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Download or read book The Control of Gene Expression by Nuclear RNA Degradation in Saccharomyces Cerevisiae written by Kevin Richard Jones Roy and published by . This book was released on 2015 with total page 164 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribonucleases play critical roles in controlling the quantity and quality of gene expression through processing and degrading RNA. An important class of evolutionarily conserved ribonucleases is the RNase III family of enzymes, which are distinguished by their specificity for cleaving double-stranded RNA (dsRNA). RNase III enzymes perform diverse functions in RNA metabolism in all eukaryotes studied, yet numerous questions remain regarding their range of natural targets in vivo, how they achieve substrate specificity, and how their cleavage activity is regulated. The model eukaryote Saccharomyces cerevisiae harbors one RNase III homolog, Rnt1p, which is responsible for all known dsRNA cleavage activity in this organism. To better understand the substrate selectivity of Rnt1p, we examined how its double-stranded RNA binding domain (dsRBD) recognizes a non-canonical substrate containing an AAGU tetraloop sequence differing from the NGNN consensus sequence. Surprisingly, we found that upon engaging the RNA, the dsRBD induces a structural change in the AAGU loop so that it closely adopts the structure of the NGNN loop. This suggested that the structures of isolated RNAs in solution are not necessarily predictive of substrate specificity. We next characterized how structural dynamics in the dsRBD mediate specific binding. We found that in order to bind substrate dsRNA with high affinity, the dsRBD must undergo a significant conformational change involving the first alpha helix and beta strand of the dsRBD. Next we implemented computational RNA secondary structure screens to scan the genome for potential Rnt1p targets. We identified a characteristic Rnt1p stem-loop in the BDF2 mRNA, which is also subject to nuclear decay by the spliceosome through a first step splicing discard pathway. Cis acting mutations in BDF2 blocking Rnt1p or spliceosome-mediated decay (SMD) conferred distinct phenotypes for each pathway, revealing that salt stress hyper-activates Rnt1p cleavage while spliceosome-mediated decay controls BDF2 expression during DNA replication stress. To globally identify RNA targets of Rnt1p cleavage, we leveraged the fact that the 5 product of Rnt1p cleavage is oligo-adenylated by Trf4/5-Air2/1-Mtr4 polyadenylation (TRAMP) complex prior to degradation by the nuclear exosome, a 3 -to-5 exonuclease complex. We mapped TRAMP poly(A) tails genome-wide by high-throughput sequencing of 3 ends of polyadenylated RNA in yeast cells lacking a nuclear exosome component. This revealed a global profile of destabilized 3 ends arising from various nuclear RNA degradation mechanisms, including Rnt1p cleavage, transcription termination by the Nrd1p-Nab3p-Sen1p (NNS) pathway and roadblock transcription termination by Reb1p and TFIIIB DNA binding factors. While the NNS pathway was known to play a prominent role in limiting pervasive RNA polymerase II, we uncovered previously unappreciated roles for roadblocks and Rnt1p in controlling Pol II transcriptional output throughout the genome, revealing how cells use a multitude of nuclear mechanisms to regulate the levels of coding and cryptic transcripts.