Proteomic and functional analysis of Argonaute‐containing mRNA–protein complexes in human cells

RNA silencing is a conserved gene‐silencing pathway that is triggered by double‐stranded RNA (dsRNA). DsRNA molecules are processed to small interfering RNAs (siRNAs) or microRNAs (miRNAs), which are then assembled into various effector complexes, including the RNA‐induced silencing complex (RISC). MiRNA precursors (pre‐miRNAs) are excised from primary transcripts by a nuclear complex containing the RNase III enzyme Drosha and the dsRNA‐binding domain protein DGCR8/Pasha. Pre‐miRNAs are subsequently exported to the cytoplasm by the export receptor exportin 5, where dsRNAs and pre‐miRNAs are further processed by the RNase III enzyme Dicer to duplexes of approximately 22 nucleotides. In a manner similar to Drosha, Dicer functions with the dsRNA‐binding domain proteins TRBP (human immunodeficiency virus transactivating response RNA binding protein) and PACT (PKR activator; reviewed by Meister & Tuschl, 2004; Filipowicz et al, 2005; Zamore & Haley, 2005). Only one strand of the siRNA–miRNA duplex intermediate is loaded into the silencing effector complex to become the guide strand. SiRNAs and miRNAs either guide the sequence‐specific degradation of complementary RNAs or inhibit the translation of partly complementary target messenger RNAs (Pillai et al, 2007). Recently, a third pathway of miRNA function has been identified in Drosophila, in which miRNAs guide the degradation of not perfectly complementary target mRNAs by recruiting de‐adenylation and de‐capping enzymes (Behm‐Ansmant et al, 2006; Wu et al, 2006).

Members of the Argonaute (Ago) protein family are crucial components of RNA silencing effector complexes. Ago proteins contain PAZ and PIWI domains, and structural studies of archaeal Ago proteins show striking similarity between the PIWI domain and RNase H. Further functional analyses have shown that some Ago proteins contain endonucleolytic activity (Parker & Barford, 2006; Peters & Meister, 2007). Recently, another Ago domain, which binds to the m⁷G cap of mRNAs, was identified (Kiriakidou et al, 2007).

Biochemical purifications of Ago1 and Ago2 complexes from HeLa cell lysates identified the DExD box protein MOV10 (Moloney leukaemia virus 10 homlogue) and a protein termed TNRC6B (trinucleotide repeat containing 6B) as new components of the Ago complex (Eulalio et al, 2007a). Interestingly, TNRC6B is highly homologous to TNRC6A (GW182), which constitutes a marker protein for cytoplasmic processing bodies (P‐bodies). P‐bodies are cellular sites of RNA metabolism, and it has been shown that Ago proteins also localize to P‐bodies (Eulalio et al, 2007a). MiRNAs and artificially bulged miRNA target mRNAs are also found in P‐bodies, and it has been suggested that localization of mRNAs to P‐bodies might prevent their translation (Liu et al, 2005; Pillai et al, 2005). Furthermore, a recent study showed that the P‐body component RCK/p54 (DEAD box polypeptide 6) is required for miRNA‐guided translational repression in human cells (Chu & Rana, 2006). Further studies showed that P‐bodies are formed as a consequence of gene silencing, but their integrity is not required for gene silencing (Eulalio et al, 2007b).

Here, we report the biochemical identification and isolation of human Ago1 and Ago2 protein complexes. We identify three distinct Ago1 and Ago2 complexes, which we refer to as Ago complexes I–III. Using a comprehensive proteomic approach, we have identified the protein composition of the Ago complexes. Among these interactors, we found RBM4 (RNA binding motif protein 4) and show that it is required for small RNA‐guided gene silencing.

Cell extracts and gradient centrifugation. Polyribosome fractionation from HEK 293 cells was carried out according to Pillai et al (2005). For complex purification and co‐immunoprecipitations, HEK 293 cells were lysed in buffer containing 25 mM Tris–HCl (pH 7.4), 150 mM KCl, 0.5% NP‐40, 2 mM EDTA, 1 mM NaF, 0.5 mM dithiothreitol and protease inhibitors (Roche, Penzberg, Germany) and centrifuged at 10,000g for 10 min at 4°C. For fractionations, gradients from 15% (w/v) to 55% (w/v) sucrose in 150 mM KCl, 25 mM Tris (pH 7.4) and 2 mM EDTA were used. Lysates were separated by centrifugation at 30,000 r.p.m. for 18 h in an SW41 rotor at 4°C. To determine indicated S values, catalase (11S), apoferritin (17S) and thyroglobin (19S) were used. For the analysis of RNA‐dependent interactions, extracts were preincubated with 100 μg/ml RNase A (Qiagen, Hilden, Germany) at 4°C for 1 h.

Immunoprecipitation of Flag/HA‐tagged AGO complexes. AGO‐containing gradient fractions were pooled and incubated with 100 μl Flag M2 agarose beads (Sigma, Taufkirchen, Germany) for 2 h at 4°C. Beads were subsequently washed with immunoprecipitation buffer (300 mM NaCl, 5 mM MgCl₂, 0.1% NP‐40 and 50 mM Tris–HCl, pH 7.5) and phosphate‐buffered saline (PBS).

Co‐immunoprecipitation experiments. Lysates were incubated with 30 μl Flag M2 agarose at 4°C for 1.5 h, and washed with IP buffer and PBS. Beads were distributed equally into two tubes and 1.5 ml PBS or PBS containing 100 μg/ml RNase A was added. After incubation at 4°C for 1.5 h, beads were washed with PBS and denatured by adding 25 μl of protein sample buffer.

Supplementary information is available at EMBO reports online (http://www.emboreports.org).

We thank S. Behrens for NF‐45/NF‐90‐specific antibodies, S. Hüttelmaier for ZBP1/3‐specific antibodies and S. Jentsch for support. Our research was supported by the Max‐Planck‐Society, Deutsche Forschungsgemeinschaft (DFG) grant ME 2064/2‐1 and European Union grant LSHG‐CT‐2005‐037900. L.W. receives a fellowship of the Boehringer Ingelheim Funds.