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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.

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Related in: MedlinePlus

Structural basis of nucleobase recognition by dPPR repeats.PPR code amino acids selectively target nucleotides by forming direct or indirect hydrogen bonds to the Watson–Crick faces of bases. The specific recognition patterns of the bases U (a), C (b), A (c) and G (d) by dPPR repeats are shown in the zoom-in view. The side chains of the 5th and 35th residues in each PPR repeat are shown in yellow. Bases are labelled and coloured according to atom type (carbon: green, oxygen: red, nitrogen: blue). The hydrogen bonds are represented by red dotted lines. Water molecules are represented by red spheres. Single-letter abbreviations for the amino acid residues and nucleobases are as follows: D, Asp; N, Asn; S, Ser; T, Thr; A, Adenine; C, Cytosine; G, Guanine and U, Uracil.
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f4: Structural basis of nucleobase recognition by dPPR repeats.PPR code amino acids selectively target nucleotides by forming direct or indirect hydrogen bonds to the Watson–Crick faces of bases. The specific recognition patterns of the bases U (a), C (b), A (c) and G (d) by dPPR repeats are shown in the zoom-in view. The side chains of the 5th and 35th residues in each PPR repeat are shown in yellow. Bases are labelled and coloured according to atom type (carbon: green, oxygen: red, nitrogen: blue). The hydrogen bonds are represented by red dotted lines. Water molecules are represented by red spheres. Single-letter abbreviations for the amino acid residues and nucleobases are as follows: D, Asp; N, Asn; S, Ser; T, Thr; A, Adenine; C, Cytosine; G, Guanine and U, Uracil.

Mentions: As observed in all four complex structures, four types of dPPR repeats recognize their corresponding targets by forming hydrogen bonds with the Watson–Crick faces of the nucleotides, which explains why PPR proteins bind ssRNAs instead of double stranded ones. The electron densities of the RNA nucleotides 5 and 6 differ remarkably from each other (Supplementary Fig. 6), providing insight into the base-recognition mechanisms of PPR repeats. Previous studies have strongly suggested that the polar amino acid at the 5th position in each repeat is the chief determinant of RNA base specificity. Serine or threonine at this position results in a preference for purines, whereas the presence of asparagine is correlated with a preference for pyrimidines. The structures of RNA-bound dPPRs provide a powerful explanation for these codes. The amide group of the Asn5 side chain donates a hydrogen bond to the O2 atom of the corresponding pyrimidine, whereas the N3 atom of purine accepts a hydrogen bond from the hydroxyl group of the corresponding amino acid (Fig. 4). The 35th residue, which is the second significant amino acid of the PPR code, is also located in close proximity to the corresponding nucleobase. We observed that water molecules between bases and PPR repeats mediate hydrogen bonds between the polar residues and the bases in the cases of uracil and cytosine recognition. This recognition pattern has not been reported in the TALE–DNA3637 or PUF–RNA interactions838. Each water molecule between the base and corresponding PPR repeat forms two hydrogen bonds: one with the N3 atom of the pyrimidine and one with the carboxyl group of Asp35 (Fig. 4a) or the hydroxyl group of Ser35 (Fig. 4b). Base selectivity is determined via ‘water bridge' polarity. The N3 atom of uracil is a hydrogen bond donor, whereas the N3 atom of cytosine is a hydrogen bond acceptor. For purine, Asn35 or Asp35 form one (Fig. 4c) or two (Fig. 4d) hydrogen bonds with adenine and guanine, respectively. The N1 atom of adenine is a hydrogen bond acceptor, whereas both the N1 and N2 atoms of guanine are hydrogen bond donors. These structures demonstrate how the amino acids asparagine and aspartate at the 35th position contribute to purine base selectivity.


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)

Structural basis of nucleobase recognition by dPPR repeats.PPR code amino acids selectively target nucleotides by forming direct or indirect hydrogen bonds to the Watson–Crick faces of bases. The specific recognition patterns of the bases U (a), C (b), A (c) and G (d) by dPPR repeats are shown in the zoom-in view. The side chains of the 5th and 35th residues in each PPR repeat are shown in yellow. Bases are labelled and coloured according to atom type (carbon: green, oxygen: red, nitrogen: blue). The hydrogen bonds are represented by red dotted lines. Water molecules are represented by red spheres. Single-letter abbreviations for the amino acid residues and nucleobases are as follows: D, Asp; N, Asn; S, Ser; T, Thr; A, Adenine; C, Cytosine; G, Guanine and U, Uracil.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Structural basis of nucleobase recognition by dPPR repeats.PPR code amino acids selectively target nucleotides by forming direct or indirect hydrogen bonds to the Watson–Crick faces of bases. The specific recognition patterns of the bases U (a), C (b), A (c) and G (d) by dPPR repeats are shown in the zoom-in view. The side chains of the 5th and 35th residues in each PPR repeat are shown in yellow. Bases are labelled and coloured according to atom type (carbon: green, oxygen: red, nitrogen: blue). The hydrogen bonds are represented by red dotted lines. Water molecules are represented by red spheres. Single-letter abbreviations for the amino acid residues and nucleobases are as follows: D, Asp; N, Asn; S, Ser; T, Thr; A, Adenine; C, Cytosine; G, Guanine and U, Uracil.
Mentions: As observed in all four complex structures, four types of dPPR repeats recognize their corresponding targets by forming hydrogen bonds with the Watson–Crick faces of the nucleotides, which explains why PPR proteins bind ssRNAs instead of double stranded ones. The electron densities of the RNA nucleotides 5 and 6 differ remarkably from each other (Supplementary Fig. 6), providing insight into the base-recognition mechanisms of PPR repeats. Previous studies have strongly suggested that the polar amino acid at the 5th position in each repeat is the chief determinant of RNA base specificity. Serine or threonine at this position results in a preference for purines, whereas the presence of asparagine is correlated with a preference for pyrimidines. The structures of RNA-bound dPPRs provide a powerful explanation for these codes. The amide group of the Asn5 side chain donates a hydrogen bond to the O2 atom of the corresponding pyrimidine, whereas the N3 atom of purine accepts a hydrogen bond from the hydroxyl group of the corresponding amino acid (Fig. 4). The 35th residue, which is the second significant amino acid of the PPR code, is also located in close proximity to the corresponding nucleobase. We observed that water molecules between bases and PPR repeats mediate hydrogen bonds between the polar residues and the bases in the cases of uracil and cytosine recognition. This recognition pattern has not been reported in the TALE–DNA3637 or PUF–RNA interactions838. Each water molecule between the base and corresponding PPR repeat forms two hydrogen bonds: one with the N3 atom of the pyrimidine and one with the carboxyl group of Asp35 (Fig. 4a) or the hydroxyl group of Ser35 (Fig. 4b). Base selectivity is determined via ‘water bridge' polarity. The N3 atom of uracil is a hydrogen bond donor, whereas the N3 atom of cytosine is a hydrogen bond acceptor. For purine, Asn35 or Asp35 form one (Fig. 4c) or two (Fig. 4d) hydrogen bonds with adenine and guanine, respectively. The N1 atom of adenine is a hydrogen bond acceptor, whereas both the N1 and N2 atoms of guanine are hydrogen bond donors. These structures demonstrate how the amino acids asparagine and aspartate at the 35th position contribute to purine base selectivity.

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.


Related in: MedlinePlus