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1.
Cell ; 149(4): 847-59, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22541070

RESUMO

Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.


Assuntos
Elementos Alu , RNA Helicases DEAD-box/metabolismo , Atrofia Geográfica/imunologia , Atrofia Geográfica/patologia , Inflamassomos/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Ribonuclease III/metabolismo , Animais , Proteínas de Transporte/metabolismo , Atrofia Geográfica/metabolismo , Humanos , Inflamassomos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Epitélio Pigmentado da Retina/patologia , Receptores Toll-Like/metabolismo
2.
EMBO J ; 32(6): 781-90, 2013 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-23395899

RESUMO

RNA polymerase II (Pol II) is a well-characterized DNA-dependent RNA polymerase, which has also been reported to have RNA-dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non-coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3'-end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α-amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3'-end.


Assuntos
RNA Polimerase II/fisiologia , Estabilidade de RNA , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Elongação da Transcrição Genética , Animais , Sequência de Bases , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Células NIH 3T3 , Conformação de Ácido Nucleico , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/fisiologia , Transcrição Gênica/genética
3.
Nature ; 471(7338): 325-30, 2011 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-21297615

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/metabolismo
4.
Proc Natl Acad Sci U S A ; 109(34): 13781-6, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22869729

RESUMO

Deficient expression of the RNase III DICER1, which leads to the accumulation of cytotoxic Alu RNA, has been implicated in degeneration of the retinal pigmented epithelium (RPE) in geographic atrophy (GA), a late stage of age-related macular degeneration that causes blindness in millions of people worldwide. Here we show increased extracellular-signal-regulated kinase (ERK) 1/2 phosphorylation in the RPE of human eyes with GA and that RPE degeneration in mouse eyes and in human cell culture induced by DICER1 depletion or Alu RNA exposure is mediated via ERK1/2 signaling. Alu RNA overexpression or DICER1 knockdown increases ERK1/2 phosphorylation in the RPE in mice and in human cell culture. Alu RNA-induced RPE degeneration in mice is rescued by intravitreous administration of PD98059, an inhibitor of the ERK1/2-activating kinase MEK1, but not by inhibitors of other MAP kinases such as p38 or JNK. These findings reveal a previously unrecognized function of ERK1/2 in the pathogenesis of GA and provide a mechanistic basis for evaluation of ERK1/2 inhibition in treatment of this disease.


Assuntos
Regulação Enzimológica da Expressão Gênica , Degeneração Macular/enzimologia , Degeneração Macular/terapia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , RNA Helicases DEAD-box/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Camundongos , Fosforilação , Epitélio Pigmentado da Retina/metabolismo , Ribonuclease III/metabolismo , Transdução de Sinais
5.
Noncoding RNA ; 1: 4-16, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26405685

RESUMO

B2 RNA is a mouse non-coding RNA that binds directly to RNA polymerase II (Pol II) and represses transcription by disrupting critical interactions between the polymerase and promoter DNA. How the structural regions within B2 RNA work together to mediate transcriptional repression is not well understood. To address this question, we systematically deleted structural regions from B2 RNA and determined the effects on transcriptional repression using a highly purified Pol II in vitro transcription system. Deletions that compromised the ability of B2 RNA to function as a transcriptional repressor were also tested for their ability to bind directly to Pol II, which enabled us to distinguish regions uniquely important for repression from those important for binding. We found that transcriptional repression requires a pattern of RNA structural motifs consisting of an extended single-stranded region bordered by two stem-loops. Hence, there is modularity in the function of the stem-loops in B2 RNA-when one stem-loop is deleted, another can take its place to enable transcriptional repression.

6.
J Mol Biol ; 425(19): 3625-38, 2013 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-23416138

RESUMO

The B2 family of short interspersed elements is transcribed into non-coding RNA by RNA polymerase III. The ~180-nt B2 RNA has been shown to potently repress mRNA transcription by binding tightly to RNA polymerase II (Pol II) and assembling with it into complexes on promoter DNA, where it keeps the polymerase from properly engaging the promoter DNA. Mammalian Pol II is an ~500-kDa complex that contains 12 different protein subunits, providing many possible surfaces for interaction with B2 RNA. We found that the carboxy-terminal domain of the largest Pol II subunit was not required for B2 RNA to bind Pol II and repress transcription in vitro. To identify the surface on Pol II to which the minimal functional region of B2 RNA binds, we coupled multi-step affinity purification, reversible formaldehyde cross-linking, peptide sequencing by mass spectrometry, and analysis of peptide enrichment. The Pol II peptides most highly recovered after cross-linking to B2 RNA mapped to the DNA binding cleft and active-site region of Pol II. These studies determine the location of a defined nucleic acid binding site on a large, native, multi-subunit complex and provide insight into the mechanism of transcriptional repression by B2 RNA.


Assuntos
Domínio Catalítico , Clivagem do DNA , Ligação Proteica/genética , RNA Polimerase II/genética , RNA não Traduzido/genética , RNA/genética , Mapeamento Cromossômico , Repressão Epigenética , Humanos , Regiões Promotoras Genéticas , Conformação Proteica , RNA/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Transcrição Gênica
7.
Curr Opin Genet Dev ; 20(2): 149-55, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20176473

RESUMO

Mammalian short interspersed elements (SINEs) are abundant retrotransposons that have long been considered junk DNA; however, RNAs transcribed from mouse B2 and human Alu SINEs have recently been found to control mRNA production at multiple levels. Upon cell stress B2 and Alu RNAs bind RNA polymerase II (Pol II) and repress transcription of some protein-encoding genes. Bi-directional transcription of a B2 SINE establishes a boundary that places the growth hormone locus in a permissive chromatin state during mouse development. Alu RNAs embedded in Pol II transcripts can promote evolution and proteome diversity through exonization via alternative splicing. Given the diverse means by which SINE encoded RNAs impact production of mRNAs, this genomic junk is proving to contain hidden gems.


Assuntos
Regulação da Expressão Gênica , RNA Mensageiro/genética , RNA/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Elementos Alu/genética , Animais , Humanos , Modelos Genéticos , RNA Mensageiro/metabolismo , Transcrição Gênica
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