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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.


Overall structure of RNA-bound dPPR-U8C2.(a) Overall structure of dPPR-U8C2 bound to its target RNA element. dPPR-U8C2 comprises 10 repeats capped by a small NTD helix (slate) and a CTD helix (cyan). The 10 dPPR repeats of dPPR-U8C2 form a right-handed superhelical assembly. Wheat and grey bundles indicate helix a and helix b, respectively. (b) Electron density of target RNA is clearly visible in the cavity of the dPPR superhelix. The electron density, contoured at 1σ, is shown in yellow. The surface electrostatic potential was calculated with PyMOL. Two perpendicular views are presented, with the ssRNA molecule depicted as sticks. All structure figures were prepared using PyMOL.
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f2: Overall structure of RNA-bound dPPR-U8C2.(a) Overall structure of dPPR-U8C2 bound to its target RNA element. dPPR-U8C2 comprises 10 repeats capped by a small NTD helix (slate) and a CTD helix (cyan). The 10 dPPR repeats of dPPR-U8C2 form a right-handed superhelical assembly. Wheat and grey bundles indicate helix a and helix b, respectively. (b) Electron density of target RNA is clearly visible in the cavity of the dPPR superhelix. The electron density, contoured at 1σ, is shown in yellow. The surface electrostatic potential was calculated with PyMOL. Two perpendicular views are presented, with the ssRNA molecule depicted as sticks. All structure figures were prepared using PyMOL.

Mentions: In the complex structure, dPPR-U8C2 has 10 dPPR repeats (residues 174–523), which are capped by NTD and CTD helices (Fig. 2a). Each repeat in dPPR-U8C2 contains 35 amino acids, forming a hairpin of α-helices that both contain four helical turns followed by a five-residue loop. The two helices, formed by residues 1–14 and 17–30 (Fig. 1a), are designated as helix a and helix b, respectively (Fig. 2a, left panel). The whole-protein molecule has an overall appearance of a solenoid with a polar axis of 75 Å and a diameter of  50 Å (Fig. 2a). The internal layer along the superhelical axis is constituted by helices a, whereas helices b outline the external layer of the superhelix. Following the assignment of dPPR proteins in the electron density maps, electron densities indicative of RNA bases, which interdigitated with PPR helices, emerged in the cavity of the superhelix (Fig. 2b and Supplementary Fig. 4c). Because of the limited quality of the electron density data, only the 10 nucleotides coordinated by repeats could be modelled. Only one complex comprising one dPPR molecule with an ssRNA target was present in each asymmetric unit, similarly to the solution complex structure of ATPH-bound PPR1030. The overall dPPR protein structure consists of repetitions of helix pairs packing against each other to form a right-handed superhelical spiral shell that embraces its target ssRNA. The ssRNA molecule forms a right-handed parallel duplex structure with an ‘outer-layer' spiral protein enclosure. All 10 nucleotides in the target RNA elements strictly exhibit the modular pattern binding to corresponding dPPR repeats.


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)

Overall structure of RNA-bound dPPR-U8C2.(a) Overall structure of dPPR-U8C2 bound to its target RNA element. dPPR-U8C2 comprises 10 repeats capped by a small NTD helix (slate) and a CTD helix (cyan). The 10 dPPR repeats of dPPR-U8C2 form a right-handed superhelical assembly. Wheat and grey bundles indicate helix a and helix b, respectively. (b) Electron density of target RNA is clearly visible in the cavity of the dPPR superhelix. The electron density, contoured at 1σ, is shown in yellow. The surface electrostatic potential was calculated with PyMOL. Two perpendicular views are presented, with the ssRNA molecule depicted as sticks. All structure figures were prepared using PyMOL.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Overall structure of RNA-bound dPPR-U8C2.(a) Overall structure of dPPR-U8C2 bound to its target RNA element. dPPR-U8C2 comprises 10 repeats capped by a small NTD helix (slate) and a CTD helix (cyan). The 10 dPPR repeats of dPPR-U8C2 form a right-handed superhelical assembly. Wheat and grey bundles indicate helix a and helix b, respectively. (b) Electron density of target RNA is clearly visible in the cavity of the dPPR superhelix. The electron density, contoured at 1σ, is shown in yellow. The surface electrostatic potential was calculated with PyMOL. Two perpendicular views are presented, with the ssRNA molecule depicted as sticks. All structure figures were prepared using PyMOL.
Mentions: In the complex structure, dPPR-U8C2 has 10 dPPR repeats (residues 174–523), which are capped by NTD and CTD helices (Fig. 2a). Each repeat in dPPR-U8C2 contains 35 amino acids, forming a hairpin of α-helices that both contain four helical turns followed by a five-residue loop. The two helices, formed by residues 1–14 and 17–30 (Fig. 1a), are designated as helix a and helix b, respectively (Fig. 2a, left panel). The whole-protein molecule has an overall appearance of a solenoid with a polar axis of 75 Å and a diameter of  50 Å (Fig. 2a). The internal layer along the superhelical axis is constituted by helices a, whereas helices b outline the external layer of the superhelix. Following the assignment of dPPR proteins in the electron density maps, electron densities indicative of RNA bases, which interdigitated with PPR helices, emerged in the cavity of the superhelix (Fig. 2b and Supplementary Fig. 4c). Because of the limited quality of the electron density data, only the 10 nucleotides coordinated by repeats could be modelled. Only one complex comprising one dPPR molecule with an ssRNA target was present in each asymmetric unit, similarly to the solution complex structure of ATPH-bound PPR1030. The overall dPPR protein structure consists of repetitions of helix pairs packing against each other to form a right-handed superhelical spiral shell that embraces its target ssRNA. The ssRNA molecule forms a right-handed parallel duplex structure with an ‘outer-layer' spiral protein enclosure. All 10 nucleotides in the target RNA elements strictly exhibit the modular pattern binding to corresponding dPPR repeats.

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.