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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.


Interaction models for more PPR codes.(a) Prediction of another 4 interaction models for PPR codes ‘NN', ‘TN' and ‘SD' with U, C, A and G, respectively. The hydrogen bonds are represented by red dotted lines. Water molecules are represented by red spheres. (b) Schematic representation of dPPR-NN, dPPR-TN, dPPR-SD and their target RNA sequences. The shaded binary amino acids are indicative of PPR repeats with different codes. NTD and CTD are from native PPR10. (c) EMSA demonstrates the specific RNA recognition of dPPR proteins with predicted codes. The final concentrations of dPPR in lanes 1–10 are 0, 0.8, 1.6, 3.2, 6.25, 12.5, 25, 50, 100 and 200 nM, respectively. The detailed Kd values are shown in Supplementary Table 1.
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f5: Interaction models for more PPR codes.(a) Prediction of another 4 interaction models for PPR codes ‘NN', ‘TN' and ‘SD' with U, C, A and G, respectively. The hydrogen bonds are represented by red dotted lines. Water molecules are represented by red spheres. (b) Schematic representation of dPPR-NN, dPPR-TN, dPPR-SD and their target RNA sequences. The shaded binary amino acids are indicative of PPR repeats with different codes. NTD and CTD are from native PPR10. (c) EMSA demonstrates the specific RNA recognition of dPPR proteins with predicted codes. The final concentrations of dPPR in lanes 1–10 are 0, 0.8, 1.6, 3.2, 6.25, 12.5, 25, 50, 100 and 200 nM, respectively. The detailed Kd values are shown in Supplementary Table 1.

Mentions: In nature, the PPR code is degenerate15252627. Multiple combinations of amino acids at positions 5 and 35 can specify the same nucleotide, but sometimes the same combination of amino acids is similarly compatible with more than one type of nucleotide. Although we have not yet obtained crystal structures of PPR repeats with all code combinations, base-recognition models for codes such as NN, TN and SD2627 can be rationally deduced from the existing structural information (Fig. 5a). For instance, the code NN has been reported to be equally compatible with uracil and cytosine. Water-mediated hydrogen bonds may connect the amino acid at the 35th position with its coordinating pyrimidine. The ability of the side chain of Asn35 to rotate is also important because it allows the amino acid to be a hydrogen bond donor or acceptor. Under these circumstances, we predict that the N3 atom of either uracil or cytosine may form a hydrogen bond with the water molecule but with the opposite polarity. We designed and purified proteins containing predicted codes NN, TN and SD for biochemical verification (Fig. 5b). The biochemical results corroborated the models, demonstrating that the code NN exhibits similar selectivity towards U and C with dissociation constants of ∼15 nM, whereas codes TN and SD specifically recognize A and G, respectively (Fig. 5c). Thus, these results provide a framework for deciphering the RNA targets of the mysterious PPR-motif code, which comprises more than 30 code combinations in nature152627.


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)

Interaction models for more PPR codes.(a) Prediction of another 4 interaction models for PPR codes ‘NN', ‘TN' and ‘SD' with U, C, A and G, respectively. The hydrogen bonds are represented by red dotted lines. Water molecules are represented by red spheres. (b) Schematic representation of dPPR-NN, dPPR-TN, dPPR-SD and their target RNA sequences. The shaded binary amino acids are indicative of PPR repeats with different codes. NTD and CTD are from native PPR10. (c) EMSA demonstrates the specific RNA recognition of dPPR proteins with predicted codes. The final concentrations of dPPR in lanes 1–10 are 0, 0.8, 1.6, 3.2, 6.25, 12.5, 25, 50, 100 and 200 nM, respectively. The detailed Kd values are shown in Supplementary Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Interaction models for more PPR codes.(a) Prediction of another 4 interaction models for PPR codes ‘NN', ‘TN' and ‘SD' with U, C, A and G, respectively. The hydrogen bonds are represented by red dotted lines. Water molecules are represented by red spheres. (b) Schematic representation of dPPR-NN, dPPR-TN, dPPR-SD and their target RNA sequences. The shaded binary amino acids are indicative of PPR repeats with different codes. NTD and CTD are from native PPR10. (c) EMSA demonstrates the specific RNA recognition of dPPR proteins with predicted codes. The final concentrations of dPPR in lanes 1–10 are 0, 0.8, 1.6, 3.2, 6.25, 12.5, 25, 50, 100 and 200 nM, respectively. The detailed Kd values are shown in Supplementary Table 1.
Mentions: In nature, the PPR code is degenerate15252627. Multiple combinations of amino acids at positions 5 and 35 can specify the same nucleotide, but sometimes the same combination of amino acids is similarly compatible with more than one type of nucleotide. Although we have not yet obtained crystal structures of PPR repeats with all code combinations, base-recognition models for codes such as NN, TN and SD2627 can be rationally deduced from the existing structural information (Fig. 5a). For instance, the code NN has been reported to be equally compatible with uracil and cytosine. Water-mediated hydrogen bonds may connect the amino acid at the 35th position with its coordinating pyrimidine. The ability of the side chain of Asn35 to rotate is also important because it allows the amino acid to be a hydrogen bond donor or acceptor. Under these circumstances, we predict that the N3 atom of either uracil or cytosine may form a hydrogen bond with the water molecule but with the opposite polarity. We designed and purified proteins containing predicted codes NN, TN and SD for biochemical verification (Fig. 5b). The biochemical results corroborated the models, demonstrating that the code NN exhibits similar selectivity towards U and C with dissociation constants of ∼15 nM, whereas codes TN and SD specifically recognize A and G, respectively (Fig. 5c). Thus, these results provide a framework for deciphering the RNA targets of the mysterious PPR-motif code, which comprises more than 30 code combinations in nature152627.

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.