Project description:In Saccharomyces cerevisiae, the maturation of both pre-rRNA and pre-small nucleolar RNAs (pre-snoRNAs) involves common factors, thereby providing a potential mechanism for the coregulation of snoRNA and rRNA synthesis. In this study, we examined the global impact of the double-stranded-RNA-specific RNase Rnt1p, which is required for pre-rRNA processing, on the maturation of all known snoRNAs. In silico searches for Rnt1p cleavage signals, and genome-wide analysis of the Rnt1p-dependent expression profile, identified seven new Rnt1p substrates. Interestingly, two of the newly identified Rnt1p-dependent snoRNAs, snR39 and snR59, are located in the introns of the ribosomal protein genes RPL7A and RPL7B. In vitro and in vivo experiments indicated that snR39 is normally processed from the lariat of RPL7A, suggesting that the expressions of RPL7A and snR39 are linked. In contrast, snR59 is produced by a direct cleavage of the RPL7B pre-mRNA, indicating that a single pre-mRNA transcript cannot be spliced to produce a mature RPL7B mRNA and processed by Rnt1p to produce a mature snR59 simultaneously. The results presented here reveal a new role of yeast RNase III in the processing of intron-encoded snoRNAs that permits independent regulation of the host mRNA and its associated snoRNA. RNA from wild type, rnt1d and rnt1ts grown at 26 C and 37 C on rich media
Project description:In Saccharomyces cerevisiae, the maturation of both pre-rRNA and pre-small nucleolar RNAs (pre-snoRNAs) involves common factors, thereby providing a potential mechanism for the coregulation of snoRNA and rRNA synthesis. In this study, we examined the global impact of the double-stranded-RNA-specific RNase Rnt1p, which is required for pre-rRNA processing, on the maturation of all known snoRNAs. In silico searches for Rnt1p cleavage signals, and genome-wide analysis of the Rnt1p-dependent expression profile, identified seven new Rnt1p substrates. Interestingly, two of the newly identified Rnt1p-dependent snoRNAs, snR39 and snR59, are located in the introns of the ribosomal protein genes RPL7A and RPL7B. In vitro and in vivo experiments indicated that snR39 is normally processed from the lariat of RPL7A, suggesting that the expressions of RPL7A and snR39 are linked. In contrast, snR59 is produced by a direct cleavage of the RPL7B pre-mRNA, indicating that a single pre-mRNA transcript cannot be spliced to produce a mature RPL7B mRNA and processed by Rnt1p to produce a mature snR59 simultaneously. The results presented here reveal a new role of yeast RNase III in the processing of intron-encoded snoRNAs that permits independent regulation of the host mRNA and its associated snoRNA.
Project description:Transcripts up- or down-regulated comparing a strain where Rnase H is ectopically overexpressed versus wild type (empty vector); assessing the effect of DNA:RNA hybrid degradation on the transcriptome. Strains harboring the RNase H1 over-expression plasmid (p425-GPD-RNase H1) or control plasmid (p425-GPD) were grown in SC-Leucine at 30M-BM-0C. For both sets of microarray experiments, duplicate cultures were analyzed. Total RNA was isolated from 1 unit of A600 mid-log phase cells using a RiboPure Yeast kit (A&B Applied Biosystems), amplified, labeled and fragmented using a Message Apm III RNA Amplification Kit (A&B Applied Biosystems) and hybridized to a GeneChIP Yeast Genome 2.0 array using the GeneChip Hybridization, Wash, and Stain Kit (Affymetrix). Arrays were scanned by the Gene Chip Scanner 3000 7G and expression data was extracted using Expression ConsoleM-bM-^DM-" Software (Affymetrix) with the MAS5.0 statistical algorithm.
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:In Saccharomyces cerevisiae, Sen1 is a 252-kDa, nuclear superfamily-1 RNA/DNA helicase that encoded by an essential gene SEN1 (Senataxin). It is an important component of the Nrd1p-Nab3p-Sen1p (NRD1) complex that regulates the transcriptional termination of most non-coding and some coding transcripts at RNA polymerase pause sites. Sen1 specifically interacts with Rnt1p (RNase III), an endoribonuclease, and with Rpb1p (Rpo21p), a subunit of RNA polymerase II, through its N-terminal domain (NTD), which is a critical element of the RNA-processing machinery. Moreover, mutations in the N-terminal tail of SETX, a human ortholog of yeast Senataxin (Sen1) reported in neurological disorders. In one of the earlier studies, we have reported that the loss of dispensable NTD in yeast Sen1 resulted in flocculation and slow growing phenotypes along with defective DNA damage repair mechanisms. So, we attempted to explore the molecular basis of functional impairment associated with the loss of Sen1 N-terminal domain through global oligonucleotide microarray analysis. Also, we investigated for functionally enriched pathways based on the altered basal level gene expression profiles upon NTD loss of Sen1. The microarray data were validated by quantitative real-time PCR wherever necessary.