Elucidation of structure–function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein

Abstract

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5′ leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein–protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.

RNAs undergo phase transitions with lower critical solution temperatures

“…5b ). We tested a range of Mg 2+ and Na + concentrations as well as buffer conditions specifically designed to promote optimal RPR folding 65, 68, 69, 70, 71, 72 . With Pfu, Mja , and Mma RPRs, we observed Mg 2+ -dependent formation of condensates ( Fig.…”

“…Most laboratories have customized folding protocols for their RNAs of interest 50, 73, 75, 76, 77 ; in this regard, RPRs are no exception 68, 69, 70, 71 . RNA folding is hierarchical with nascent secondary structures transitioning through discrete steps to generate a tertiary fold.…”

Coevolution of RNA and protein subunits in RNase P and RNase MRP, two RNA processing enzymes

“…In yeast, Pop3 enhances the interaction with Rpp21, thereby stabilizing the RNA structure even in the absence of the kink-turn, whereas in humans, Rpp38 recognizes the kink-turn and stabilizes the RNA conformation similar to L7Ae’s role in archaeal RNase P ( Fig. 3 , A and B ) ( 27 , 28 , 29 , 49 , 56 ). Of note, human Rpp38 also possesses a novel functional domain required for subnucleolar localization, highlighting how this protein was adapted to acquire new functions during evolution ( 57 ).…”

RNAs undergo phase transitions with lower critical solution temperatures

“…Here, we investigated archaeal RPRs from two thermophiles [Pyrococcus furiosus (Pfu) and Methanocaldococcus jannaschii (Mja)] and a mesophile [Methanococcus maripaludus (Mma)]. While RNase P from these three archaea have been extensively characterized 68,71,72,73,74,75 , the Pfu RPR is of special interest given that its activity in the absence of protein cofactors has been studied under different conditions 74,76 .…”