Kinetic logic of uridylation-mediated RNA decay

Summary 3′-terminal uridylation marks structured non-coding RNAs for cytoplasmic decay, yet how uridylation is quantitatively coupled to exonucleolytic degradation remains unclear. Here, we dissect the kinetic logic of uridylation-mediated RNA surveillance in Drosophila melanogaster . Using biochemical reconstitution together with high-throughput enzymology and quantitative modeling, we show that the terminal uridylyl transferase Tailor generates discrete oligo(U) intermediates through product-dependent kinetic tuning, while co-substrate promiscuity suppresses sustained processivity under physiological nucleotide conditions. Massively parallel binding and decay assays further reveal how the 3′-to-5′ exoribonuclease Dis3l2 selectively degrades Tailor-primed RNAs by integrating 3′-proximal uridine content and defined 3′-end accessibility—features encoded by short, kinetically tuned oligo(U) intermediates centered on four nucleotides—to enable productive threading of RNA substrates along an extended RNA-binding path to the catalytic site. Together, our findings establish a quantitative framework in which uridylation encodes decay competence through transient RNA 3′-end states that are matched to the mechanistic requirements for decay. Highlights The TUTase Tailor kinetically tunes uridylation to generate short, discrete oligo(U) intermediates Mixed nucleotide availability suppresses sustained processive uridylation Dis3l2 decodes 3′-proximal uridine content and end accessibility to commit RNAs to decay Short oligo(U) tails encode RNA decay competence through transient RNA 3′-end states