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Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208


Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.

Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
J Cell Biol ; 218(2): 559-579, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30538140


Cellular differentiation involves remodeling cellular architecture to transform one cell type to another. By investigating mitochondrial dynamics during meiotic differentiation in budding yeast, we sought to understand how organelle morphogenesis is developmentally controlled in a system where regulators of differentiation and organelle architecture are known, but the interface between them remains unexplored. We analyzed the regulation of mitochondrial detachment from the cell cortex, a known meiotic alteration to mitochondrial morphology. We found that mitochondrial detachment is enabled by the programmed destruction of the mitochondria-endoplasmic reticulum-cortex anchor (MECA), an organelle tether that bridges mitochondria and the plasma membrane. MECA regulation is governed by a meiotic transcription factor, Ndt80, which promotes the activation of a conserved kinase, Ime2. We further present evidence for Ime2-dependent phosphorylation and degradation of MECA in a temporally controlled manner. Our study defines a key mechanism that coordinates mitochondrial morphogenesis with the landmark events of meiosis and demonstrates that cells can developmentally regulate tethering to induce organelle remodeling.

BMC Cell Biol ; 18(1): 16, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28335714


BACKGROUND: In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure. RESULTS: The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits. CONCLUSIONS: We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Morfogênese , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Testículo/embriologia , Animais , Drosophila melanogaster/enzimologia , Evolução Molecular , Voo Animal/fisiologia , Técnicas de Silenciamento de Genes , Genes de Insetos , Proteínas de Fluorescência Verde/metabolismo , Masculino , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Modelos Biológicos , Músculo Esquelético/metabolismo , Mutação/genética , Especificidade de Órgãos , Fenótipo , Filogenia , Multimerização Proteica , Subunidades Proteicas/genética , Interferência de RNA , Espermátides/metabolismo , Espermatogênese