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

Abstract

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 defined minimal functional cores of ~70–100 nucleotides, and computational modeling suggests that they adopt novel structural architectures. Our work establishes a powerful strategy for transcriptome-wide mining of catalytic RNA, expands the catalogue of human ribozymes from 6 to 10, and reveals new layers of functional complexity in the human RNA world.