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PMID:16357216

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Citation

Maniataki, E and Mourelatos, Z (2005) A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev. 19:2979-90

Abstract

RNA interference (RNAi) is mediated by RNA-induced silencing complexes (RISCs), which are guided by microRNAs (miRNAs) or short interfering RNAs (siRNAs) to cognate RNA targets. In humans, the catalytic engine of RISC is a ribonucleoprotein formed by the Argonaute2 (Ago2) protein and either miRNA (miRNP) or siRNA (siRNP). The Dicer nuclease produces mature miRNAs and siRNAs from pre-miRNAs and double-stranded RNA (dsRNA), respectively, and associates with Ago2. Here, we studied the assembly of human RISC by presenting pre-miRNA to immunopurified complexes that contain Ago2, Dicer, and TRBP. Mature miRNAs were produced in an ATP-independent manner and guided specific cleavage of cognate RNA targets in a pattern that is typical of RISC. This de novo formed RISC activity dissociated from Dicer. The asymmetry of the RISC loading process was fully recapitulated in this system, which, however, could not efficiently assemble RISC from siRNA duplexes. Our findings demonstrate that, in humans, a miRNA loading complex (miRLC) is formed by Ago2 and Dicer prior to their encounter with pre-miRNA. We suggest that the miRLC couples the processing of the pre-miRNA substrate to the unwinding of the product and that after loading of the mature miRNA to Ago2, the miRLC disassembles and the miRNP is released.

Links

PubMed PMC1315402 Online version:10.1101/gad.1384005

Keywords

Animals; Argonaute Proteins; Cell Line; Eukaryotic Initiation Factor-2; Humans; MicroRNAs/metabolism; Peptide Initiation Factors/metabolism; RNA Precursors/metabolism; RNA Processing, Post-Transcriptional/physiology; RNA, Small Interfering/metabolism; RNA-Induced Silencing Complex/metabolism; Ribonuclease III/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

MOUSE:AGO2

involved_in

GO:0031054: pre-miRNA processing

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:AGO2

part_of

GO:0070578: RISC-loading complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:AGO2

part_of

GO:0016442: RISC complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:AGO2

involved_in

GO:0035279: mRNA cleavage involved in gene silencing by miRNA

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:DICER

NOT|part_of

GO:0016442: RISC complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:DICER

part_of

GO:0070578: RISC-loading complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:DICER

involved_in

GO:0031054: pre-miRNA processing

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TRBP2

involved_in

GO:0031054: pre-miRNA processing

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TRBP2

part_of

GO:0070578: RISC-loading complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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