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

A conversation with authors John Ridenour and Rafal Donczew
An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae
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BioMed Central
BioMed Central BioMed Central

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

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.

Join Gabriel Gasque, Head of Outreach at protocols.io, as he interviews John Ridenour and Rafal Donczew, the authors of a recently published protocol in BMC Methods, presenting an end-to-end workflow to deplete proteins of interest and measure newly synthesized RNA in Saccharomyces cerevisiae.
 
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Molecular Biology
Life Sciences > Biological Sciences > Molecular Biology
Gene Transcription
Physical Sciences > Chemistry > Biological Chemistry > Nucleic Acid > Gene Transcription
Saccharomyces cerevisiae
Life Sciences > Biological Sciences > Biological Techniques > Experimental Organisms > Model Fungi > Saccharomyces cerevisiae
RNA Sequencing
Life Sciences > Biological Sciences > Biological Techniques > Genomic Analysis > Sequencing > RNA Sequencing
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Cell communication analysis technology

Cell communication is a fundamental process that underpins numerous biological functions, from immune responses to tissue repair. The intricate signaling pathways that govern how cells communicate are crucial for maintaining homeostasis and regulating physiological responses. Recent technological advances, including single-cell RNA sequencing, mass cytometry, and high-resolution imaging, have dramatically improved our ability to study these networks and understand how cells coordinate their behavior in response to external cues. Computational tools can now infer intercellular signaling from single-cell and spatial transcriptomics data, identifying key ligand-receptor pairs and signaling pathways. Spatially informed approaches further map these interactions with high resolution, revealing how a cell’s location shapes its communication with neighbors. Complementing these computational methods, experimental technologies such as genetic marking systems can directly label contacting cells in vivo, allowing researchers to track interaction history and study how cellular crosstalk drives development and disease progression.

Future advancements may include the integration of artificial intelligence and machine learning to analyze complex datasets, the creation of real-time monitoring systems for live-cell interactions, and the establishment of standardized protocols for cell communication analysis. These innovations could significantly enhance our ability to dissect cellular communication networks and their roles in health and disease.

In light of these developments, BMC Methods is opening a collection on “Cell communication analysis technology.” Topics of interest include:

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Submissions should present an experimental or computational method for the analysis of cell–cell communication. The method may be completely new or offer an improved version of an existing method, but in either case it must represent a clear advance over what is currently available, be tested and validated, and include a discussion of its advantages and limitations relative to alternative approaches. Protocols describing routine or well-established methods without methodological innovation will not be considered for publication.

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Deadline: Nov 09, 2026