RESUMO
MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that regulate gene expression on the level of translation and/or mRNA stability. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences in the 3' untranslated region (UTR) of specific target mRNAs. Computer algorithms based on factors such as free binding energy or sequence conservation have been used to predict miRNA target mRNAs. Based on such predictions, up to one third of all mammalian mRNAs seem to be under miRNA regulation. However, due to the low degree of complementarity between the miRNA and its target, such computer programs are often imprecise and therefore not very reliable. Here we report the first biochemical identification approach of miRNA targets from human cells. Using highly specific monoclonal antibodies against members of the Ago protein family, we co-immunoprecipitate Ago-bound mRNAs and identify them by cloning. Interestingly, most of the identified targets are also predicted by different computer programs. Moreover, we randomly analyzed six different target candidates and were able to experimentally validate five as miRNA targets. Our data clearly indicate that miRNA targets can be experimentally identified from Ago complexes and therefore provide a new tool to directly analyze miRNA function.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/genética , Animais , Proteínas Argonautas , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/isolamento & purificação , Fatores de Iniciação em Eucariotos/isolamento & purificação , Humanos , MicroRNAs/genética , MicroRNAs/isolamento & purificação , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , RatosRESUMO
Small regulatory RNAs such as short interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi interacting RNAs (piRNAs) have been discovered in the past, and it is becoming more and more apparent that these small molecules have key regulatory functions. Small RNAs are found in all higher eukaryotes and play important roles in cellular processes as diverse as development, stress response, or transposon silencing. Soon after the discovery of small regulatory RNAs, members of the Argonaute protein family were identified as their major cellular protein interactors. This review focuses on the various cellular functions of mammalian Argonaute proteins in conjunction with the different small RNA species that are known today.
Assuntos
Inativação Gênica , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Animais , Drosophila , Humanos , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Biossíntese de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espermatogênese/genética , Espermatogênese/fisiologia , Frações Subcelulares/metabolismo , Transcrição GênicaRESUMO
RNA silencing processes are guided by small RNAs known as siRNAs and microRNAs (miRNAs) . They reside in ribonucleoprotein complexes, which guide the cleavage of complementary mRNAs or affect stability and translation of partial complementary mRNAs . Argonaute (Ago) proteins are at the heart of silencing effector complexes and bind the single-stranded siRNA and miRNA . Our biochemical analysis revealed that Ago2 is present in a pre-miRNA processing complex that is able to transfer the miRNA into a target-mRNA cleaving complex. To gain insight into the function and composition of RNA silencing complexes, we purified Ago1- and Ago2-containing complexes from human cells. Several known Ago1- and/or Ago2-associated proteins including Dicer were identified, but also two novel factors, the putative RNA helicase MOV10, and the RNA recognition motif (RRM)-containing protein TNRC6B/KIAA1093. The new proteins localize, similar to Ago proteins, to mRNA-degrading cytoplasmic P bodies, and they are functionally required to mediate miRNA-guided mRNA cleavage.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , RNA Helicases/genética , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas Argonautas , Northern Blotting , Western Blotting , Linhagem Celular , Fator de Iniciação 2 em Eucariotos , Fatores de Iniciação em Eucariotos/metabolismo , Imunofluorescência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espectrometria de Massas , MicroRNAs/metabolismo , Oligonucleotídeos , Fatores de Iniciação de Peptídeos/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , Complexo de Inativação Induzido por RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease III/metabolismoRESUMO
Cellular signal transduction proceeds through a complex network of molecular interactions and enzymatic activities. The timing of these molecular events is critical for the propagation of a signal and the generation of a specific cellular response. To define the timing of signalling events, we introduce the combination of high-resolution confocal microscopy with the application of small-molecule inhibitors at various stages of signal transduction in T cells. Inhibitors of Src-family tyrosine kinases and actin dynamics were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent contacts. Anti-CD3epsilon-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 were detected by high-resolution confocal microscopy. The analysis revealed that at 5 min after receptor engagement, Lck activity was required for maintenance of contacts. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts.
