Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates

Abstract

Small interfering RNAs (siRNAs) direct Argonaute proteins, the core components of the RNA-induced silencing complex (RISC), to cleave complementary target RNAs. Here, we describe a method to purify active RISC containing a single, unique small RNA guide sequence. We begin by capturing RISC using a complementary 2'-O-methyl oligonucleotide tethered to beads. Unlike other methods that capture RISC but do not allow its recovery, our strategy purifies active, soluble RISC in good yield. The method takes advantage of the finding that RISC partially paired to a target through its siRNA guide dissociates more than 300 times faster than a fully paired siRNA in RISC. We use this strategy to purify fly Ago1- and Ago2-RISC, as well as mouse AGO2-RISC. The method can discriminate among RISCs programmed with different guide strands, making it possible to deplete and recover specific RISC populations. Endogenous microRNA:Argonaute complexes can also be purified from cell lysates. Our method scales readily and takes less than a day to complete.

FIGURE 1.

Strategy to purify active RISC.

FIGURE 2.

Active RISC can be eluted from a capture oligo partially complementary to the small RNA guide. let-7-programmed Drosophila Ago2-RISC, assembled in Drosophila embryo lysate, was incubated with a capture oligo fully complementary to let-7, to the let-7 seed plus 3′ supplementary region, or only to the let-7 seed sequence. The RISC assembly reaction (Input), the supernatant after capture (Sup), the washes (five with 100 mM and five with 2 M potassium acetate, pooled and concentrated to the volume of the original RISC assembly reaction, 100 µL; Wash), and the eluate from the capture oligos were incubated with 100 nM target RNA for 5 min at 25°C to detect Ago2-RISC activity. (Ø) No incubation.

FIGURE 3.

Drosophila Ago1- and Ago2-RISC, as well as mouse AGO2-RISC, can be purified using a partially complementary capture oligo. (A) A let-7/let-7* duplex was loaded into Drosophila Ago1 or (B) a let-7 siRNA was loaded into Drosophila Ago2 by incubation in Drosophila embryo lysate and purified with a partially complementary capture oligo. The RISC assembly reaction (Input), the supernatant after capture (Sup), the first wash, and the eluates were incubated with 100 nM let-7 complementary target RNA for 10 min (Ago1 and Ago2) and 180 min (Ago1) at 25°C. (C) Tenfold concentrated, purified Drosophila Ago1- and Ago2-RISC, mouse AGO2, and control samples were analyzed by quantitative mass spectrometry to determine their protein composition. The enrichment of each Argonaute protein in the purified RISC was calculated as the ratio of let-7-programmed, purified samples to control samples in which the small RNA duplex was omitted (background). (D) let-7-programmed mouse AGO2-RISC was assembled in S100 cytosolic extract from Ago2^−/− MEFs overexpressing mouse AGO2, then purified using either a fully or partially complementary capture oligo. Target cleaving activity was tested using 100 nM target RNA for 5 min at 37°C.

FIGURE 4.

The purification method separates RISCs programmed with different siRNA guides. (A) Experimental strategy. (Red) Guide; (blue) passenger strand. (B) The activity of the samples was measured by incubating them with target RNAs (100 nM) complementary to let-7 (186 nt) and luciferase (506 nt) guide siRNA strands for 5 min (Input, Sup, Eluate) or 30 min (Eluate) at 25°C. (Ø) No incubation.

FIGURE 5.

Purification of endogenous miRNA-RISC complexes. (A) S100 cytosolic extract from Ago2^−/− MEFs overexpressing mouse AGO2 was concentrated 10-fold and then purified using a capture oligo partially complementary to mouse miR-21. Target cleavage assays were performed by incubating samples with 100 nM target RNA for 5 min (Input, Sup, Eluate) or 30 min (Eluate) at 25°C. (Ø) No incubation. (B) Drosophila miR-286 was purified from embryo lysate using a partially complementary capture oligo. Total RNA from each sample was resolved by denaturing electrophoresis and miR-286 was detected by Northern hybridization.