RESUMO
Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.
Assuntos
Regulação da Expressão Gênica , Técnicas Genéticas , MicroRNAs/genética , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Testiculares/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Testes Genéticos/métodos , Humanos , Masculino , Camundongos , Camundongos Nus , MicroRNAs/química , Dados de Sequência Molecular , Transplante de Neoplasias , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas ras/metabolismoRESUMO
White Spot Syndrome Virus, the type species of the virus family Nimaviridae, is a large dsDNA virus infecting shrimp and other crustaceans. Genomic analysis of three completely sequenced WSSV isolates identified two major polymorphic loci, "variable region ORF14/15" and "variable region ORF23/24". Here, we characterize a WSSV isolate originating from shrimp collected in Thailand in 1996 (TH-96-II). This isolate contains the largest WSSV genome ( approximately 312 kb) identified so far, mainly because of its sequences in both major polymorphic loci. Analysis of "variable region ORF14/15" suggests that TH-96-II may be ancestral to the WSSV isolates described to date. A comparison for virulence was made between TH-96-II and WSSV-TH, a well characterized isolate containing the smallest genome ( approximately 293 kb) identified at present. After injection of the isolates into Penaeus monodon the mortality rates showed that the median lethal time (LT50) of TH-96-II was approximately 14 days, compared to 3.5 days for WSSV-TH. When both isolates were mixed in equal amounts and serially passaged in shrimp, WSSV-TH outcompeted TH-96-II within four passages. These data suggest a higher virulence of WSSV-TH compared to TH-96-II. The molecular basis for the difference in virulence remains unclear, but a replication advantage of the 19 kb smaller WSSV-TH genome could play a role.
Assuntos
Penaeidae/virologia , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Animais , DNA Viral/química , DNA Viral/genética , Modelos Animais de Doenças , Dados de Sequência Molecular , Fases de Leitura Aberta , Polimorfismo Genético , Análise de Sequência de DNA , Tailândia , Virulência , Vírus da Síndrome da Mancha Branca 1/isolamento & purificaçãoRESUMO
MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the miRNA-targeted mRNAs. Thus, our data unravel a novel role of Dnd1 in protecting certain mRNAs from miRNA-mediated repression.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Células Germinativas/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Proteínas de Peixe-Zebra/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Conexina 43/genética , Conexina 43/metabolismo , Sequência Conservada , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Ribonucleico , Transfecção , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumor-suppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.