Assuntos
Biotecnologia/métodos , Sistemas CRISPR-Cas/genética , Microscopia Crioeletrônica/métodos , Desoxirribonucleases/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Interferência de RNA , Animais , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Biotecnologia/tendências , HumanosRESUMO
Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute 2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex, which also requires Argonaute 2 for assembly into RISC. We have compared R2 to the RISC-loading complex and show that the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. We have examined the requirements for RISC formation and show that the siRNA 5'-termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2'-position of the 5'-terminal nucleotide also affects RISC assembly, because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference pathway.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Interferência de RNA , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Animais , Proteínas Argonautas , Dimerização , Proteínas de Drosophila/química , Modelos Biológicos , Complexos Multiproteicos , Conformação de Ácido Nucleico , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease IIIRESUMO
An array of gene silencing pathways share a common early step: Dicer cleaves double-stranded RNA (dsRNA) into approximately 20-25 nucleotide fragments that direct the silencing machinery to specific targets. A recent report in Cell reveals how Dicer's two RNase III domains collaborate during dsRNA processing and sets the stage for a deeper understanding of Dicer's roles in later phases of silencing complex assembly.
Assuntos
RNA Interferente Pequeno , Animais , Inativação Gênica , Humanos , Biossíntese de Proteínas/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
2'-aminonucleosides are commonly used as sites of post-synthetic chemical modification within nucleic acids. As part of a larger cross-linking strategy, we appended alkyl groups onto the N2' position of 2'-amino-modified RNAs via 2'-ureido and 2'-amido linkages. We have characterized the thermodynamics of 2'-amino, 2'-alkylamido and 2'-alkylureido-modified RNA duplexes and show that 2'-ureido-modified RNAs are significantly more stable than analogous 2'-amido-modified RNAs. Using NMR spectroscopy and NMR-based molecular modeling of 2'-modified RNA duplexes, we examined the effects that 2'-nitrogen modifications have on RNA helices. Our data suggest that the 2'-ureido group forms a specific intra-nucleoside interaction that cannot occur within 2'-amido-modified helices. These results indicate that 2'-ureido modifications are superior to analogous 2'-amido ones for applications that require stable base pairing.
Assuntos
Nitrogênio/química , RNA de Cadeia Dupla/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estabilidade de RNA , Compostos de Sulfidrila/química , TermodinâmicaRESUMO
The RNase III enzyme Dicer processes RNA into siRNAs and miRNAs, which direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutations in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of miRNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). We find that Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 but not Dicer-2 is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Anormalidades do Olho/genética , MicroRNAs/genética , RNA Helicases/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismo , Animais , Proteínas de Drosophila/genética , Feminino , Masculino , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mutação/genética , Biossíntese de Proteínas/genética , Estrutura Terciária de Proteína/fisiologia , RNA Helicases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease III/genética , Homologia de Sequência de AminoácidosRESUMO
We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.
