The expressions of chloroplast and mitochondria genes are tightly controlled by numerous nuclear-encoded proteins, mainly at the post-transcriptional level. Recent analyses have identified a large, plant-specific family of pentatricopeptide repeat (PPR) motif-containing proteins that are exclusively involved in RNA metabolism of organelle genes via sequence-specific RNA binding. A tandem array of PPR motifs within the protein is believed to facilitate the RNA interaction, although little is known of the mechanism. Here, we describe the RNA interacting framework of a PPR protein, Arabidopsis HCF152. First, we demonstrated that a Pfam model could be relevant to the PPR motif function. A series of proteins with two PPR motifs showed significant differences in their RNA binding affinities, indicating functional differences among PPR motifs. Mutagenesis and informatics analysis putatively identified five amino acids organizing its RNA binding surface [the 1st, 4th, 8th, 12th and ‘ii’(-2nd) amino acids] and their complex connections. SELEX (Systematic evolution of ligands by exponential enrichment) and nucleobase preference assays determined the nucleobases with high affinity for HCF152 and suggested several characteristic amino acids that may be involved in determining specificity and/or affinity of the PPR/RNA interaction.

During liver development, biliary epithelial cells differentiated from bipotential hepatic progenitor cells (hepatoblasts) form a cell layer, called the ductal plate surrounding portal veins (PVs), and develop into intrahepatic bile ducts (IBDs) following developmental programs. Because IBDs make duct structures in the liver, it is necessary to perform sequential and three-dimensional (3D) analyses from the early stages of liver development to address the process of morphogenesis in detail. However, to date, the development of IBDs has mainly been investigated using tissue sections in two-dimensional planes, and examinations of the 3D morphogenesis and quantitative analyses based on morphometrics have not been performed. Therefore, in this study, we simulated the solid structures of IBDs from mouse embryos to adults in silico, analyzed the subjects for the length and number of developing duct structures, number of predicted connections, and discrete distance from the PV, and examined the developmental process of the IBD in detail in a quantitative manner. Conclusions: Through quantitative analyses with spatiotemporal observations using a 3D structural reconstruction model and morphometrics, we succeeded in constructing a 3D dynamic model of bile duct formation. Because the 3D reconstruction technique used in this study is available for analyzing solid structures in tissues that are difficult to approach, it shows promise for wide use in the fields of biology and medicine. (HEPATOLOGY 2015;61:1003-1011

Programmable RNA editing offers significant therapeutic potential for a wide range of genetic diseases. Currently, several deaminase enzymes, including ADAR and APOBEC, can perform programmable adenosine-to-inosine or cytidine-to-uridine RNA correction. However, enzymes to perform guanosine-to-adenosine and uridine-to-cytidine (U-to-C) editing are still lacking to complete the set of transition reactions. It is believed that the DYW:KP proteins, specific to seedless plants, catalyze the U-to-C reactions in mitochondria and chloroplasts. In this study, we designed seven DYW:KP domains based on consensus sequences and fused them to a designer RNA-binding pentatricopeptide repeat (PPR) domain. We show that three of these PPR-DYW:KP proteins edit targeted uridine to cytidine in bacteria and human cells. In addition, we show that these proteins have a 5′ but not apparent 3′ preference for neighboring nucleotides. Our results establish the DYW:KP aminase domain as a potential candidate for the development of a U-to-C editing tool in human cells.