An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae

Abstract

Abstract Background Gene transcription by RNA polymerase II is a fundamental process in eukaryotic cells. The precise regulation of transcription is necessary for cellular growth and development and requires the coordinated activity of numerous proteins and protein complexes. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unresolved. Accurately defining the direct effects of transcriptional regulators is critical to addressing these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly synthesized RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly synthesized RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we assembled an end-to-end workflow to deplete proteins of interest using the AID system and measure newly synthesized RNA using SLAM-seq in the model eukaryote, Saccharomyces cerevisiae. Methods We provide an open-source, step-by-step protocol to support the rapid implementation of this workflow. We include methods for targeted protein degradation, 4-thiouracil (4tU) incorporation, rapid methanol fixation, RNA purification, RNA alkylation, 3´ mRNA-seq library construction, and data analysis. Additionally, we demonstrate that this workflow can help define the direct effects of transcriptional regulators using the bromodomain and extra-terminal domain (BET) proteins, Bdf1 and Bdf2, as an example. Discussion We demonstrate that data generated using this workflow effectively quantifies 4tU-labeled transcripts and is robust to normalization using whole-cell spike-in or, at least in the case of Bdf1 and Bdf2 depletion, total read counts. We additionally demonstrate that this data correlates well with 4tU-seq data and identifies extensive differential expression due to the depletion of Bdf1 and Bdf2. Lastly, the workflow is modular and readily adaptable to other systems. Taken together, this workflow and supporting protocol will help address outstanding questions underlying the molecular basis of transcriptional regulation and other processes in S. cerevisiae and other eukaryotes.