High-resolution mapping of CCR4-NOT recruitment elements reveals transcriptome-wide drivers of mRNA decay

The CCR4-NOT complex is a central regulator of gene expression, orchestrating mRNA turnover through interactions with RNA-binding proteins (RBPs) and the microRNA (miRNA)-induced silencing complex. However, identifying which RBP- and miRNA-associated RNA elements recruit CCR4-NOT remains challenging, due in part to the multiple modes by which the complex can be recruited. To address this, we developed TRACER (targeted RNA association with CCR4-NOT and element recovery), a high-throughput method for transcriptome-wide identification of RNA elements that recruit the CCR4-NOT to target RNAs. TRACER analysis in human epithelial cells uncovers thousands of CCR4-NOT-associated elements, including many that map to known and/or predicted RBP and miRNA target sites. We show that TRACER-identified elements drive mRNA repression and decay, and disrupting elements via gene editing or antisense oligonucleotides can relieve repression, boosting target gene expression. This positions TRACER as a powerful discovery platform for identifying regulatory RNA elements that can be targeted to control gene expression.