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Structural basis for specific single-stranded RNA recognition by designer pentatricopeptide repeat proteins.

Shen C, Zhang D, Guan Z, Liu Y, Yang Z, Yang Y, Wang X, Wang Q, Zhang Q, Fan S, Zou T, Yin P - Nat Commun (2016)

Bottom Line: The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA.Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G.These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China.

ABSTRACT
As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes. Binding to their target single-stranded RNAs (ssRNAs) in a modular and base-specific fashion, PPR proteins can serve as designable modules for gene manipulation. However, the structural basis for nucleotide-specific recognition by designer PPR (dPPR) proteins remains to be elucidated. Here, we report four crystal structures of dPPR proteins in complex with their respective ssRNA targets. The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA. Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G. These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

No MeSH data available.


Design of dPPR proteins with RNA-recognition specificity.(a) Sequence of dPPR motif containing 35 amino acids. The secondary structural elements of a typical PPR motif are shown above. PPR codes comprising two residues located at the 5th and 35th positions are labelled in distinct colours (‘ND', ‘NS', ‘SN' and ‘TD', which recognize uracil, cytosine, adenine and guanine, respectively, are coloured green, lilac, yellow and cyan, respectively). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; S, Ser; T, Thr; V, Val and Y, Tyr. (b) Schematic representation of dPPR-U10, dPPR-U8C2, dPPR-U8A2 and dPPR-U8G2, and their targeting of specific RNA sequences. The shaded binary amino acids indicate PPR repeats with different codes. The NTD and CTD are from native PPR10. (c) Specific RNA target binding of dPPRs. In the RNA EMSA, 50 nM purified dPPRs were mixed with 2 nM 32P-labelled RNA, respectively. The sequence of the RNA probe is listed below each panel. Fig. 1a is reprinted from, Shen et al.28 with permission from Elsevier.
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f1: Design of dPPR proteins with RNA-recognition specificity.(a) Sequence of dPPR motif containing 35 amino acids. The secondary structural elements of a typical PPR motif are shown above. PPR codes comprising two residues located at the 5th and 35th positions are labelled in distinct colours (‘ND', ‘NS', ‘SN' and ‘TD', which recognize uracil, cytosine, adenine and guanine, respectively, are coloured green, lilac, yellow and cyan, respectively). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; S, Ser; T, Thr; V, Val and Y, Tyr. (b) Schematic representation of dPPR-U10, dPPR-U8C2, dPPR-U8A2 and dPPR-U8G2, and their targeting of specific RNA sequences. The shaded binary amino acids indicate PPR repeats with different codes. The NTD and CTD are from native PPR10. (c) Specific RNA target binding of dPPRs. In the RNA EMSA, 50 nM purified dPPRs were mixed with 2 nM 32P-labelled RNA, respectively. The sequence of the RNA probe is listed below each panel. Fig. 1a is reprinted from, Shen et al.28 with permission from Elsevier.

Mentions: On the basis of previous investigation28, we used dPPR scaffolds with four different codes: ND, NS, SN and TD (Fig. 1a). Each of the dPPR repeat scaffolds contains 35 amino acids, and those at positions 5 and 35 are referred to as PPR code amino acids. Parts of PPR10 from Z. mays were fused onto the amino and carboxyl termini of a series of tandem dPPR repeats as amino-terminal domain (NTD) and carboxyl-terminal domain (CTD), to enhance the solubility of the engineered protein (Fig. 1b and Supplementary Fig. 1). To verify the RNA selectivity, the dPPR proteins dPPR-U8N2 (in which N indicates any nucleotide) comprising 10 dPPR repeats with different 5th and 6th repeats with different PPR codes were constructed and purified to homogeneity. Each of the four dPPR-U8N2 proteins specifically bound to its respective target ssRNA with a dissociation constant of ∼20 –75 nM, as estimated on the basis of the results of electrophoretic mobility shift assay (EMSA) (Fig. 1c; Supplementary Fig. 2 and Supplementary Table 1). The substitution of any target RNA base with another led to a notable reduction in or complete abrogation of dPPR-U8N2 binding (Fig. 1c and Supplementary Fig. 2). For instance, the substitution of cytosine at positions 5 and 6 with the pyrimidine uracil or the purines adenine and guanine resulted in the dissociation of the dPPR–RNA complex.


Structural basis for specific single-stranded RNA recognition by designer pentatricopeptide repeat proteins.

Shen C, Zhang D, Guan Z, Liu Y, Yang Z, Yang Y, Wang X, Wang Q, Zhang Q, Fan S, Zou T, Yin P - Nat Commun (2016)

Design of dPPR proteins with RNA-recognition specificity.(a) Sequence of dPPR motif containing 35 amino acids. The secondary structural elements of a typical PPR motif are shown above. PPR codes comprising two residues located at the 5th and 35th positions are labelled in distinct colours (‘ND', ‘NS', ‘SN' and ‘TD', which recognize uracil, cytosine, adenine and guanine, respectively, are coloured green, lilac, yellow and cyan, respectively). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; S, Ser; T, Thr; V, Val and Y, Tyr. (b) Schematic representation of dPPR-U10, dPPR-U8C2, dPPR-U8A2 and dPPR-U8G2, and their targeting of specific RNA sequences. The shaded binary amino acids indicate PPR repeats with different codes. The NTD and CTD are from native PPR10. (c) Specific RNA target binding of dPPRs. In the RNA EMSA, 50 nM purified dPPRs were mixed with 2 nM 32P-labelled RNA, respectively. The sequence of the RNA probe is listed below each panel. Fig. 1a is reprinted from, Shen et al.28 with permission from Elsevier.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837458&req=5

f1: Design of dPPR proteins with RNA-recognition specificity.(a) Sequence of dPPR motif containing 35 amino acids. The secondary structural elements of a typical PPR motif are shown above. PPR codes comprising two residues located at the 5th and 35th positions are labelled in distinct colours (‘ND', ‘NS', ‘SN' and ‘TD', which recognize uracil, cytosine, adenine and guanine, respectively, are coloured green, lilac, yellow and cyan, respectively). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; S, Ser; T, Thr; V, Val and Y, Tyr. (b) Schematic representation of dPPR-U10, dPPR-U8C2, dPPR-U8A2 and dPPR-U8G2, and their targeting of specific RNA sequences. The shaded binary amino acids indicate PPR repeats with different codes. The NTD and CTD are from native PPR10. (c) Specific RNA target binding of dPPRs. In the RNA EMSA, 50 nM purified dPPRs were mixed with 2 nM 32P-labelled RNA, respectively. The sequence of the RNA probe is listed below each panel. Fig. 1a is reprinted from, Shen et al.28 with permission from Elsevier.
Mentions: On the basis of previous investigation28, we used dPPR scaffolds with four different codes: ND, NS, SN and TD (Fig. 1a). Each of the dPPR repeat scaffolds contains 35 amino acids, and those at positions 5 and 35 are referred to as PPR code amino acids. Parts of PPR10 from Z. mays were fused onto the amino and carboxyl termini of a series of tandem dPPR repeats as amino-terminal domain (NTD) and carboxyl-terminal domain (CTD), to enhance the solubility of the engineered protein (Fig. 1b and Supplementary Fig. 1). To verify the RNA selectivity, the dPPR proteins dPPR-U8N2 (in which N indicates any nucleotide) comprising 10 dPPR repeats with different 5th and 6th repeats with different PPR codes were constructed and purified to homogeneity. Each of the four dPPR-U8N2 proteins specifically bound to its respective target ssRNA with a dissociation constant of ∼20 –75 nM, as estimated on the basis of the results of electrophoretic mobility shift assay (EMSA) (Fig. 1c; Supplementary Fig. 2 and Supplementary Table 1). The substitution of any target RNA base with another led to a notable reduction in or complete abrogation of dPPR-U8N2 binding (Fig. 1c and Supplementary Fig. 2). For instance, the substitution of cytosine at positions 5 and 6 with the pyrimidine uracil or the purines adenine and guanine resulted in the dissociation of the dPPR–RNA complex.

Bottom Line: The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA.Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G.These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China.

ABSTRACT
As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes. Binding to their target single-stranded RNAs (ssRNAs) in a modular and base-specific fashion, PPR proteins can serve as designable modules for gene manipulation. However, the structural basis for nucleotide-specific recognition by designer PPR (dPPR) proteins remains to be elucidated. Here, we report four crystal structures of dPPR proteins in complex with their respective ssRNA targets. The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA. Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G. These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

No MeSH data available.