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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.

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

  • 实验方案

  • 发表日期：

    2025年

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  • Sophie Martin,
  • Christopher Y. Kim,
  • Jeff Coller

摘要

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.