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1.
Cell ; 165(2): 488-96, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26997482

RESUMO

RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.


Assuntos
RNA/análise , Sistemas CRISPR-Cas , Grânulos Citoplasmáticos/química , Endonucleases/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Proteínas de Fluorescência Verde/análise , Humanos , RNA Guia de Cinetoplastídeos/análise , RNA Mensageiro/análise
2.
PLoS Genet ; 9(2): e1003279, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23408911

RESUMO

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.


Assuntos
Autofagia/genética , Proteínas de Ciclo Celular , Ribossomos , Serina-Treonina Quinases TOR , Proteína Supressora de Tumor p53 , Proteínas de Peixe-Zebra , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular , Genes Letais/genética , Mutação , Biossíntese de Proteínas/genética , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
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