Effects of lipid membranes on RNA catalytic activity and stability

Czerniak, T.; Sáenz, J. P. (2025) Biology of the Cell, 117(2), e202400115. DOI: 10.1111/boc.202400115

Summary

Extending our earlier discovery that lipid membranes modulate artificial ribozyme (R3C) activity, we examined the effects of lipid membranes on two naturally occurring ribozymes: the hepatitis delta virus (HDV) ribozyme and the hammerhead (HH) ribozyme. We found that gel-phase lipid membranes decreased HDV ribozyme activity but increased hammerhead ribozyme activity. Furthermore, liquid-phase membrane surfaces promoted RNA degradation, with greater degradation in single-stranded regions. These results show that different combinations of ribozyme type and membrane phase state produce distinct regulatory outcomes.

Key Findings

• Gel-phase membranes (ordered, rigid) decreased HDV ribozyme self-cleavage activity but increased hammerhead ribozyme activity — demonstrating that the effect of lipid membranes on RNA catalysis is ribozyme-specific and membrane-phase-dependent.
• Liquid-phase membranes (fluid) triggered RNA degradation, with greater degradation occurring in single-stranded regions of RNA — suggesting that fluid membranes can destabilize RNA structure.
• The lipid-to-nucleotide ratio modulates the effect, with dose-dependent changes in ribozyme activity.
• These findings expand the repertoire of RNA-lipid regulatory interactions beyond the artificial R3C system studied earlier (Czerniak & Sáenz, 2022, PNAS).

Methods

• HDV and hammerhead ribozyme assays: gel-based self-cleavage assays in the presence of liposomes of varying composition and phase state.
• RNA degradation analysis: gel electrophoresis to quantify intact vs. degraded RNA after incubation with liposomes.
• Lipid compositions: gel-phase (DPPC) and liquid-phase (DOPC) liposomes to compare membrane state effects.

Significance

By demonstrating that membrane phase state differentially affects natural ribozymes, this paper strengthened the case that RNA-lipid interactions are a general phenomenon with diverse functional outcomes. The finding that fluid membranes can degrade single-stranded RNA adds RNA stability to the list of membrane-modulated RNA properties, alongside catalytic activity.