SLAM-Drop-seq reveals mRNA kinetic rates throughout the cell cycle

• Published in: Molecular systems biology Vol. 19; no. 10; pp. 1 - 23
• Main Authors: Liu, Haiyue, Arsiè, Roberto, Schwabe, Daniel, Schilling, Marcel, Minia, Igor, Alles, Jonathan, Boltengagen, Anastasiya, Kocks, Christine, Falcke, Martin, Friedman, Nir, Landthaler, Markus, Rajewsky, Nikolaus
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 12.10.2023; EMBO Press; John Wiley and Sons Inc; Springer Nature
• Subjects: Alkylation; Biological activity; Cell cycle; Cell Cycle - genetics; Correlation analysis; Cycles; Degradation; Gene expression; Genes; Genomes; Humans; Kinetics; Life Sciences; Metabolism; Method; mRNA kinetics; Nucleotides; Ribonucleic acid; RNA; RNA processing; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Analysis, RNA - methods; single cells; Systems Biology; temporal regulation; transcription and degradation; temporal regulation; single cells; mRNA kinetics; cell cycle; transcription and degradation
• ISSN: 1744-4292; 1744-4292
• DOI: 10.15252/msb.202211427

RNA abundance is tightly regulated in eukaryotic cells by modulating the kinetic rates of RNA production, processing, and degradation. To date, little is known about time-dependent kinetic rates during dynamic processes. Here, we present SLAM-Drop-seq, a method that combines RNA metabolic labeling and alkylation of modified nucleotides in methanol-fixed cells with droplet-based sequencing to detect newly synthesized and preexisting mRNAs in single cells. As a first application, we sequenced 7280 HEK293 cells and calculated gene-specific kinetic rates during the cell cycle using the novel package Eskrate. Of the 377 robust-cycling genes that we identified, only a minor fraction is regulated solely by either dynamic transcription or degradation (6 and 4%, respectively). By contrast, the vast majority (89%) exhibit dynamically regulated transcription and degradation rates during the cell cycle. Our study thus shows that temporally regulated mRNA degradation is fundamental for the correct expression of a majority of cycling genes. SLAM-Drop-seq, combined with Eskrate, is a powerful approach to understanding the underlying mRNA kinetics of single-cell gene expression dynamics in continuous biological processes. Synopsis A novel approach (SLAM-Drop-seq) is coupled to a robust computational analysis method (Eskrate) to discriminate new and preexisting transcripts in single cells and to calculate their time-dependent kinetic rates of synthesis and decay during the cell cycle. SLAM-Drop-seq identifies newly transcribed and preexisting mRNAs in single cells via 4sU incubation, efficient iodoacetamide alkylation, and droplet-based single-cell RNAseq. Eskrate is a newly developed R-package that estimates temporally resolved RNA kinetic rates from 4sU-labeled single cell data. During the HEK293 cell cycle, oscillating mRNA levels of hundreds of cell cycle genes are regulated by time-dependent dynamic transcription, dynamic degradation, or both.