Human Pumilio (hPUM) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1′ and R8′, however, remain unclear. Here, we report how the repeats contribute to overall RNA binding. We first prepared three mutants in which R1′ and/or R8′ were deleted and then analyzed RNA binding using gel shift assays. The assays showed that all deletion mutants bound to their target less than the original hPUM, but that R1′ contributed more than R8′, unlike Drosophila PUM. We next investigated which amino acid residues of R1′ or R8′ were responsible for RNA binding. With detailed analysis of the protein tertiary structure, we found a hydrophobic core in each of the repeats. We therefore mutated all hydrophobic amino residues in each core to alanine. The gel shift assays with the resulting mutants revealed that both hydrophobic cores contributed to the RNA binding: especially the hydrophobic core of R1′ had a significant influence. In the present study, we demonstrated that the flanking R1′ and R8′ repeats are indispensable for RNA binding of hPUM and suggest that hydrophobic R1′–R1 interactions may stabilize the whole hPUM structure.

No abstract

Previously, we reported that our designed artificial DNA‐cleaving enzyme which comprises our artificial DNA‐binding protein and a DNA‐cleaving domain inhibited DNA replication of human papilloma virus (HPV) in mammalian cells by cleaving its target HPV ori plasmid sequence‐specifically. Our artificial DNA‐binding proteins have much higher affinity and selectivity than other DNA‐binding proteins such as CRISPR‐dCas9. In addition, our artificial DNA‐cleaving enzymes cleave target DNA with multiple turnovers while CRISPR acts as a single‐turnover enzyme. Thereafter, we applied this methodology for inactivating DNA viruses to RNA viruses. We have recently developed artificial RNA‐binding proteins with higher affinity and selectivity than other RNA‐binding proteins such as CRISPR‐Cas13. In the present study, in order to inhibit RNA replication of influenza virus, we developed artificial RNA‐cleaving enzymes which comprise our artificial RNA‐binding protein and an RNA‐cleaving domain. After we confirmed that an artificial RNA‐binding protein specifically bound to its target RNA, we demonstrated that the artificial RNA‐cleaving enzymes site‐selectively and efficiently cleaved their target RNA sequence in an influenza viral genome in vitro. And we also demonstrated that the artificial RNA‐cleaving enzyme effectively inhibited RNA replication of influenza virus in mammalian cells due to cleavage of target RNA. We will present the detail in this conference.