RISC is a 5' phosphomonoester-producing RNA endonuclease

Abstract

Gene silencing in the process of RNA interference is mediated by a ribonucleoprotein complex referred to as RNA-induced silencing complex (RISC). Here we describe the molecular mechanism of target RNA cleavage using affinity-purified minimal RISC from human cells. Cleavage proceeds via hydrolysis and the release of a 3'-hydroxyl and a 5'-phosphate terminus. Substitution of the 2'-hydroxyl group at the cleavage site by 2'-deoxy had no significant effect, suggesting that product release and/or a conformational transition rather than a chemical step is rate-limiting. Substitution by 2'-O-methyl at the cleavage site substantially reduced cleavage, which is presumably due to steric interference. Mutational analysis of the target RNA revealed that mismatches across from the 5' or the 3' end of the siRNA had little effect and that substrate RNAs as short as 15 nucleotides were cleaved by RISC.

Figure 1.

Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. (A) Preparation of site-specifically labeled substrates and cleavage assay. 5′-³²P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′-³²P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. (B) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′-³²P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2, respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5, respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. (C) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ ³²pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.

Figure 2.

Substrates as short as 15-nt are cleaved by RISC. (A) Sequences of the truncated substrates. The 13-nt core sequence required for RISC-guided cleavage is shaded. (B) PhosphorImaging of cleavage reactions incubated for 1 h at 30°C and resolved on a 15% denaturing polyacrylamide gel. Substrate RNAs were radiolabeled only at the 5′ terminus. Arrows indicate the 7- and 9-nt 5′ cleavage product. The fraction of cleaved material is indicated at the bottom of the gel.

Figure 3.

Specificity of target RNA cleavage. (A) Sequences of modified substrate RNAs. (B) PhosphorImaging of 1 h at 30°C cleavage reactions resolved on a 15% denaturing polyacrylamide gel. Substrate RNAs were radiolabeled at the 5′ terminus. The arrow indicates the 9-nt cleavage product, and the arrow with the dotted line indicates miscleavage guided by substrate S4 that contains a bulge over the cleavage site. The fraction of cleaved material is indicated at the bottom of the gel.

Figure 4.

Cleavage analysis of 2′-modified substrate RNAs. (A) Sequence and position of 2′-deoxy and 2′-O-methyl modified substrates. (B) PhosphorImaging of cleavage reactions incubated for 1 h at 30°C and resolved on a 15% denaturing polyacrylamide gel. Substrate RNAs were radiolabeled at the 5′ terminus. The arrow indicates the 9-nt cleavage product. The fraction of cleaved material is indicated at the bottom of the gel.