Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants, with more than 400 members in most species. Over the past decade, much has been learned about the molecular functions of these proteins, where they act in the cell, and what physiological roles they play during plant growth and development. A typical PPR protein is targeted to mitochondria or chloroplasts, binds one or several organellar transcripts, and influences their expression by altering RNA sequence, turnover, processing, or translation. Their combined action has profound effects on organelle biogenesis and function and, consequently, on photosynthesis, respiration, plant development, and environmental responses. Recent breakthroughs in understanding how PPR proteins recognize RNA sequences through modular base-specific contacts will help match proteins to potential binding sites and provide a pathway toward designing synthetic RNA-binding proteins aimed at desired targets.

Pentatricopeptide repeat proteins in plants

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

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Programmable RNA recognition and cleavage by CRISPR/Cas9

The highly divergent ribosomes of human mitochondria (mitoribosomes) synthesize 13 essential proteins of oxidative phosphorylation complexes. We have determined the structure of the intact mitoribosome to 3.5 angstrom resolution by means of single-particle electron cryogenic microscopy. It reveals 80 extensively interconnected proteins, 36 of which are specific to mitochondria, and three ribosomal RNA molecules. The head domain of the small subunit, particularly the messenger (mRNA) channel, is highly remodeled. Many intersubunit bridges are specific to the mitoribosome, which adopts conformations involving ratcheting or rolling of the small subunit that are distinct from those seen in bacteria or eukaryotes. An intrinsic guanosine triphosphatase mediates a contact between the head and central protuberance. The structure provides a reference for analysis of mutations that cause severe pathologies and for future drug design.

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The structure of the human mitochondrial ribosome

The pentatricopeptide repeat (PPR) proteins form one of the largest protein families in land plants. They are characterised by tandem 30-40 amino acid motifs that form an extended binding surface capable of sequence-specific recognition of RNA strands. Almost all of them are post-translationally targeted to plastids and mitochondria, where they play important roles in post-transcriptional processes including splicing, RNA editing and the initiation of translation. A code describing how PPR proteins recognise their RNA targets promises to accelerate research on these proteins, but making use of this code requires accurate definition and annotation of all of the various nucleotide-binding motifs in each protein. We have used a structural modelling approach to define 10 different variants of the PPR motif found in plant proteins, in addition to the putative deaminase motif that is found at the C-terminus of many RNA-editing factors. We show that the super-helical RNA-binding surface of RNA-editing factors is potentially longer than previously recognised. We used the redefined motifs to develop accurate and consistent annotations of PPR sequences from 109 genomes. We report a high error rate in PPR gene models in many public plant proteomes, due to gene fusions and insertions of spurious introns. These consistently annotated datasets across a wide range of species are valuable resources for future comparative genomics studies, and an essential pre-requisite for accurate large-scale computational predictions of PPR targets. We have created a web portal (http://www.plantppr.com) that provides open access to these resources for the community.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/tpj.13121

Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants.

Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate production in eukaryotic cells. Throughout evolution, mitoribosomes have become functionally specialized for synthesizing mitochondrial membrane proteins, and this has been accompanied by large changes to their structure and composition. We review recent high-resolution structural data that have provided unprecedented insight into the structure and function of mitoribosomes in mammals and fungi.

Structure and Function of the Mitochondrial Ribosome

Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants(1-5), recognize single-stranded RNA (ssRNA) in a modular fashion(6-8). The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPI-ATPH and PSAJ-RPL33 intergenic regions, referred to as ATPH and PSAJ, respectively(9,10). By protecting the target RNA elements from 5' or 3' exonucleases, PPR10 defines the corresponding 5' and 3' messenger RNA termini(9-11). Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45 angstrom, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.

Structural basis for the modular recognition of single-stranded RNA by PPR proteins

Specific RNA Recognition by Designer Pentatricopeptide Repeat Protein

NleC is one of the virulence factors that is injected into infected host cells by enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) via a needle-like protein complex called the type III secretion system (T3SS). The cytosolic NleC specifically cleaves the p65 subunit of NF-­κB in the p65–p50 heterodimeric complex just after the Cys38 site in its N-terminal domain. The degradation of the remainder of the p65 C-terminal domain by the proteasome disrupts the NF-κB signalling pathway, thus dampening the host inflammatory response. Here, the crystal structure of NleC is reported at 1.55 A resolution. In conjunction with biochemical analyses, the structure reveals that NleC is a member of the zincin zinc protease family and that the configuration of the NleC active site resembles that of the metzincin clan of metallopeptidases but without the canonical Met turn of astacin. The extended zinc-binding motif of NleC (HEXXHXXTXXXD) includes three metal ligands. The fifth zinc ligand, a conserved tyrosine (a bound water molecule is the fourth ligand), lies 45 residues downstream of the zincin motif. Furthermore, the electrostatic potential complementarity between NleC and p65 also contributes to the cleavage activity of the protease. These results not only provide important insights into the mechanism of how NleC recognizes its substrates, but also shed light on the design of new antibiotics for the food-borne diseases arising from EPEC and EHEC.

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Quanxiu Li

Papers

Structural basis for the modular recognition of single-stranded RNA by PPR proteins

Specific RNA Recognition by Designer Pentatricopeptide Repeat Protein

Structure and mechanism of a type III secretion protease, NleC

Examination of the Dimerization States of the Single-stranded RNA Recognition Protein Pentatricopeptide Repeat 10 (PPR10)