BioProject

Crm1 and Nup2 bind upstream of and at the promoters of many genes in a manner that correlates with Pol II binding. More...

Crm1 and Nup2 bind upstream of and at the promoters of many genes in a manner that correlates with Pol II binding. To see if the loss of any one of these proteins can impact the transcriptome, we inhibited Crm1 or degraded Nup2 and examined their effects on nascent and total RNA using thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq). Yeast cells were grown in synthetic complete medium containing glucose (SDC) before Crm1 was inhibited by treating the cultures with 185 nM leptomycin B (LMB) in ethanol for 30 min at 30C. Nup2 was degraded by treating cultures with 500 uM indole-3-acetic acid (3-IAA) in ethanol for 16 hours at 30C. Following treatment, cultures were treated with 0.2 mM 4sU for 6 min at 30C. Cells were spun down and snap frozen. Total RNA was extracted using Phenol:Chloroform:IAA (25:24:1) extraction. 5 ug total RNA was treated with 10 mM iodoacetamide for 15 min at 50C. Libraries were made using the QuantSeq 3mRNA-seq Library Prep Kit for Illumina (FWD, Lexogen) following manufacturer recommendations. All experiments had at least three biological replicates. Less...