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

Cuicui Shen,Yan Yang,Yexing Liu,Qiang Wang,Qunxia Zhang,Ping Yin,Tingting Zou,Shilong Fan,Xiang Wang,Zeyuan Guan,Zhao Yang,Delin Zhang

doi:10.1038/ncomms11285

Cuicui Shen, Yan Yang + Show 10 more

Open Access

https://doi.org/10.1038/ncomms11285

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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.

Highlights

As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes
Each of the four designer PPR (dPPR)-U8N2 proteins bound to its respective target single-stranded RNA (ssRNA) with a dissociation constant of B20 –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)

Summary

Introduction

As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes. Similar results and crystal structures of artificially engineered PPR proteins free of target RNA have been reported by other groups[29,35] These results suggest that PPR scaffolds are amenable to the engineering of designer RNAbinding domains, as promising tools to achieve specific RNA recognition in vitro. Despite these advances, the lack of a structure of a dPPR–RNA complex has hindered the elucidation of RNA recognition by dPPR proteins and, more importantly, has restricted the development of more efficient RNA manipulation tools

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Results

Conclusion