A distinct pseudoknot fold defines the hovlinc self-cleaving ribozyme in human lncRNA
Abstract
The hovlinc ribozyme is the first ribozyme identified within a human very long intergenic noncoding RNA (vlincRNA), uncovering a previously unexplored layer of functional RNA biology in the human genome. To elucidate its tertiary organization, we determined the crystal structure of a catalytically active truncated hovlinc ribozyme, which adopts an unanticipated homodimeric architecture composed of two functional units. Within each functional unit, the catalytic core is organized and stabilized by a previously unrecognized Rainbow-type (R-type) pseudoknot, defining a new RNA topological class distinct from all known pseudoknot architectures. At the cleavage site, A6-C7 adopts a splayed-apart conformation precisely organized by base-pairing and stacking interactions. Structure-based comparative analysis of 22 homologs, including an inactive gorilla variant, provides a structural rationale for widespread evolutionary loss of activity. Upon retaining the catalytic domain, we performed systematic truncation, sequence engineering, and SELEX-based selection to probe the variable regions, uncovering a broad landscape of sequence plasticity that expands the functional variant space, identifying activity-enhancing ribozyme variants. Structural modeling of the full-length ribozyme further reveals how peripheral elements modulate global folding and fine-tune catalytic efficiency. Collectively, these findings establish the first structural framework of a human lincRNA-encoded ribozyme and uncover a new RNA folding motif, revealing the structural principles underlying hovlinc ribozyme catalysis, demonstrating how subtle evolutionary variations tune RNA catalysis, and providing a framework for the discovery and engineering of functional RNA elements.
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