The role of PACT in the RNA silencing pathway

Abstract

Small RNA-mediated gene silencing (RNA silencing) has emerged as a major regulatory pathway in eukaryotes. Identification of the key factors involved in this pathway has been a subject of rigorous investigation in recent years. In humans, small RNAs are generated by Dicer and assembled into the effector complex known as RNA-induced silencing complex (RISC) by multiple factors including hAgo2, the mRNA-targeting endonuclease, and TRBP (HIV-1 TAR RNA-binding protein), a dsRNA-binding protein that interacts with both Dicer and hAgo2. Here we describe an additional dsRNA-binding protein known as PACT, which is significant in RNA silencing. PACT is associated with an approximately 500 kDa complex that contains Dicer, hAgo2, and TRBP. The interaction with Dicer involves the third dsRNA-binding domain (dsRBD) of PACT and the N-terminal region of Dicer containing the helicase motif. Like TRBP, PACT is not required for the pre-microRNA (miRNA) cleavage reaction step. However, the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference. Our study indicates that, unlike other RNase III type proteins, human Dicer may employ two different dsRBD-containing proteins that facilitate RISC assembly.

Figure 1

PACT interacts with Dicer, but does not facilitate the cleavage activity. (A) Immunoprecipitation followed by Western blotting. V5-Dicer protein was coexpressed in HEK293T cells with FLAG-PACT, or FLAG-TRBP protein. Immunoprecipitation was carried out using anti-FLAG antibody and the protein was visualized using either anti-V5 or anti-FLAG antibody. (B) In vitro pre-miRNA processing assay. Total HEK293 T extract (total lysate) or immunoprecipitates (FLAG-IP) of FLAG-Dicer, FLAG-TRBP, or FLAG-PACT were incubated with 5′ end radiolabeled pre-let-7a-1. The RNA was extracted by phenol extraction method, followed by separating on 15% urea-PAGE. (C) In vitro processing assay using recombinant human Dicer and PACT. In all, 200 ng of recombinant PACT prepared from E. coli and 0.25 U of recombinant human Dicer (Stratagene) were used in each reaction. Pre-let-7a-1 (left panel) and long dsRNA of 80 bp (right panel) were used as substrates.

Figure 2

Domains responsible for the interaction of PACT and TRBP with Dicer. (A) Schematic representation of a series of mutants of Dicer and PACT. (B) Immunoprecipitation followed by Western blotting. FLAG-Dicer protein was coexpressed in HEK293T cells with PACT mutant proteins fused to a V5 tag. Immunoprecipitation was carried out using anti-FLAG antibody and the protein was visualized using either anti-V5 or anti-FLAG antibody. (C) The same experiment as described in (B), except for using FLAG-Dicer mutants and V5-PACT (wild type). (D) The same experiment as described in (A), except for using FLAG-Dicer mutants and V5-TRBP (wild type). (E) In vitro pre-miRNA processing assay using FLAG-Dicer mutants. Immunoprecipitates of FLAG-Dicer mutants were incubated with 5′ end radiolabeled pre-miR30a followed by RNA extraction and separation on 15% urea-PAGE (upper panel). The amount of FLAG-Dicer mutants used in in vitro pre-miRNA processing reactions was visualized using anti-FLAG antibody (lower panel).

Figure 3

PACT binds to hAgo2. (A) Immunoprecipitation followed by Western blotting. FLAG-PACT or FLAG-TRBP proteins, V5-Dicer and V5-hAgo2 protein were coexpressed in HEK293T cells. Immunoprecipitation was carried out using anti-FLAG antibody and the protein was visualized using either anti-V5 or anti-FLAG antibody. (B) The same experiment as described in (A), except for using FLAG-PACT mutants and V5-hAgo2 (wild type). In this experiment, V5-Dicer was not coexpressed.

Figure 4

Both PACT and TRBP are associated with the slicer activity in vitro. (A) In vitro pre-miRNA processing assay and the detection of mature miRNA bound to the RISC components. Dicer, hAgo2, TRBP, or PACT fused to FLAG was expressed in HEK293T cells and immunoprecipitated in RNAi reaction buffer. The immunoprecipitates were incubated with 5′-end radiolabeled pre-miR-30a. The left panel shows the RNA extracted from each reaction tube. After in vitro processing, the beads were washed with RNAi reaction buffer and treated with phenol to extract the RNA associated with the proteins (right panel). (B) In vitro RNAi assay. Dicer, hAgo2, TRBP, or PACT fused to FLAG was expressed in HEK293T cells and immunoprecipitated in RNAi reaction buffer. The immunoprecipitates were incubated with cold pre-miR-30a or 27mer siRNA. After washing, the immunoprecipitates were incubated with 5′-end radiolabeled target RNA that is complementary to miR-30a-5p.

Figure 5

PACT and TRBP coexist in the ∼500 kDa RISC. Total extract was prepared from HEK293T cells transiently transfected with FLAG-PACT, V5-Dicer, and V5-hAgo2 expression vectors and fractionated through a Sephacryl-S300 HR column. Each fraction was subject to immunoprecipitation with anti-FLAG antibody. The immunoprecipitate was used for (A) Western blot analysis using anti-TRBP or anti-FLAG antibody, or (B) in vitro RNAi assay using miR-30a-5p target RNA. The fraction number and the protein molecular mass standard (Sigma) are indicated at the top of the figures.

Figure 6

PACT is required for siRNA-induced RNAi. (A) HeLa cells were transiently transfected with siRNA targeting Dicer, PACT, or TRBP along with siRNA against firefly luciferase, firefly luciferase-expression plasmid, and renilla luciferase-expression plasmid. The firefly luciferase activity was normalized against renilla luciferase activity. This experiment was performed in triplicate. Error bars indicate standard deviations. (B) Western blot analysis. siRNA targeting GFP, Dicer, hAgo2, TRBP, or PACT was transfected into HeLa cells. Following transfection (48 h), total extract was prepared in lysis buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 500 mM NaCl, 0.5% Triton-X100). In all, 120 μg of total extract was loaded on 10% SDS–PAGE. Endogenous Dicer, TRBP, or PACT was visualized using anti-Dicer, anti-TRBP, or anti-PACT antibody.

Figure 7

PACT is required for the accumulation of mature miRNA. (A) Northern blot analysis. siRNA targeting Dicer, TRBP, or PACT was transfected into a HeLa cell line that expresses primary miR-30a under the control of the tetracycline-inducible promoter. Following transfection (48 h), doxycycline (3 μg/ml) was added to the medium to induce the expression of miR-30a. After 24 h, the cells were harvested for Northern blotting (the left panel). After normalization for loading, the relative ratios for mature miR-30a were calculated and normalized to no doxycycline-treated experiment (lane 1). The average value obtained from two independent experiments was presented in the right panel. (B) The same experiment as described in (A), except for using pri-miR-124a inducible HeLa cell line. The left panel shows a representative Northern blot result. The right panel presents the mean value of relative expression level for mature miR-124a obtained from two independent experiments. (C) RT–PCR. Total RNA used in (A) was reverse-transcribed and amplified by PCR using gene-specific primers.