RESUMO
Heterochromatic regions of the genome are epigenetically regulated to maintain a heritable '"silent state"'. In fission yeast and other organisms, epigenetic silencing is guided by nascent transcripts, which are targeted by the RNA interference pathway. The key effector complex of the RNA interference pathway consists of small interfering RNA molecules (siRNAs) associated with Argonaute, assembled into the RNA-induced transcriptional silencing (RITS) complex. This review focuses on our current understanding of how RITS promotes heterochromatin formation, and in particular on the role of Argonaute-containing complexes in many other functions such as quelling, release of RNA polymerases, cellular quiescence and genome defense.
Assuntos
Proteínas Argonautas/genética , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/química , RNA/química , Proteínas Argonautas/química , Ciclo Celular/genética , Genoma Fúngico/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , RNA/genética , RNA/ultraestrutura , RNA Interferente Pequeno/genética , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/ultraestrutura , Schizosaccharomyces/genéticaRESUMO
Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DExH/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.
Assuntos
Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas Argonautas , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/ultraestrutura , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Microscopia Eletrônica , Modelos Biológicos , Ligação Proteica/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Complexo de Inativação Induzido por RNA/ultraestrutura , Ribonuclease III/genética , Ribonuclease III/ultraestruturaRESUMO
RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5' end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5' end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.
Assuntos
Modelos Químicos , Modelos Genéticos , Interferência de RNA/fisiologia , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/genética , Animais , Células Cultivadas , Simulação por Computador , Drosophila melanogaster , Modelos Moleculares , Complexo de Inativação Induzido por RNA/ultraestruturaRESUMO
BACKGROUND: The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. RESULTS: The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes - an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. CONCLUSION: Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference.
