GSE134660
The Dynamics of Cytoplasmic mRNA Metabolism
For all but a few mRNAs, the dynamics of metabolism are unknown. Here, we developed an experimental and analytical framework for examining these dynamics for mRNAs from thousands of genes. mRNAs of mouse fibroblasts exit the nucleus with diverse intragenic and intergenic poly(A)-tail lengths. Once in the cytoplasm, they have a broad (1000-fold) range of deadenylation rates, which correspond to cytoplasmic lifetimes. Indeed, degradation appears to occur primarily through deadenylation-linked mechanisms, with little contribution from endonucleolytic cleavage or deadenylation-independent decapping. Most mRNA molecules degrade only after their tail lengths fall below 25 nt. Decay rates of short-tailed mRNAs vary broadly (1000-fold) and are more rapid for short-tailed mRNAs that had previously undergone more rapid deadenylation. This coupling helps clear rapidly deadenylated mRNAs, enabling the large range in deadenylation rates to impart a similarly large range in stabilities. Overall design: The experiments consist of ribosome profiling libraries (small RNA-seq and RPF reads) from two microRNA-inducible 3T3 cell lines (miR-1 and miR-155) with or without microRNA induction. These samples were prepared for 5 and 6 metabolic-labeling time points for the miR-1 and miR-155 lines, respectively. In addition, a metabolic labeling/transcriptional shutoff experiment was performed which generated 12 sequences samples made with the NEXTflex RNA-seq kit. Finally, 38 PAL-seq samples from these time courses are also included to measure poly(A)-tail lengths.