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

Abstract

ABSTRACTAccurate regulation of gene transcription by RNA polymerase II is essential for the growth and development of eukaryotic cells. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unanswered. Defining the direct effects of transcriptional regulators is critically important to answering these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly transcribed 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 transcribed RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we engineered an end-to-end workflow inSaccharomyces cerevisiaeto deplete proteins of interest using the AID system and measure newly transcribed RNA using SLAM-seq. We provide a step-by-step protocol to support rapid implementation and demonstrate that the workflow can help define the direct effects of transcriptional regulators using the BET proteins Bdf1 and Bdf2 as a test case. This workflow will help address outstanding questions underlying the molecular basis of transcription and other biological processes inS. cerevisiaeand other systems.