RESUMO
Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.
Assuntos
Capsídeo , Vetores Genéticos , Animais , Camundongos , Capsídeo/metabolismo , Vetores Genéticos/genética , Dependovirus/genética , Proteínas do Capsídeo/genética , Fígado/metabolismoRESUMO
4E-BP (eIF4E-BP) represses translation initiation by binding to the 5' cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity, and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used HyperTRIBE (targets of RNA binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets more substantially compared to nontargets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.
Assuntos
Drosophila , Fator de Iniciação 4E em Eucariotos , Animais , Drosophila/genética , Drosophila/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Drosophila NonA and its mammalian ortholog NONO are members of the Drosophila behavior and human splicing (DBHS) family. NONO also has a strong circadian connection: it associates with the circadian repressor protein PERIOD (PER) and contributes to circadian timekeeping. Here, we investigate NonA, which is required for proper levels of evening locomotor activity as well as a normal free-running period in Drosophila. NonA is associated with the positive transcription factor CLOCK/CYCLE (CLK/CYC), interacts directly with complexin (cpx) pre-mRNA, and upregulates gene expression, including the gene cpx. Downregulation of cpx expression in circadian neurons phenocopies NonA downregulation, whereas cpx overexpression rescues the nonA RNAi phenotypes, indicating that cpx is an important NonA target gene. As the cpx protein contributes to proper neurotransmitter and neuropeptide release in response to calcium, these results and others indicate that this control is important for the normal circadian regulation of locomotor activity.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Proteínas de Drosophila/biossíntese , Locomoção/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas Nucleares/biossíntese , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/genética , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genéticaRESUMO
RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of an RBP are imperfect and not amenable to examining small numbers of cells. To address these issues, we developed TRIBE (targets of RNA-binding proteins identified by editing), a technique that couples an RBP to the catalytic domain of the Drosophila RNA-editing enzyme ADAR and expresses the fusion protein in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. We have used TRIBE to identify the targets of three RBPs (Hrp48, dFMR1, and NonA). TRIBE compares favorably to other methods, including CLIP, and we have identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.
