Poster abstracts
Poster number 64 submitted by Walter Zahurancik
Modulation of Archaeal Ribonuclease P Ribozyme Activity by Temperature- and Metal-ion–dependent Phase Separation
Walter J. Zahurancik (Department of Chemistry and Biochemistry, The Ohio State University), Gable M. Wadsworth, Paul Pullara, Priya R. Banerjee (Department of Physics, The State University of New York at Buffalo), Xiangze Zeng (Department of Physics, Hong Kong Baptist University), Lien B. Lai, Vaishnavi Sidharthan, Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University), Rohit V. Pappu (Department of Biomedical Engineering, Washington University in St. Louis)
Abstract:
Phase separation of proteins and nucleic acids is a ubiquitous mechanism for cellular organization. Studies examining phase-separated ribonucleoprotein (RNP) condensates have led to a growing appreciation of RNAs as co-drivers, and recent reports highlight protein-free RNA phase separation in the presence of polyamines (e.g., spermine) or divalent cations (e.g., Mg2+). However, the molecular forces driving RNA condensation are not fully understood. To address this gap, we selected as a tractable model ribonuclease (RNase P), an essential and ubiquitous RNP that catalyzes removal of the 5' leader from precursor (pre)-tRNAs. Our choice was inspired by the availability of functional RNase P RNAs (RPRs) that could be assayed for pre-tRNA cleavage in the absence of protein co-factors. Here, we focused on archaeal RNase P to parse the interplay between phase separation and RPR activity. We studied in vitro transcribed RPRs from three different archaea: Pyrococcus furiosus (Pfu), Methanocaldococcus jannaschii, and Methanococcus maripaludis. Upon heating, these RPRs underwent a Mg2+-dependent phase transition, a finding inconsistent with the current understanding that RNA condensation is driven primarily by intermolecular base pairing and base stacking. Phase separation also drastically decreased the cleavage activity of Pfu RPR. The phase-separated Pfu RPR formed micron-size condensates that were reversed under stringent denaturing conditions or upon incubation with EDTA, consistent with a Mg2+ ion-dependent bridging mechanism of phase separation. Our findings suggest that phase separation may play a critical role in regulating non-coding RNA function and offer a reversible mechanism that could be fine-tuned by key cellular metabolites (e.g., citrate).
References:
Wadsworth GM, Zahurancik WJ, Zeng X, Pullara P, Lai LB, Sidharthan V, Pappu RV, Gopalan V, and Banerjee PR. (2023) RNAs undergo phase transitions with lower critical solution temperatures. Nat Chem, 15: 1693-1704.
Keywords: Phase separation, Ribozyme, RNase P