RESUMO
Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.
Assuntos
Elementos Alu/genética , RNA Helicases DEAD-box/deficiência , Degeneração Macular/genética , Degeneração Macular/patologia , RNA/genética , RNA/metabolismo , Ribonuclease III/deficiência , Animais , Morte Celular , Sobrevivência Celular , Células Cultivadas , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Fenótipo , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
Previously we established Zygote Electroporation of Nucleases (ZEN) technology as an efficient and high-throughput way to generate genetically modified mouse models. However, there were significant variations of the targeting efficiency among different genomic loci using our previously published protocol. In this study, we improved the ZEN technology by delivering Cas9 protein into mouse zygotes through a series of electroporation. Using this approach, we were able to introduce precise nucleotide substitutions, large segment deletion and short segment insertion into targeted loci with high efficiency.
Assuntos
Desoxirribonuclease I/metabolismo , Eletroporação , Zigoto/metabolismo , Animais , Camundongos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
RNA editing is a process that modifies RNA nucleotides and changes the efficiency and fidelity of the central dogma. Enzymes that catalyze RNA editing are required for life, and defects in RNA editing are associated with many diseases. Recent advances in sequencing have enabled the genome-wide identification of RNA editing sites in mammalian transcriptomes. Here, we demonstrate that canonical RNA editing (A-to-I and C-to-U) occurs in liver, white adipose, and bone tissues of the laboratory mouse, and we show that apparent non-canonical editing (all other possible base substitutions) is an artifact of current high-throughput sequencing technology. Further, we report that high-confidence canonical RNA editing sites can cause non-synonymous amino acid changes and are significantly enriched in 3' UTRs, specifically at microRNA target sites, suggesting both regulatory and functional consequences for RNA editing.