Assessment of mRNA Decay and Calculation of Codon Occurrence to mRNA Stability Correlation Coefficients after 5-EU Metabolic Labeling

Sophie Martin; Christopher Y Kim; Jeff Coller

doi:10.1007/978-1-0716-4176-7_11

Assessment of mRNA Decay and Calculation of Codon Occurrence to mRNA Stability Correlation Coefficients after 5-EU Metabolic Labeling

Methods Mol Biol. 2025:2863:151-182. doi: 10.1007/978-1-0716-4176-7_11.

Authors

Sophie Martin¹, Christopher Y Kim², Jeff Coller^{3

4

5

6}

Affiliations

¹ Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. smart164@jhmi.edu.
² Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
³ Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. jmcoller@jhmi.edu.
⁴ RNA Innovation Center, Johns Hopkins University, Baltimore, MD, USA. jmcoller@jhmi.edu.
⁵ Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA. jmcoller@jhmi.edu.
⁶ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, United States. jmcoller@jhmi.edu.

PMID: 39535710
DOI: 10.1007/978-1-0716-4176-7_11

Abstract

mRNA translation and decay are tightly connected. This chapter describes a method to assess the influence of each codon identity on mRNA stability in cultured cells. The technique involves metabolic labeling of the nascent mRNAs by addition of the nucleoside analog 5-ethynyluridine (5-EU), purification of the RNA at different time-points after chase of the 5-EU, then biotinylation with Click chemistry, pull-down, and sequencing. The transcripts' half-lives are calculated from the expression level of each mRNA at the different time-points. Finally, the method describes the calculation of the Codon occurrence to mRNA Stability correlation Coefficient, or CSC, as a correlation between the codon occurrence in a transcript and the transcript half-life, for each codon.

Keywords: CSC; Codon optimality; Transcriptomics; mRNA decay; Metabolic labeling.

MeSH terms

Biotinylation
Click Chemistry / methods
Codon* / genetics
Humans
Protein Biosynthesis
RNA Stability*
RNA, Messenger* / genetics
RNA, Messenger* / metabolism
Uridine* / analogs & derivatives
Uridine* / chemistry
Uridine* / metabolism

Substances

RNA, Messenger
Uridine
Codon