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Structural basis for RNA recognition by a dimeric PPR-protein complex

Nature Structural & Molecular Biology volume 20, pages 1377–1382 (2013)Cite this article

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Abstract

Thylakoid assembly 8 (THA8) is a pentatricopeptide repeat (PPR) RNA-binding protein required for the splicing of the transcript of ycf3, a gene involved in chloroplast thylakoid-membrane biogenesis. Here we report the identification of multiple THA8-binding sites in the ycf3 intron and present crystal structures of Brachypodium distachyon THA8 either free of RNA or bound to two of the identified RNA sites. The apostructure reveals a THA8 monomer with five tandem PPR repeats arranged in a planar fold. The complexes of THA8 bound to the two short RNA fragments surprisingly reveal asymmetric THA8 dimers with the bound RNAs at the dimeric interface. RNA binding induces THA8 dimerization, with a conserved G nucleotide of the bound RNAs making extensive contacts with both monomers. Together, these results establish a new model of RNA recognition by RNA-induced formation of an asymmetric dimer of a PPR protein.

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Figure 1: The structure of THA8 in complex with a 13-nucleotide Zm-4 RNA.
Figure 2: RNA induces conformational changes and dimerization of THA8.
Figure 3: Zm4 RNA induces THA8 protein dimerization and oligomerization.

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Acknowledgements

We thank staff members of the Life Science Collaborative Access Team of the Advanced Photon Source (APS) for assistance in data collection at the beamlines of sector 21, which is in part funded by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). Use of APS was supported by the Office of Science of the US Department of Energy, under contract no. DE-AC02-06CH11357. This work was supported by the Jay and Betty Van Andel Foundation, the Ministry of Science and Technology (China) (grants 2012ZX09301001-005 and 2012CB910403), Amway (China), the US National Institutes of Health (grant R01 DK071662 to H.E.X.) and by the Chinese Academy of Sciences (J.-K.Z.).

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Author notes

  1. Jiyuan Ke, Run-Ze Chen and Ting Ban: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Structural Sciences, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, Grand Rapids, Michigan, USA

    Jiyuan Ke, X Edward Zhou, Xin Gu, M H Eileen Tan, Chen Chen, Yanyong Kang, Karsten Melcher & H Eric Xu

  2. Van Andel Research Institute–Shanghai Institute of Materia Medica (VARI-SIMM) Center, Center for Structure and Function of Drug Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Run-Ze Chen, Ting Ban & H Eric Xu

  3. Shanghai Center for Plant Stress Biology, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Ting Ban & Jian-Kang Zhu

  4. Department of Obstetrics & Gynecology, National University Hospital, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    M H Eileen Tan & Chen Chen

  5. Life Sciences Collaborative Access Team, Synchrotron Research Center, Northwestern University, Argonne, Illinois, USA

    Joseph S Brunzelle

  6. Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, USA

    Jian-Kang Zhu

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  1. Jiyuan Ke
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Contributions

H.E.X. and J.-K.Z. conceived of the study; H.E.X., J.K., J.-K.Z. and K.M. supervised the study; J.K., R.-Z.C., T.B., X.G., M.H.E.T., C.C. and Y.K. performed experiments of RNA binding, protein expression, purification and crystallization; J.K. and J.S.B. carried out data collection; J.K. and X.E.Z. performed model building, refinement and data analysis; and H.E.X. and J.K. wrote the manuscript with contribution from all authors.

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Correspondence to H Eric Xu.

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Integrated supplementary information

Supplementary Figure 1 Crystal structure and sequence alignment of THA8.

a, A cartoon diagram of THA8 is shown with rainbow color scheme from N-terminus (blue) to C-terminus (red). b, Superposition of THA8 (green, PDB code: 4ME2) and THA8L (magenta, PDB code: 4LEU) structures. The ribbon diagrams are shown for both structures. c, Sequence alignment of THA8 proteins from different species. BdTHA8, OsI-THA8, OsJ-THA8, ZmTHA8, SiTHA8 are THA8 protein sequences from Brachypodium distachyon, Oryza sativa Indica Group, Oryza sativa Japonica Group, Zea mays, and Setaria italica, respectively. Sequence alignment was performed by using ClustalW with manual adjustments. The secondary structure elements for BdTHA8 are noted on the top of the alignment. The conserved regions are highlighted in yellow. Residues making specific contacts with the G nucleotide are denoted with a black star; residues making contacts with the RNA backbone are denoted with a green star. Residues mediating critical dimer interactions are denoted with a magenta star. d, The BdTHA8 amino acid sequence is arranged as five PPR motifs. The predicted code residues in BdTHA8 are highlighted in blue (position 1 or 1') and yellow (position 6). The residues whose mutation severely impaired protein-RNA binding are colored in red whereas the residues whose mutation did not affect protein-RNA interaction are colored in green. The combination of T172 and D203 in positions 6 and 1' of motif 4 generates a specific code for G nucleotide.

Supplementary Figure 2 Sequence alignment of the ycf3 intron 2 from different species.

Sequence alignment was performed by using ClustalW with ycf3 intron 2 sequences from Zea mays, Brachypodium distachyon, Oryza sativa Indica Group, Oryza sativa Hassawi, Hordeum vulgare, Chasmanthium latifolium, Pennisetum glaucum. The conserved regions are highlighted in yellow. Multiple sites containing the conserved AGAAA core sequence are indicated by black triangles. The RNA fragments (1a, 2, 4) which are bound by THA8 protein are indicated at the top of the sequence alignment.

Supplementary Figure 3 Identification of a short ycf3-intron RNA sequence for THA8 interaction.

a, A schematic diagram for the principle of the AlphaScreen binding assay. b, The binding between 10 nM His6-THA8 and 10 nM of different biotin tagged RNAs was measured by AlphaScreen binding assay (n=3, error bars=SD). RNAzm1a is derived from the maize ycf3 intron 2, whereas RNACTL1 and RNACTL2 are two control RNAs that bind CRP1 and HCF152 (two other PPR proteins), respectively. The binding assay was repeated once with similar results. c, The minimal region of RNAzm1a that binds to THA8 protein. A competition assay was performed using 4 μM of different truncations of Zm-1a RNA to compete the interaction between biotin-RNAzm1a and His6-THA8 protein by AlphaScreen (n=3, error bars=SD). The competition was repeated once with similar results. d, Sequence alignment of 13-nucleotide RNAs from different species. The arrow indicates the decreasing order of affinities of BdTHA8 for different RNAs. e, Bindings between 10 nM His6-BdTHA8 and 10 nM biotin-RNAzm-1a were competed with 13-nucleotide homologous RNAs from different species using AlphaScreen assay (n=3, error bars=SD). THA8 binds to RNA sequences from different species with a preference of Zm-4 ≈ Os > Zm-1a ≈ Zm2 > Cs ≈ At. f, Bindings between 10 nM His6-BdTHA8 and 10 nM biotin-RNAZm-1a were competed with an increasing concentration of untagged Zm1a-5, Os or Zm4 RNA (n=3, error bars=SD). The IC50 values were calculated by curve fitting using Graphpad Prism. g, The minimal region of Zm-4 RNA that binds to THA8 protein. A competition assay was performed using 1 μM of different truncations of Zm-4 RNA to compete the interaction between 10 nM biotin-RNAzm1a and 10 nM His6-THA8 protein by AlphaScreen (n=3, error bars=SD). The competition was repeated once with similar results.

Supplementary Figure 4 THA8 protein prefers single-stranded RNA (ssRNA) and charge-complementary interactions are important for THA8 and RNA interactions.

a, THA8 protein binds to Zm4 ssRNA preferentially. Bindings between 10 nM His6-BdTHA8 and 10 nM biotin-Zm1a RNA were competed with untagged Zm4 ssRNA, ssDNA, dsRNA, RNA/DNA hybrid, or dsDNA (n=3, error bars=SD). THA8 protein binds Zm4 with a preference order of ssRNA > dsRNA ≈ DNA-RNA hybrid > dsDNA > ssDNA. The IC50 values were calculated by curve fitting using Graphpad Prism. b, The Zm4 RNA 2-OH group of ribose interacts with THA8 dimer protein through H-bonds. c, Mutational effects of the THA8 positively charged residues on THA8 and Zm1a RNA interaction. The binding between 10 nM biotin-Zm1a RNA and 50 nM His6-THA8 wild type or mutant proteins were measured by AlphaScreen assay (n=3, error bars=SD). The mutants with strongly reduced binding affinity are indicated by asterisks. The binding assay was repeated once with similar results. d, Mapping the positively charged residues onto the THA8 dimeric structure (shown in stick models). The positively charged residues that significantly reduced protein-RNA interaction are colored in magenta whereas those did not significantly affect protein-RNA interaction are colored in yellow. The RNA molecule is shown as a cartoon diagram.

Supplementary Figure 5 RNA induces THA8 protein dimerization and oligomerization, as measured by dynamic light scattering and gel-filtration chromatography.

a, Zm4 RNA induces THA8 dimerization in wild type, but not mutant proteins. The dynamic light scattering experiments were performed on THA8 wild type, dimerization mutant (S99R) or RNA-binding mutant (Y169S) proteins in the absence or presence of Zm4 RNA or control RNA (n=3). b, Analytic HPLC gel filtration profiles of THA8 wild type, dimerization mutant (S99R) and RNA-binding mutant (Y169S) in the presence or absence of Zm4 RNA.

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Ke, J., Chen, RZ., Ban, T. et al. Structural basis for RNA recognition by a dimeric PPR-protein complex. Nat Struct Mol Biol 20, 1377–1382 (2013). https://doi.org/10.1038/nsmb.2710

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