An end-to-end workflowto study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae v2

In this protocol, we describe an end-to-end workflow for rapidly degrading a target protein using the AID system and quantifying newly synthesized mRNA using SLAM-seq in Saccharomyces cerevisiae. We describe methods for targeted protein degradation, 4-thiouracil (4tU) incorporation, rapid methanol fixation, RNA purification, RNA alkylation, 3´ mRNA-seq library construction, and data analysis. Although the individual methods described in this protocol are not novel per se, this workflow provides a complete resource for turnkey implementation of these methods, which will benefit others working with S. cerevisiae. In addition, this workflow is modular and readily adaptable to other systems, including industrial, pathogenic, or other model fungi, which will benefit the larger research community. REFERENCES Alalam H, Zepeda-Martínez JA, Sunnerhagen P. (2022). Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets. RNA, 28(6), 905-915. https://doi.org/10.1261/rna.079077.121 Alexander RD, Barrass JD, Dichtl B, Kos M, Obtulowicz T, Robert MC, Koper M, Karkusiewicz I, Mariconti L, Tollervey D, Dichtl B, Kufel J, Bertrand E, Beggs JD. (2010). RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3′-end processing in Saccharomyces cerevisiae.RNA,16(12), 2570-2580. https://doi.org/10.1261/rna.2162610 Aslanzadeh V, Huang Y, Sanguinetti G, Beggs JD. (2018). Transcription rate strongly affects splicing fidelity and cotranscriptionality in budding yeast. Genome Research, 28(2), 203-213. https://doi.org/10.1101/gr.225615.117 Barrass JD, Beggs JD (2019). Extremely rapid and specific metabolic labelling of RNA in vivo with 4-thiouracil (Ers4tu).JoVE (Journal of Visualized Experiments), 150, e59952. https://doi.org/10.3791/59952 Barrass JD, Reid JE, Huang Y, Hector RD, Sanguinetti G, Beggs JD, Granneman S. (2015). Transcriptome-wide RNA processing kinetics revealed using extremely short 4tU labeling. Genome Biology, 16(1), 1-17. https://doi.org/10.1186/s13059-015-0848-1 Chappleboim,A, Joseph-Strauss D, Gershon O, Friedman N. (2022). Transcription feedback dynamics in the wake of cytoplasmic mRNA degradation shutdown.Nucleic Acids Research,50(10), 5864-5880. https://doi.org/10.1093/nar/gkac411 Donczew R, Hahn S. (2021). BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae.eLife,10, e69619. https://doi.org/10.7554/eLife.69619 Donczew R, Warfield L, Pacheco D, Erijman A, Hahn S. (2020). Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA.eLife,9, e50109. https://doi.org/10.7554/eLife.50109 Foley JW, Zhu C, Jolivet P, Zhu SX, Lu P, Meaney MJ, West RB. (2019). Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ. Genome Research, 29(11), 1816-1825. https://doi.org/10.1101/gr.234807.118 Glenn TC, Nilsen RA, Kieran TJ, Sanders JG, Bayona-Vásquez NJ, Finger JW, Pierson TW, Bentley KE, Hoffberg SL, Louha S, Garcia-De Leon FJ, Del Rio Portilla MA, Reed KD, Anderson JL, Meece JK, Aggrey SE, Rekaya R, Alabady M, Belanger M, Winker K, Faircloth BC. (2019). Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext).PeerJ,7. https://doi.org/10.7717/peerj.7755 Herzog VA, Reichholf B, Neumann T, Rescheneder P, Bhat P, Burkard TR, Wlotzka W, von Haeseler A, Zuber J, Ameres SL. (2017). Thiol-linked alkylation of RNA to assess expression dynamics. Nature Methods, 14(12), 1198-1204. https://doi.org/10.1038/nmeth.4435 Neumann T, Herzog VA, Muhar M, von Haeseler A, Zuber J, Ameres SL, Rescheneder P (2019). Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets.BMC Bioinformatics,20, 1-16. https://doi.org/10.1186/s12859-019-2849-7 Veeranagouda Y, Remaury A, Guillemot JC, Didier M. (2019). RNA Fragmentation and Sequencing (RF‐Seq): Cost‐Effective, Time‐Efficient, and High‐Throughput 3′ mRNA Sequencing Library Construction in a Single Tube. Current Protocols in Molecular Biology, 129(1). https://doi.org/10.1002/cpmb.109