Synthetic PPR proteins as tools for sequence-specific targeting of RNA.

In native systems, gene expression is regulated by RNA binding proteins. Such proteins have been the target of a great deal of recent research interest, due to the potential for harnessing these regulatory effects for the construction of new biotechnological tools. In particular, focus has been targeted on building synthetic RNA binding proteins for sequence-specific targeting of new RNA transcripts. Pentatricopeptide repeat (PPR) proteins make compelling candidates as synthetic RNA binding proteins to target and bind RNA transcripts of interest, due to their defined RNA binding "code", modular structure, and native capability to deliver catalytic C-terminal domains. In this review, we present a summary of up-to-date understanding of RNA site recognition by PPR proteins, progress towards the design of synthetic PPR proteins for RNA targeting in vitro and in vivo, highlight key areas for further research around these proteins and present an outlook for future applications for synthetic PPR proteins as biotechnological tools.

The Expansion and Diversification of Pentatricopeptide Repeat RNA-Editing Factors in Plants.

The RNA-binding pentatricopeptide repeat (PPR) family comprises hundreds to thousands of genes in most plants, but only a few dozen in algae, indicating massive gene expansions during land plant evolution. The nature and timing of these expansions has not been well defined due to the sparse sequence data available from early-diverging land plant lineages. In this study, we exploit the comprehensive OneKP datasets of over 1000 transcriptomes from diverse plants and algae toward establishing a clear picture of the evolution of this massive gene family, focusing on the proteins typically associated with RNA editing, which show the most spectacular variation in numbers and domain composition across the plant kingdom. We characterize over 2 250 000 PPR motifs in over 400 000 proteins. In lycophytes, polypod ferns, and hornworts, nearly 10% of expressed protein-coding genes encode putative PPR editing factors, whereas they are absent from algae and complex-thalloid liverworts. We show that rather than a single expansion, most land plant lineages with high numbers of editing factors have continued to generate novel sequence diversity. We identify sequence variations that imply functional differences between PPR proteins in seed plants versus non-seed plants and variations we propose to be linked to seed-plant-specific editing co-factors. Finally, using the sequence variations across the datasets, we develop a structural model of the catalytic DYW domain associated with C-to-U editing and identify a clade of unique DYW variants that are strong candidates as U-to-C RNA-editing factors, given their phylogenetic distribution and sequence characteristics.

Towards a plant model for enigmatic U-to-C RNA editing: the organelle genomes, transcriptomes, editomes and candidate RNA editing factors in the hornwort Anthoceros agrestis.

Hornworts are crucial to understand the phylogeny of early land plants. The emergence of 'reverse' U-to-C RNA editing accompanying the widespread C-to-U RNA editing in plant chloroplasts and mitochondria may be a molecular synapomorphy of a hornwort-tracheophyte clade. C-to-U RNA editing is well understood after identification of many editing factors in models like Arabidopsis thaliana and Physcomitrella patens, but there is no plant model yet to investigate U-to-C RNA editing. The hornwort Anthoceros agrestis is now emerging as such a model system. We report on the assembly and analyses of the A. agrestis chloroplast and mitochondrial genomes, their transcriptomes and editomes, and a large nuclear gene family encoding pentatricopeptide repeat (PPR) proteins likely acting as RNA editing factors. Both organelles in A. agrestis feature high amounts of RNA editing, with altogether > 1100 sites of C-to-U and 1300 sites of U-to-C editing. The nuclear genome reveals > 1400 genes for PPR proteins with variable carboxyterminal DYW domains. We observe significant variants of the 'classic' DYW domain, in the meantime confirmed as the cytidine deaminase for C-to-U editing, and discuss the first attractive candidates for reverse editing factors given their excellent matches to U-to-C editing targets according to the PPR-RNA binding code.

Fern genomes elucidate land plant evolution and cyanobacterial symbioses.

Ferns are the closest sister group to all seed plants, yet little is known about their genomes other than that they are generally colossal. Here, we report on the genomes of Azolla filiculoides and Salvinia cucullata (Salviniales) and present evidence for episodic whole-genome duplication in ferns-one at the base of 'core leptosporangiates' and one specific to Azolla. One fern-specific gene that we identified, recently shown to confer high insect resistance, seems to have been derived from bacteria through horizontal gene transfer. Azolla coexists in a unique symbiosis with N2-fixing cyanobacteria, and we demonstrate a clear pattern of cospeciation between the two partners. Furthermore, the Azolla genome lacks genes that are common to arbuscular mycorrhizal and root nodule symbioses, and we identify several putative transporter genes specific to Azolla-cyanobacterial symbiosis. These genomic resources will help in exploring the biotechnological potential of Azolla and address fundamental questions in the evolution of plant life.