsion of transcription profiles in S. cerevisiae strains
Project description:Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthesis of second-strand cDNA during reverse transcription reactions. Strand-specific hybridization signals obtained from Saccharomyces cerevisiae tiling arrays were compared between samples prepared with and without ActD. Use of ActD removed about half of the detectable antisense transcripts, consistent with their being artifacts, while sense expression levels and about 200 antisense transcripts were not affected. Our findings thus facilitate a more accurate assessment of the extent and position of antisense transcription, towards a better understanding of its role in cells.
Project description:Set3 complex (Set3C) binds histone H3 dimethylated at lysine 4 (H3K4me2) to mediate deacetylation of histones in 5Õ transcribed regions. To discern how Set3C affects gene expression, genome-wide transcription was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3 primarily caused changes during transition periods, as genes were induced or repressed. Surprisingly, a majority of Set3-affected genes are overlapped by non-coding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2 instead of me3 over promoter regions, due either to reduced H3K4me3 or ncRNA transcription from distal or anti-sense promoters. The resulting deacetylation by Set3C slows gene induction. Set3C represses internal cryptic promoters, but in different regions of genes than the Set2/Rpd3S pathway. Finally, Set3C stimulates some genes by repressing an overlapping antagonistic anti-sense transcript. These results suggest that Set3C and overlapping non-coding transcription can cooperate to fine tune the response kinetics of individual genes.
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:Prp45 is a budding yeast NineTeen-Complex associated factor, which plays a role in pre-mRNA splicing. In addition to its documented involvement in the second step of splicing, here we show that it is important in the early stages of co-transcriptional spliceosome assembly as well. To determine the overall splicing efficiency of prp45 mutant cells and to reveal how the efficiency depends on the sequences which define introns (whether they are consensual or non-consensual), we performed RNA-seq analysis of prp45(1-169) and corresponding wild-type cells (two biological replicates each). Total RNA was isolated by combining phenol-chlorophorm extraction with MasterPure Yeast RNA Purification Kit (Epicentre). Ribodepletion, library preparation and sequencing were performed by BGI Genomics.
Project description:Most Saccharomyces cerevisiae ribosomal protein genes (RPGs) are present as two paralogous copies that emerged during a whole genome duplication. The preservation of two copies of the same gene during the past ~100 million years enabled diversification of the regulation of their expression, and also partially of their functional properties. One example of such paralogous genes are RPL22A and RPL22B that are asymmetrically expressed and their protein sequences represent one of the most divergent pairs among budding yeast RPG paralogs. It was shown that introns in the RPL22A and RPL22B genes are important for regulation of their expression levels. To investigate functional differences between RPL22A and RPL22B we performed RNA-seq analysis of strains with complete deletions of either RPL22A or RPL22B, and also of strains with intron deletions in the RPL22 genes, in comparison with a wild-type strain. Cells were grown to mid-exponential phase in the rich YPAD medium. Total RNA was isolated by combining phenol-chlorophorm extraction with MasterPure Yeast RNA Purification Kit (Epicentre). Ribodepletion, library preparation and sequencing were performed by BGI Genomics.
Project description:Prp45 is a budding yeast NineTeen-Complex associated factor, which plays a role in pre-mRNA splicing. Because its human ortholog, SNW1/SKIP, is known to functionally and physically interact with factors involved in transcription elongation and chromatin modifications, it was suggested to be one of the coupling factors which functionally connect splicing and transcription. To determine whether the yeast Prp45 has this function as well we tested genetic interactions between prp45(1-169) allele and deletions of histone modifiers and transcription elongation regulators. RNA-seq analysis was performed to address the changes in transcription and/or splicing in strains deleted in rad6, a kinase phosphorylating serine 2 of the C-terminal domain of RNA Polymerase II and an E2 enzyme for H2BK123 monoubiquitination, respectively, and the corresponding double mutant with prp45(1-169). Total RNA was isolated by combining phenol-chlorophorm extraction with MasterPure Yeast RNA Purification Kit (Epicentre). Ribodepletion, library preparation and sequencing were performed by BGI Genomics.