In vitro reconstitution and analysis of eukaryotic RNase P RNPs

Abstract

RNase P is a ubiquitous site-specific endoribonuclease primarily responsible for the maturation of tRNA. Throughout the three domains of life, the canonical form of RNase P is a ribonucleoprotein (RNP) built around a catalytic RNA. The core RNA is well conserved from bacteria to eukaryotes, whereas the protein parts vary significantly. The most complex and the least understood form of RNase P is found in eukaryotes, where multiple essential proteins playing largely unknown roles constitute the bulk of the enzyme. Eukaryotic RNase P was considered intractable to in vitro reconstitution, mostly due to insolubility of its protein components, which hindered its studies. We have developed a robust approach to the in vitro reconstitution of Saccharomyces cerevisiae RNase P RNPs and used it to analyze the interplay and roles of RNase P components. The results eliminate the major obstacle to biochemical and structural studies of eukaryotic RNase P, identify components required for the activation of the catalytic RNA, reveal roles of proteins in the enzyme stability, localize proteins on RNase P RNA, and demonstrate the interdependence of the binding of RNase P protein modules to the core RNA.

Biogenesis of RNase P RNA from an intron requires co-assembly with cognate protein subunits

“…Individual depletion of Pop5, Rpp14, Rpp30 or Rpp21, which had little or no effect on RPR stability, also affected RNase P activity and showed accumulation of pre-tRNAs (Figure 3F and G). Rpp21 had the least effect on activity, consistent with the observation that in vitro reconstituted yeast RNase P did not require Rpp21 for activity (7).…”

“…RPR biogenesis requires association with a specific subset of Rpps: Pop1, Pop4, Rpp20 and Rpp25 (Figures 2–4). The importance of these Rpps can be rationalized based on the high-resolution structures of yeast and human RNase P and in vitro reconstitution of yeast RNase P (7,9,11). Pop1 (∼100 kDa protein) in yeast and human RNase P makes extensive contacts with the RPR and stabilizes the helical core of the RPR’s catalytic domain ((9,11) and Supplementary Figure S6).…”

Cryo-EM structure of catalytic ribonucleoprotein complex RNase MRP

“…Proteins Pop6 and Pop7 form a heterodimer that helps to engage Pop1 in both RNase MRP (Fig. 4a ) and RNase P 21 , 22 , 26 , 37 . Pop6 and Pop7 interact with P3 RNA regions that adopt similar folds in RNase MRP and RNase P and are folded similarly in the two enzymes (RMSD = 1.8 Å and 1.3 Å, respectively).…”

“…S. cerevisiae RNase MRP and RNase P share eight proteins (Pop1, Pop3, Pop4, Pop5, Pop6, Pop7, Pop8, and Rpp1 (two copies)); RNase MRP protein Snm1 23 has a homolog in RNase P (Rpr2), while Rmp1 24 is found only in RNase MRP. Shared proteins bind to both C- and S-domains 21 , 25 , 26 . Yeast RNase MRP proteins Pop1, Pop3, Pop4, Pop5, Pop6, Pop7, Pop8 have homologs in human RNase P 3 , 4 , 18 , 22 , while Pop3, Pop4, Pop5, and Rpp1 have homologs in archaeal RNases P 3 , 4 , 27 .…”

Stability and nuclear localization of yeast telomerase depend on protein components of RNase P/MRP

“…7a, b) revealed that in 24°C grown pop cells, the level of TLC1 association with Est1 was only~14% of the level in WT cells. In vitro studies suggest that the secondary structures of NME1 and RPR1 are altered in the absence of Pop proteins 24,34 . Therefore, we considered the possibility that the structure of the Est1 and (to a lesser extent) the Est2 binding sites in TLC1 might be Pop protein dependent.…”