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

Although self-cleaving ribozymes are widespread in microbial and viral genomes, their existence and functional roles in humans remain largely unknown. 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.