Mechanisms linking cytoplasmic decay of translation-defective mRNA to transcriptional adaptation [Cross-linked RIP-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: Cross-linked RIP was performed using the Magna Nuclear RIP (Cross-Linked) Nuclear RNA-Binding Protein Immunoprecipitation Kit (Millipore Sigma) according to the manufacturer's protocol using at least 2x 107 WT or rescued Actg1-NSD MEFs per replicate. For the cross-linked RIP, fixed nuclei were subjected to sonication using Bioruptor (Diagenode) to generate fragments of 200–600 bp in size prior to IP. Enriched 'input' samples were generated by reserving 10% of the starting lysate; the remaining volume was subjected to IP using the with 10 µg Anti-ILF3 antibody (abcam; ab92355 and BD Biosciences; Clone 21/DRBP76) coated onto protein A/G magnetic beads as described in the Magna RIP technical manual. RNA purified from both IP and input samples was concentrated by ethanol precipitation and resuspended in equivalent volumes of RNase-free water to be used to generate RNA-seq libraries. For the cross-linked total RNA RIP-seq, sequencing libraries were generated using 10 ng of RNA from input and IP samples using the SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Clontech).