Mechanisms linking cytoplasmic decay of translation-defective mRNA to transcriptional adaptation [TT-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: TT-seq For each replicate, 10 million cells were seeded into two 15-cm dishes one day prior to the experiment. Two replicates were performed per genotype, resulting in a total of four plates per genotype. On the day of the experiment, one plate per genotype was processed at a time to avoid handling more than four plates simultaneously. The media was aspirated and replaced with fresh media containing 500 µM 4-thiouridine (4sU) for 20 minutes. Following incubation, the media was aspirated again, and cells were rapidly washed with PBS. Two milliliters of TRIzol were then added per plate, and the cells were scraped and collected into tubes, which were flash-frozen in liquid nitrogen. TRIzol samples were then handled to the Nascent Transcriptomics Core at Harvard Medical school which performed the TT-seq library preparation and sequencing as described before (Vlaming et al., NSMB, 2022 & Stein et al., Mol Cell, 2022).