Human RISC assembly is uncoupled from dicing and dependent on ATP. (a) Small-RNA duplexes, a pre-miRNA–like hairpin RNA, and single-stranded RNAs used in b–e. miR-luc (guide strand) is shown in red. The dicing site of pre–miR-luc is indicated by a black line. (b) RISC assembly is uncoupled from dicing in HeLa cells. The dual luciferase reporter assay was performed 24 h after transfection. miR-luc–miR-luc* duplex and luc siRNA duplex repressed the perfectly complementary reporter, much better than pre–miR-luc. Mock refers to no siRNA treatment. (c) RISC assembly is uncoupled from dicing in HeLa cell lysate, but immunopurified Ago2 complex and purified recombinant Ago2 represent a bypass pathway that can use only single-stranded RNAs. Target cleavage by miR-luc–miR-luc* duplex, luc siRNA duplex and pre–miR-luc were monitored. (d) Purified recombinant Ago2 can use single-stranded RNAs that cannot form hairpin structure. Target cleavage by miR-luc–miR-luc* duplex and 21-nt and 58-nt single-stranded RNAs were monitored. (e) ATP facilitates RISC assembly in HeLa cell lysate, but not in immunopurified Ago2 complex or purified recombinant Ago2. Target cleavage by miR-luc–miR-luc* duplex was monitored in the presence or absence of ATP. The energy regenerating system was omitted, and ATP was substituted with EDTA for the –ATP condition.

ATP facilitates RISC loading but not unwinding. (a) Formation of pre–Ago2-RISC is promoted by ATP. HeLa cell lysate was incubated with radiolabeled duplex C at 15 °C in the presence or absence of ATP. Pre–Ago2-RISC formation was markedly impaired in the absence of ATP. (b) Conversion of pre–Ago2-RISC into mature–Ago2-RISC does not require ATP. Pre–Ago2-RISC was formed by incubating radiolabeled duplex A in HeLa cell lysate in presence of ATP. ATP was then depleted by hexokinase and glucose, a 20-fold excess nonradiolabeld duplex was added, and complex formation was monitored. Pre–Ago2-RISC was chased into mature Ago2-RISC regardless of the presence of ATP.

Four human AGO proteins have similar structural preferences for small-RNA duplexes for RISC loading and unwinding. (a) RISC loading. The mm series of duplexes (mm1–mm17) were incubated at 15 °C in lysate from HEK 293T cells expressing Flag-tagged Ago1, Ago2, Ago3 or Ago4. (b) Unwinding. The MM10-mm series of duplexes (MM10-mm1 to MM10-mm17) were similarly incubated at 25 °C.

Complex I and complex II are pre–Ago2 RISC and mature–Ago2 RISC, respectively. (a) Small-RNA duplexes used in this study. Each duplex contained an identical guide strand sequence and a 5′ ³²P radiolabel (indicated by an asterisk). (b) Agarose native gel analysis of wild-type and ago2(RNAi) HeLa cell lysate. Six distinct complexes were detected, and complexes I and II disappeared in ago2(RNAi) lysate. Anti-Ago2 western blotting showed that >95% of endogenous Ago2 was depleted by RNAi. (c) Complexes I and II contain Ago2. The reaction mixtures were photo-cross-linked and separated by an agarose native gel electrophoresis. The complexes were cut out from the gel and then analyzed by SDS-PAGE. The ~100-kDa bands detected in complexes I and II corresponded to Ago2. (d) Complex I contained double-stranded small RNA, whereas complex II contained single-stranded guide annealed with the target ASO. The complexes were cut out from the gel, and RNA was extracted and analyzed by 15% native PAGE. The identity of the bands were assigned by their comigration with the corresponding synthetic markers. (e) Complex I is a precursor of complex II. Complex I was formed with radiolabeled duplex A at 15 °C, a 20-fold excess nonradiolabeled duplex was added and complex formation was further monitored at 25 °C. Complex I was efficiently ‘chased’ into complex II.

Central mismatches promote Ago2-RISC loading, and seed and 3′-mid mismatches facilitate unwinding. (a) HeLa cell lysate was incubated with mm series of 17 small-RNA duplexes (mm1–mm17) at 15 °C. Pre–Ago2-RISC formation was enhanced with duplexes containing central mismatches. (b) HeLa cell lysate was incubated with MM10-mm series of small-RNA duplexes (MM10-mm1 to MM10-mm17) at 25 °C. Conversion of pre–Ago2-RISC into mature–Ago2-RISC was promoted by seed or 3′-mid mismatches.

Ago1–4 can use miRNA-like duplexes, whereas only Ago2 can efficiently unwind siRNA duplexes. (a) Lysates from HEK 293T cells expressing Flag-tagged Ago1, Ago2, Ago3 or Ago4 were incubated with radiolabeled duplexes A–C and complex formation was monitored. Ago1–4 could unwind miRNA-like duplex A but not central-bulged duplex C. Only Ago2 could unwind perfectly complementary duplex B via passenger-strand cleavage. (b) A noncatalytic mutant of Ago2 (D597A) cannot unwind perfectly complementary duplex B, indicating that this process is mediated by passenger-strand cleavage.