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: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.
Project description:Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome and encompass wide variety of sequence indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes and their impact on RNA expression is linked to growth conditions. Together the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions.
Project description:RNase III is an important and highly conserved endoribonuclease known to impact rRNA, mRNA and ncRNA abundances by RNA processing. In this study we analyzed the effects of an inactivation of RNase III (inactivated through substitution of two strictly conserved amino acids within the active enzyme center) on the transcriptome of the facultative phototrophic model organism Rhodobacter sphaeroides.
Project description:Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome and encompass wide variety of sequence indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes and their impact on RNA expression is linked to growth conditions. Together the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions. Total RNA and whole cell extract from rnt1d strain was divided in two samples. One sample was treated with purified Rnt1p. The other was only incubated with buffer. The two samples were then treated with recombinant Xrn1p.
