Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from...

Area of Science:

• Molecular Biology
• Cell Biology
• Genomics

Background:

• Asynchronous cell cycling complicates phase-specific gene expression analysis.
• Existing methods like RNAseq lack resolution for dynamic gene regulatory networks.
• Single cell RNAseq (scRNAseq) is limited by cell type and requires sufficient cycling cells.

Purpose of the Study:

• To develop a novel method for isolating cell cycle phase-specific mRNA.
• To overcome limitations of current gene expression profiling techniques in cycling cells.
• To enable accurate characterization of dynamic gene regulatory networks.

Main Methods:

• Merged Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) degron technology (CDT1 and GMNN) with RiboTrap/Tag ribosomal protein epitope tagging.
• Developed cell cycle dependent, tagged ribosomal proteins (ccTaggedRP).
• Utilized ccTaggedRP to isolate and sequence cell cycle phase-specific RNA.

Main Results:

• ccTaggedRP were differentially expressed across the cell cycle.
• ccTaggedRP exhibited similar subcellular localization to endogenous ribosomal proteins.
• ccTaggedRP incorporated into ribosomes and polysomes, facilitating RNA recovery.
• Successfully recovered cell cycle phase-specific RNA for sequencing.

Conclusions:

• ccTaggedRP is a powerful new strategy for isolating cell cycle phase-specific mRNA.
• This method enhances the study of dynamic gene expression in complex cell populations.
• ccTaggedRP offers broad applications for cell cycle research and transcriptomics.