Kinetic logic of uridylation-mediated RNA decay

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. HighlightsO_LIThe TUTase Tailor kinetically tunes uridylation to generate short, discrete oligo(U) intermediates C_LIO_LIMixed nucleotide availability suppresses sustained processive uridylation C_LIO_LIDis3l2 decodes 3'-proximal uridine content and end accessibility to commit RNAs to decay C_LIO_LIShort oligo(U) tails encode RNA decay competence through transient RNA 3'-end states C_LI