TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing - PubMed (original) (raw)

TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing

Thimmaiah P Chendrimada et al. Nature. 2005.

Abstract

MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.

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Figures

Figure 1

Figure 1. Isolation of a Dicer–TRBP-containing complex

a, Flag-Dicer isolated from a HEK293-derived cytoplasmic extract (S100) resolved by SDS–PAGE and visualized by silver staining (left panel), and western blot analysis using anti-Flag antibodies (right panel). Arrows point to the bands containing Dicer and TRBP proteins. The molecular masses of marker proteins (kDa) are indicated (right). Asterisks denote contaminating polypeptides that were also present in control immunoprecipitation (IP) from naive cells (Mock). b, Diagrammatic representation of the human TRBP protein and alignment with orthologous proteins. Conserved dsRBDs are depicted as black squares. Protein accession numbers (NCBI) are: AAB50581 (human TRBP), NP_003681 (human PRKRA), NP_03345 (mouse PRBP), AAH55390 (Danio rerio), MGC82499 (Xenopus), NP_609646 (Drosophila). aa, amino acids. c, The Dicer–TRBP complex isolated from S100 by Flag-TRBP, fractionated by Superose 6 gel filtration, and visualized by silver staining and western blot analysis. INP denotes the input to the column; asterisks denote the common contaminants (SKB1 and MEP50). Fractions of the column are shown along the top; molecular mass markers are indicated along the bottom. d, Silver stains of recombinant Dicer (rDicer) and recombinant Dicer–TRBP complex (rDicer + rTRBP) reconstituted and purified by gel filtration. e, Silver stain analysis of recombinant TRBP fractionated by gel filtration.

Figure 2

Figure 2. The Dicer–TRBP complex processes miRNAs and associates with siRNA to form a ternary complex with Ago2

a, Pre-miRNA processing assay (left panel). The pre-miRNAs generated by the Microprocessor complex were subjected to a second step of processing using recombinant Dicer, recombinant Dicer–TRBP, or fraction 28 of the Superose 6 column of immunopurified TRBP complex (Flag-TRBP) to yield a 22-nucleotide mature miRNA. The right panel shows the long double-stranded RNA processing assay. Double-stranded RNA was incubated with recombinant Dicer or recombinant Dicer–TRBP to yield a 22-nucleotide mature miRNA. b, EMSA performed using radiolabelled double-stranded siRNA. Recombinant Dicer and recombinant Dicer–TRBP (fraction 28 of the Superose 6 column) was analysed for complex formation with siRNA. 1× corresponds to 20 ng of Dicer and 10 ng of recombinant Ago2. c, EMSA performed using radiolabelled siRNA. Recombinant Dicer–TRBP (fraction 28 of the Superose 6 column) and recombinant TRBP (fraction 32 of the Superose 6 column) were analysed for complex formation with siRNA. 1× corresponds to 20 ng for each protein. d, EMSA performed with Dicer–TRBP (fraction 28 of the Superose 6 column corresponding to Flag-TRBP) and addition of recombinant Ago2. 1 × corresponds to 5 ng of native complex and 2.5 ng of recombinant Ago2. Arrows indicate the two ribonucleoprotein complexes.

Figure 3

Figure 3. Stable association of Dicer–TRBP with Ago2

a, Western blot analysis of Flag-TRBP affinity eluate fractionated by Superose 6 gel filtration. Column fractions were analysed using the antibodies indicated. b, Western blot analysis of Flag-Ago2 affinity eluate fractionated by Superose 6 gel filtration. Column fractions were analysed using the antibodies indicated.

Figure 4

Figure 4. The Dicer–TRBP complex is required for miRNA biogenesis and post-transcriptional gene silencing

a, Analysis of the transcript levels of TRBP, Dicer and β-actin by RT–PCR, after treatment of HeLa cells with a representative siRNA to TFII-I (control), TRBP and Dicer. b, Western blot analysis of Flag-Ago2 immunoprecipitate after siRNA-mediated knockdown of TFII-I (control), TRBP and Dicer. c, Northern blot analysis of miR-16, miR-23, let-7a-1 and miR-20 after treatment of HeLa cells with the indicated siRNA. d, HeLa cells co-transfected with firefly and Renilla luciferase plasmids and a combination of siRNA targeting firefly luciferase and one of the siRNAs shown in the bottom of the figure. Firefly luciferase activity was normalized relative to that of Renilla luciferase. Error bars represent standard error of the mean for three independent experiments.

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