Genome-Wide Dual-Selection Unveils Novel Self-Cleaving Ribozymes in the Human Genome

The landscape of catalytic RNA in complex eukaryotes remains poorly charted. Although self-cleaving ribozymes are widespread in microbial and viral genomes, their existence and functional roles in humans remain limited. Here, we introduce a generalizable, genome-wide discovery platform that integrates high-throughput signals from two complementary adapter ligation assays—3P-seq and 5OH-seq—to specifically capture RNA fragments bearing cleavage signatures (2′,3′-cyclic phosphate/3′-phosphate and 5′-hydroxyl termini). By applying a dedicated computational scoring algorithm to human genomic data, we systematically identified four previously unrecognized self-cleaving ribozymes. These ribozymes localize to diverse genomic features: an exon of WDFY1, an intron of PLD5 embedded within a repetitive element, an LTR retrotransposon, and the antisense strand of an SYNJ2BP intron. Truncation analyses and mutational profiling defined minimal functional cores of 21–55 nucleotides, and experiment-assisted secondary structure prediction suggests that they adopt simple structural architectures. Despite their short sequences and simple architectures, we demonstrated that these ribozymes possess robust cleavage activity under multiple biochemical conditions. Most importantly, our experimental results indicate that three of them exhibit significant self-cleavage activity within the cellular environment. Our work establishes a powerful strategy for genome-wide mining of self-cleaving ribozymes, expands the catalogue of human ribozymes from 6 to 10, and reveals new layers of functional complexity in the human RNA world.