Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from asynchronously cycling cells

Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey the steady state gene expression across the cell cycle and are inherently limited by their inability to resolve dynamic gene regulatory netwo...

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Published inScientific reports Vol. 14; no. 1; p. 1623
Main Authors Cochran, Jesse D, Leathers, Tess A, Maldosevic, Emir, Siejda, Klara W, Vitello, Julian, Lee, Haesol, Bradley, Leigh A, Young, Alex, Jomaa, Ahmad, Wolf, Matthew J
Format Journal Article
LanguageEnglish
Published England Nature Publishing Group 18.01.2024
Nature Portfolio
Subjects
Cell cycle
Cell Cycle - genetics
Cell Cycle Proteins - genetics
Epitopes
Gene expression
Polyribosomes
Proteins
Quorum sensing
Ribosomal proteins
Ribosomal Proteins - genetics
Ribosomes
Ribosomes - genetics
Transcriptome
Transcriptomes
Ubiquitination
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Summary:Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey the steady state gene expression across the cell cycle and are inherently limited by their inability to resolve dynamic gene regulatory networks. Single cell RNAseq (scRNAseq) can identify different cell cycle transcriptomes if enough cycling cells are present, however some cells are not amenable to scRNAseq. Therefore, we merged two powerful strategies, the CDT1 and GMNN degrons used in Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cell cycle sensors and the ribosomal protein epitope tagging used in RiboTrap/Tag technologies to isolate cell cycle phase-specific mRNA for sequencing. The resulting cell cycle dependent, tagged ribosomal proteins (ccTaggedRP) were differentially expressed during the cell cycle, had similar subcellular locations as endogenous ribosomal proteins, incorporated into ribosomes and polysomes, and facilitated the recovery of cell cycle phase-specific RNA for sequencing. ccTaggedRP has broad applications to investigate phase-specific gene expression in complex cell populations.
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ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-024-52085-5