Mechanisms linking cytoplasmic decay of translation-defective mRNA to transcriptional adaptation [PRO-seq]

Transcriptional adaptation (TA) is a genetic robustness mechanism through which mutant mRNA decay induces sequence-dependent upregulation of so-called adapting genes. How cytoplasmically generated mRNA fragments impact nuclear transcription remains poorly understood. Using genome-wide CRISPR screens, we uncover ILF3 as an RNA-binding protein connecting cytoplasmic mRNA decay and transcription during TA, and show it is required for a range of TA substrates. ILF3 is enriched at adapting genes' RNAs, and its artificial recruitment via dCas13 promotes gene expression. Using tiling oligonucleotide screens, we identify trigger RNA fragments that activate adapting genes when introduced into cells. Further functional dissection reveals critical role for homology between trigger and target sequences. These findings enhance our molecular understanding of TA and inform design of programmable oligonucleotides for gene expression augmentation. Overall design: PRO-seq was performed in three different batches. The first was WT versus Actg1-NSD MEFs, the second was Actg1-NSD MEFs versus Actg1-NSD; ?Ilf3 cells, and the third was WT versus WT;?Ilf3 cells. PRO-seq library preparation and sequencing was performed with the nascent transcriptomics core at Harvard Medical school. For each sample, we provided 2 million cells that were permeabilized, followed by library construction as described before (Mahat et al., 2016; Mimoso and Adelman, 2023).