Expanded RNA-binding activities of mammalian Argonaute 2 - PubMed (original) (raw)

Expanded RNA-binding activities of mammalian Argonaute 2

Grace S Tan et al. Nucleic Acids Res. 2009 Dec.

Abstract

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells.

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Figures

Figure 1.

Recombinant mouse GST-Ago2 and let-7a form active RISC in vitro. 15 nM of either wild-type or catalytically inactive (D669A) GST-Ago2 was loaded with 0.02 μM synthetic 5′P-let-7a and then incubated with 13.6 nmol 5′-32P target RNA (TD). Reactions were analyzed by 15% Urea PAGE and detected by autoradiography. pBR322/MspI DNA marker (M) and a synthetic 10 nt oligo (G10) were radiolabeled and included as size markers.

Figure 2.

GST-Ago2:miRNA binding analysis. 5′-32P-let-7a (10.8 nM) was incubated with increasing concentrations of GST-Ago2 or GST-D669A. GST-Ago2:let-7a (A) or GST-D669A:let-7a (C) complexes were analyzed by native gel electrophoresis. The fraction of let-7a bound to GST-Ago2 (B) or GST-D669A (D) was plotted against the concentration of the recombinant proteins. Panels show a representative of three independent experiments.

Figure 3.

Endogenous Dicer activity from Sf9 cells co purifies with GST-Ago2. (A) Silver stained NuPAGE showing fractions obtained from anion exchange chromatography. GST-Ago2 was present in fractions with NaCl concentrations ranging from 200 to 400 mM. (B) GST-Ago2 containing fractions A10, A11, B3, and B9 were tested for pre-miRNA processing activity by incubation with 5′-32P radiolabeled pre-let-7a-3. Reactions were then analyzed by 15% Urea PAGE and detected by autoradiography. Fractions A10 and A11 were Dicer-free. Cleavage of 5′-32P-target TD by A10, A11, B3, and B9 fractions of purified GST-Ago2 pre-incubated with 5′P-let-7a. Reactions were analyzed on the same 15% Urea PAGE gel.

Figure 4.

Purified, recombinant GST-Ago2 does not process pre-let-7a-3 in vitro and cleaves target TD in the absence of Dicer. (A) GST-Ago2, GST-D669A or recombinant human Dicer was incubated with 5′-32P-pre-let-7a-3. Reaction products were analyzed by 15% Urea PAGE. (B) Purified, Dicer-free, GST-Ago2, GST-D669A or Dicer was pre-incubated with 5′P-pre-let-7a-3 before adding 5′-32P target TD and analyzed by 15% Urea PAGE.

Figure 5.

Purified, Dicer-free recombinant GST-Ago2 binds directly to pre-miRNAs or unstructured RNAs longer than miRNAs or siRNAs and forms active RISC. (A) A 2000 c.p.m. of 3′-32P pCp labeled indicated RNA oligo was incubated with ∼16.7 nM of recombinant, Dicer-free GST-Ago2, and then irradiated with 254 nM UV light. Photo-crosslinked samples were then analyzed by NuPAGE and detected by autoradiography. In competition experiments, 10×, 50× and 100× molar excess of unlabeled indicated RNA oligos or unlabeled tRNA were included. (B) 15 nM of GST-Ago2 was pre-incubated with 0.02 μM of 5′P-pre-let-7a-3, pre-let-7a-3, 5′P-73 nt unstructured single stranded RNA or mature 5′P-let-7a, followed by addition of 13.6 nmol 5′-32P target TL (100 nt). Reaction products were analyzed by 10% Urea PAGE with Decade RNA size marker (M1) and detected by autoradiography. (C) Sequences of 5′P-pre-let-7a-3, 5′P-let-7a, 5′P-73 nt ssRNA oligo and target RNA (TL).

Figure 6.

Endogenous human Ago2 binds directly to pre-let-7a-3. (A) Western blot of human Ago2 immunoprecipitates from total HeLa cell lysates. Non-immune mouse serum (NMS) was used as negative control. (B) Beads containing immunopurified Ago2 were washed with Empigen and then incubated with 5′-32P-radiolabeled pre-let-7a-3. Recombinant Dicer and NMS were used as positive and negative controls for the processing reaction, respectively. (C) RNA isolated from immunopurified, Empigen-washed, Ago2 or from NMS was probed on Northern blot with an LNA probe against let-7a. The three distinct pre-let-7a signals correspond to the three isoforms of pre-let-7a (pre-let-7a-1, pre-let-7a-2 and pre-let-7a-3), detected by the LNA probe.

Figure 7.

Accumulation of pre-miRNAs bound to Ago in Dicer-null mouse embryonic fibroblasts (MEFs). (A) Western blot showing similar amounts of Ago immunoprecipitated from Dicer+/− or Dicer−/− MEFs. The asterisk indicates cross-reaction of 2A8 antibody with radixin (27). (B) Western blots showing comparable expression of Agos in Dicer+/− and Dicer−/− MEFs and absence of Dicer in Dicer−/− MEF lysates. (C) RNA was isolated from Ago immunoprecipitated with anti-Ago 2A8 antibody or non-immune mouse serum (NMS) from Dicer+/− or Dicer−/− MEFs, 3′-end labeled with 32P-pCp and resolved on a 15% Urea PAGE gel. Accumulation of pre-miRNAs concomitant with abrogation of miRNAs was detected in the anti-Ago IP from Dicer−/− MEFs. (D) Experiment was repeated as described in (A–C), except northern blot analysis of isolated RNA was performed and probed with LNA-let-7a. A total of 0.1 nM synthetic 5′P-pre-let-7a-3 was included as positive control.

Figure 8.

Endogenous human Ago2 is localized in the cytoplasm and nuclei of HeLa cells. (A) Differential interference contrast (DIC) image show the morphology of fixed and immunostained HeLa cells. (B) Endogenous Ago2 was immunostained with Ago2-specific antibody (4G8). The images were captured using deconvolution microscopy. (C) Nuclei of HeLa cells were co-stained with DAPI. (D) Merged images of (B) and (C), showing endogenous Ago2 in the cytoplasm and in the DAPI-stained nuclei.

Figure 9.

Nuclear and cytoplasmic human Ago2 associate with pre-miR-21. (A) Nuclear and cytoplasmic fractions of HeLa cells were probed on western blot with antibodies against indicated proteins. The same membrane was used with all antibodies. (B) Western blot of Ago2 immunoprecipitates from nuclear and cytoplasmic HeLa cell fractions. (C) Beads containing immunopurified Ago2 (or NMS) from nuclear and cytoplasmic fractions were washed with Empigen and then incubated with 5′-32P-radiolabeled pre-miR-30a. Recombinant Dicer was used as positive control for the processing reaction. (D) RNA was isolated from nuclear and cytoplasmic Ago2 immunoprecipitates that had been washed with Empigen and probed on northern blot with an LNA probe against miR-21.

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Expanded RNA-binding activities of mammalian Argonaute 2 - PubMed (original) (raw)