Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA-binding proteins that are commonly found in plants. Organelle transcript processing and stability are mediated by PPR proteins in a gene-specific manner through recognition by tandem arrays of degenerate 35-amino-acid repeating units, the PPR motifs. However, the sequence-specific RNA recognition mechanism of the PPR protein remains largely unknown. Here, we show the principle underlying RNA recognition for PPR proteins involved in RNA editing. The distance between the PPR-RNA alignment and the editable C was shown to be conserved. Amino acid variation at 3 particular positions within the motif determined recognition of a specific RNA in a programmable manner, with a 1-motif to 1-nucleotide correspondence, with no gap sequence. Data from the decoded nucleotide frequencies for these 3 amino acids were used to assign accurate interacting sites to several PPR proteins for RNA editing and to predict the target site for an uncharacterized PPR protein.

Summary: During early chloroplast differentiation, the regulation of the plastid genetic system including transcription and translation differs greatly from that in the mature chloroplast, suggesting the existence of a stage‐dependent mechanism that regulates the chloroplast genetic system during this period. The virescent‐1 (v₁) mutant of rice (Oryza sativa) is temperature‐conditional and develops chlorotic leaves under low‐temperature conditions. We reported previously that leaf chlorosis in the v₁ mutant is caused by blockage of the activation of the chloroplast genetic system during early leaf development. Here we identify the V₁ gene, which encodes a chloroplast‐localized protein NUS1. Accumulation of NUS1 specifically occurred in the pre‐emerged immature leaves, and is enhanced by low‐temperature treatment. The C‐terminus of NUS1 shows structural similarity to the bacterial antitermination factor NusB, which is known to play roles in the regulation of ribosomal RNA transcription. The RNA‐immunoprecipitation and gel mobility shift assays indicated that NUS1 binds to several regions of chloroplast RNA including the upstream leader region of the 16S rRNA precursor. In the leaves of the NUS1‐deficient mutant, accumulation of chloroplast rRNA during early leaf development was impaired and chloroplast translation/transcription capacity was severely suppressed under low temperature. Our results suggest that NUS1 is involved in the regulation of chloroplast RNA metabolism and promotes the establishment of the plastid genetic system during early chloroplast development under cold stress conditions.

The pentatricopeptide repeat (PPR) protein family is highly expanded in terrestrial plants. Arabidopsis contains 450 PPR genes, which represents 2% of the total protein-coding genes. PPR proteins are eukaryote-specific RNA-binding proteins implicated in multiple aspects of RNA metabolism of organellar genes. Most PPR proteins affect a single or small subset of gene(s), acting in a gene-specific manner. Studies over the last 10 years have revealed the significance of this protein family in coordinated gene expression in different compartments: the nucleus, chloroplast and mitochondrion. Here, we summarize recent studies addressing the mechanistic aspect of PPR proteins.

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

Regulations of proliferation and quiescence in response to nutritional cues are important for medicine and basic biology. The fission yeast Schizosaccharomyces pombe serves as a model, owing to the shift of proliferating cells to the metabolically active quiescence (designate G0 phase hereafter) by responding to low nitrogen source. S. pombe G0 phase cells keep alive for months without growth and division. Nitrogen replenishment reinstates vegetative proliferation phase (designate VEG). Some 40 genes required for G0 maintenance were identified, but many more remain to be identified. We here show, using mutants, that the proteasome is required for maintaining G0 quiescence. Functional outcomes of proteasome in G0 and VEG phases appear to be distinct. Upon proteasome dysfunction, a number of antioxidant proteins and compounds responsive to ROS (reactive oxygen species) are produced. In addition, autophagy-mediated destruction of mitochondria occurs, which suppresses the loss of viability by eliminating ROS-generating mitochondria. These defensive responses are found in G0 but not in VEG, suggesting that the main function of proteasome in G0 phase homeostasis is to minimize ROS. Proteasome and autophagy are thus collaborative to support the lifespan of S. pombe G0 phase.

C-to-U RNA editing has been widely observed in organellar RNAs in terrestrial plants. Recent research has revealed the significance of a large, plant-specific family of pentatricopeptide repeat (PPR) proteins for RNA editing and other RNA processing events in plant mitochondria and chloroplasts. PPR protein is a sequence-specific RNA-binding protein that identifies specific C residues for editing. Discovery of the RNA recognition code for PPR motifs, including verification and prediction of the individual RNA editing site and its corresponding PPR protein, expanded our understanding of the molecular function of PPR proteins in plant organellar RNA editing. Using this knowledge and the co-expression database, we have identified two new PPR proteins that mediate chloroplast RNA editing. Further, computational target assignment using the PPR RNA recognition codes suggests a distinct, unknown mode-of-action, by which PPR proteins serve a function beyond site recognition in RNA editing.