Monitoring translation-dependent and -independent mRNA decay in CD4+ T lymphocytes. (A) Schematic representation of the procedure to monitor overall and translation-dependent mRNA degradation. Purified CD4+ T lymphocytes, as shown by flow cytometry analysis of CD3 and CD4 surface expression in cells obtained from spleen and lymph nodes before and after purification by negative selection, are incubated with transcription inhibitors (triptolide or DRB). Fifteen minutes following addition of transcription inhibitors, translation inhibitors (cycloheximide or harringtonine) are added to cells. Cells are collected at 0, 1, and 3 h following transcription inhibition to monitor transcript expression by RNA-seq. Ribosome profiling and poly(A)-site sequence (PAS-seq) were also performed at the time 0 h in absence of transcription inhibitors. (B) Fold-change in transcript abundance (compared with the 0-h time point) upon blocking transcription only or when blocking both transcription and translation with different drug combinations. (C) Details of the calculation of the TDD and TID indexes illustrated by the expression dynamics of the transcript coding for CCT3 (ENSMUSG00000001416) upon incubation with transcription and translation inhibitors. (D) mRNA half-life measurement of TDD and TID targets by quantitative PCR in resting T cells incubated with triptolide or triptolide + cycloheximide. (E) Comparison of the TDDindex (left) or TIDindex (right), obtained using triptolide and cycloheximide, for protein-coding transcripts (mRNAs), long intergenic noncoding RNAs (lincRNAs), and UPF2-regulated transcripts in resting T cells. Displayed P-values correspond to the mean P-value of a Wilcoxon test performed 1000 independent times on samples of the same size for each compared group. (F) Distribution of the TDDindex and TIDindex calculated from resting T cells incubated for 3 h in the presence of triptolide and cycloheximide.
