Structural basis for the modular recognition of single-stranded RNA by PPR proteins

Abstract

Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants1,2,3,4,5, recognize single-stranded RNA (ssRNA) in a modular fashion6,7,8. The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPIATPH and PSAJRPL33 intergenic regions, referred to as ATPH and PSAJ, respectively9,10. By protecting the target RNA elements from 5′ or 3′ exonucleases, PPR10 defines the corresponding 5′ and 3′ messenger RNA termini9,10,11. Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45 Å, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.

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Figure 1: Crystal structure of RNA-free PPR10.
Figure 2: Structure of PPR10 bound to an 18-nt PSAJ RNA element.
Figure 3: Base-specific recognition of ssRNA by PPR10 repeats.
Figure 4: Coordination of the 3′-end segment of the PSAJ RNA by PPR10.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors of RNA-free and RNA-bound PPR10 have been deposited in the Protein Data Bank (PDB) with the accession codes 4M57 and 4M59, respectively.

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Acknowledgements

We thank X. Yu and Y. Chen at the Institute of Biophysics, Chinese Academy of Sciences, for technical support. We thank K. Hasegawa and T. Kumasaka at the SPring-8 beamline BL41XU for on-site assistance. This work was supported by funds from the Ministry of Science and Technology (grant number 2011CB910501 for N.Y.), and Projects 91017011 (N.Y.), 31070644 (N.Y.), 31021002 (Y.S., N.Y., J.W.) and 31200567 (P.Y.) of the National Natural Science Foundation of China. The research of N.Y. was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute.

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P.Y., Q.L., J.-K.Z., Y.S. and N.Y. designed all experiments. P.Y., Q.L., C.Y., Y.L., J.L., F.Y., Z.W., J.L., J.H., H.-W.W., J.W. and N.Y. performed the experiments. All authors analysed the data and contributed to manuscript preparation. N.Y. wrote the manuscript.

Corresponding author

Correspondence to Nieng Yan.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequence alignment of the 19 repeats of PPR10.

a, PPR10 from maize specifically recognizes two RNA elements. The cartoon above illustrates the predicted domain organization of PPR10. 1 and 19 refer to the repeat numbers. CTP, chloroplast transit peptide. The blue brick with ‘?’ represents a fragment of approximately 30 amino acids whose function remains to be characterized. The minimal RNA elements of PSAJ and ATPH that are targeted by PPR10 are shown below the cartoon. b, Sequence alignment of 19 repeats in PPR10. The secondary structural elements of a typical PPR motif are shown above. The residues at the 2nd, 5th and 35th positions which were predicted to be the molecular determinants for RNA-binding specificity are highlighted in magenta. The RNA sequences that can be recognized by PPR10 are listed on the right, 5′ to 3′ from top to bottom. The nucleotides which are recognized by PPR10 in a modular fashion in the PSAJ–PPR10 structure are shaded grey. c, The three numbering systems for a PPR motif. 1 is being used by Lurin et al.12, Barkan et al.6 and others; 2 is adopted by the Pfam database and being used by Kobayashi et al.28, Yagi et al.7 and others; and 3 is our proposed, structure-based numbering system. The residues that are predicted to specifically recognize RNA are coloured magenta.

Extended Data Figure 2 AUC-SE of PPR10 (residues 37–786, C256S/C279S/C430S/C449S) in the absence or presence of the target RNA elements.

The molar concentrations of PPR10 are indicated above each panel. PPR10 and the RNA oligonucleotides were mixed at a stoichiometric ratio of approximately 1:1.5. Details of the experiments are described in Methods.

Extended Data Figure 3 The two protomers of the RNA-bound PPR10 dimer exhibit similar conformations.

a, The two protomers can be superimposed with a root-mean-squared deviation of 1.31 Å over 629 Cα atoms. b, c, The two ssRNA segments are coordinated by the PPR10 dimer similarly. The 5′ and 3′ segments of the bound PSAJ RNA are separately coordinated by the N-terminal repeats of one protomer (b), and the C-terminal repeats of the other protomer (c). Stereo-views are shown for all panels.

Extended Data Figure 4 Electron density maps for a bound ssRNA segment.

a, The 2Fo – Fc electron density for one segment of the bound PSAJ RNA. The electron density, contoured at 1σ and coloured blue, is displayed in stereo. b, c, The anomalous signals for bromine in the structures where the highlighted nucleotides were substituted with 5-bromouracil (5-BrU). The anomalous signals, shown in magenta mesh, are contoured at 5σ.

Extended Data Figure 5 Coordination of the bound ssRNA by PPR10.

a, The 5′ and 3′ portions of the PSAJ RNA element are separately bound by the N-terminal and C-terminal repeats of the two PPR10 protomers. Shown here is a close-up view of the binding of PSAJ by one end of the PPR10 dimer. b, The nucleotides U5–A10, which form a U-turn in the ssRNA, are uncoordinated in the cavity of the PPR10 dimer. The two protomers of PPR10 are shown in semi-transparent surface contour. c, The RNA backbone is coordinated by polar or charged residues through hydrogen bonds. The hydrogen bonds are represented by red dotted lines. The two protomers of PPR10 are coloured light purple and grey.

Extended Data Figure 6 Mutational analysis of PPR10 residues that may be important for PSAJ.

a, EMSA analysis of the interaction between PPR10 (residues 37–786, C256S/C279S/C430S/C449S) and PSAJ (5′-GUAUUCUUUAAUUAUUUC-3′). PPR10 was added with increasing concentrations of 0, 2, 4, 8, 16, 31, 63, 125, 250, 500, 1,000 nM in lanes 1–11 with approximately 40 pM 32P-labelled PSAJ in each lane. b, Mutational analysis of the 5th residues of the indicated PPR motifs. The indicated point mutations were introduced to PPR10 (residues 37–786, C256S/C279S/C430S/C449S). c, Examination of the 2nd residues in repeats 3 and 5. d, Examination of the 35th residue of repeat 6. Note that the side group of Asp 314 is hydrogen bonded to the side chain of Asn 284, the 5th residue of repeat 6. The same structural feature is also seen in repeat 4 (Fig. 3b).

Extended Data Figure 7 The predicted coordination of base C by an Asn at the 5th position of a PPR motif.

Left, the coordination of base U by Asn observed in the structure. Right, the coordination of base C by Asn at the 5th position of a PPR motif modelled on the basis of the structure shown on the left.

Extended Data Figure 8 Comparison of ssRNA coordination by PUF and PPR proteins.

a, The structure of the human PUF protein PUM1 (also known as HSPUM) bound to the RNA element NRE1-19 (PDB accession code, 1M8W)24. The PUF repeats constitute an arc with 8-nt ssRNA bound to the concave side. Notably, the orientations of the bound RNA and the protein are antiparallel, namely the 5′ end is close to the C terminus of PUF. b, The structure of a PUF repeat. One canonical PUF repeat contains three helices, of which a short helix precedes a helical hairpin. c, Representative recognition of the RNA bases G, U, A by PUF repeats as seen in the structure of PUM1 bound to NRE1-19. The amino acids are labelled by the repeat number (r5, r6, r7, r8) followed by its one-letter code and position on the 2nd helix within a PUF repeat (S3, N4, E8, and so on). The same scheme applies to d. d, The coordination of RNA bases G, U, A by PPR10. It is noteworthy that PUF and PPR proteins share several common features for RNA binding: (1) the ssRNA elements are coordinated by the helices on the inner layer; and (2) the base is sandwiched mostly by hydrophobic residues or Arg. Yet the differences are evident between the two families of repeat proteins. As seen in c, the RNA base is usually coordinated by two residues that are located at the 4th and the 7th positions on helix 2 within a PUF repeat. By contrast, the base is mainly coordinated by the 5th residue of a PPR motif. The 35th residue, the last residue of a PPR motif that is located at a loop region preceding the next PPR motif, also contributes to base recognition.

Extended Data Table 1 Statistics of data collection and refinement
Extended Data Table 2 Statistics of data collection

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Yin, P., Li, Q., Yan, C. et al. Structural basis for the modular recognition of single-stranded RNA by PPR proteins. Nature 504, 168–171 (2013). https://doi.org/10.1038/nature12651

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