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 in | Scientific reports Vol. 14; no. 1; p. 1623 |
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| Main Authors | , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
England
Nature Publishing Group
18.01.2024
Nature Portfolio |
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| Abstract | 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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| AbstractList | 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. Abstract 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. Abstract 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. |
| ArticleNumber | 1623 |
| Author | Young, Alex Siejda, Klara W Leathers, Tess A Wolf, Matthew J Cochran, Jesse D Maldosevic, Emir Jomaa, Ahmad Lee, Haesol Vitello, Julian Bradley, Leigh A |
| Author_xml | – sequence: 1 givenname: Jesse D surname: Cochran fullname: Cochran, Jesse D organization: Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA – sequence: 2 givenname: Tess A surname: Leathers fullname: Leathers, Tess A organization: Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, USA – sequence: 3 givenname: Emir surname: Maldosevic fullname: Maldosevic, Emir organization: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, USA – sequence: 4 givenname: Klara W surname: Siejda fullname: Siejda, Klara W organization: Department of Medicine, University of Virginia, Charlottesville, VA, USA – sequence: 5 givenname: Julian surname: Vitello fullname: Vitello, Julian organization: Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA – sequence: 6 givenname: Haesol surname: Lee fullname: Lee, Haesol organization: Department of Medicine, University of Virginia, Charlottesville, VA, USA – sequence: 7 givenname: Leigh A surname: Bradley fullname: Bradley, Leigh A organization: Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA, USA – sequence: 8 givenname: Alex surname: Young fullname: Young, Alex organization: Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA, USA – sequence: 9 givenname: Ahmad surname: Jomaa fullname: Jomaa, Ahmad organization: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, USA – sequence: 10 givenname: Matthew J surname: Wolf fullname: Wolf, Matthew J email: MJW5MC@hscmail.mcc.virginia.edu, MJW5MC@hscmail.mcc.virginia.edu, MJW5MC@hscmail.mcc.virginia.edu organization: Division of Cardiology, University of Virginia, Medical Research Building 5 (MR5), Room G213, 415 Lane Road, Charlottesville, VA, 22908, USA. MJW5MC@hscmail.mcc.virginia.edu |
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| Snippet | Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey... Abstract Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including... Abstract Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including... |
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| StartPage | 1623 |
| SubjectTerms | 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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| Title | Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from asynchronously cycling cells |
| URI | https://www.ncbi.nlm.nih.gov/pubmed/38238470 https://www.proquest.com/docview/2916280553/abstract/ https://search.proquest.com/docview/2917553543 https://doaj.org/article/d14e63c98abf4685a1e7bb9896c3afd6 |
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