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, 46 (13), 6857-6868

In Vitro Reconstitution and Analysis of Eukaryotic RNase P RNPs

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In Vitro Reconstitution and Analysis of Eukaryotic RNase P RNPs

Anna Perederina et al. Nucleic Acids Res.

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.

Figures

Figure 1.
Figure 1.
(A) Secondary structure diagram of S. cerevisiae RNase P RNA (4). The nomenclature of structural elements is based on (13). (B) Protein components of archaeal RNase P from P. furiosus (Pfu) (12) and eukaryotic RNase P from S. cerevisiae (14). Arrows indicate homology between archaeal and yeast proteins.
Figure 2.
Figure 2.
(A) Stepwise assembly of the reconstituted RNase P RNP (lanes 1–5) and binding of RNase P proteins to RNase P RNA (lanes 6–9). (B) The addition of Pop4 does not result in a mobility shift unless the Rpp1/Pop5/Pop8 protein subcomplex is already present. (C) The presence of Pop6/Pop7 is required for the structural stability of the reconstituted RNP. (D) RNP assembly with the Rpp1/Pop5 complex substituting for Rpp1/Pop5/Pop8. Lanes 1, 10, 18: RNase P RNA alone; other lanes: electrophoretic mobility shifts upon the addition of protein components as indicated above the gel. Protein components are added to RNase P RNA at a 1:1 molar ratio; the resulting RNP complexes are resolved on a native polyacrylamide gel. RNA is stained with Toluidine Blue. Pop6 and Pop7, as well as Rpp1, Pop5, Pop8 or Rpp1, Pop5 formed subcomplexes and were co-expressed and co-purified together.
Figure 3.
Figure 3.
Catalytic activity of reconstituted RNPs assayed under conditions of moderate ionic strength. The insert below the main gel shows the overexposed boxed section of the gel. 4 pmol (0.36 μM) of RNase P RNA was used to form RNPs; proteins were added to RNA at a 1:1 molar ratio. 40 pmol (3.6 μM) of 5′-end 32P-labeled S. cerevisiae pre-tRNAThr(AGT) substrate was incubated with the reconstituted RNPs for 30 min at 30°C in 50 mM HEPES-NaOH pH 7.8, 100 mM ammonium acetate, 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5% glycerol, 0.5 μg/ml BSA. The protein combinations that resulted in activity (highlighted in gray below the gel) were assayed with the ‘wild type’ RNase P RNA (labeled as ‘W’ in the ‘RNase P RNA’ description lane, bottom part of the figure), as well as with a mutated RNA that lacked catalytic core nucleotides A90, U93 essential for catalysis (labeled as ‘m’ in the ‘RNase P RNA’ description lane), which served as a negative control. Lane 1: pre-tRNA substrate before cleavage. Lane 2: pre-tRNA substrate partially digested with RNase A. Lane 3: pre-tRNA substrate digested in alkali. Lane 4: pre-tRNA substrate digested with the endogenous RNase P (control). Lanes 5: incubation with RNase P RNA alone. Lanes 6–25: activities of the reconstituted RNPs with compositions as indicated below the gel.
Figure 4.
Figure 4.
Footprinting data for RNase P RNP assemblies. (A) Quantification of hydroxyl radicals (Fe(II)-EDTA) footprinting assays. The grey horizontal axes correspond to no additional protection (same reactivity as the RNA-only reference); the elevation above an axis reflects the degree of the protection (compared to RNA-only reference) in the presence of proteins. Protein compositions of the assayed RNP complexes are color-coded as shown on top of the figure; the proteins present in the RNP are underscored. Thick black line: proteins Pop6/Pop7, Pop1, Rpp1/Pop5/Pop8, Pop4. Red line: Pop6/Pop7, Pop1, Rpp1/Pop5/Pop8. Sand line: Pop1, Rpp1/Pop5/Pop8, Pop4. Green line: Pop6/Pop7, Pop1. Blue line: Pop6/Pop7. Sequence and secondary structure elements of the RNase P RNA (Figure 1A) are shown below the graphs. Helical stems are shown by thick solid lines; terminal loops are shown by thin solid lines; large internal loops and conserved elements are shown by dotted lines. See Supplementary Figure S8 for gels. (B–E) Changes in RNase P RNA reactivity induced by protein binding during RNase P RNP assembly. (B) Protection upon the binding of Pop6/Pop7 to RNA (blue). (C) Additional protection (green) and hypersensitivity (violet) upon the binding of Pop1 to the RNA-Pop6/Pop7 RNP. (D) Additional protection (red) and hypersensitivity (violet) upon the binding of Rpp1/Pop5/Pop8 to the RNA-Pop6/Pop7/Pop1 RNP. (E) Additional protection (black) and hypersensitivity (violet) upon the binding of Pop4 to the RNA-Pop6/Pop7/Pop1/Rpp1/Pop5/Pop8 RNP.

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