Fragile X-related protein and VIG associate with the RNA interference machinery

Abstract

RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous genes. Here, we report the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs). We identify two putative RNA-binding proteins, the Drosophila homolog of the fragile X mental retardation protein (FMRP), dFXR, and VIG (Vasa intronic gene), through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that dFXR, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease.

Figure 1

Identification of dFXR [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)], and VIG as RISC components. (A) Coomassie-stained polyacrylamide gel showing purified RISC fractions from a Source Q column. (B) Peptides obtained from microsequencing are indicated in red on the dFXR protein sequence. The KH (hnRNP K homology) domains are boxed in black and the RGG box is indicated in blue (accession no. dFXR NP_611645). (C) Peptides obtained from microsequencing are indicated in red on the VIG protein sequence; the RGG box is indicated in blue. At right, a comparison of the domain structure of VIG to the human homolog PAI-RBP-1 (accession nos. VIG NP_523572 and PAI-RBP-1 NP_056455).

Figure 1

Identification of dFXR [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)], and VIG as RISC components. (A) Coomassie-stained polyacrylamide gel showing purified RISC fractions from a Source Q column. (B) Peptides obtained from microsequencing are indicated in red on the dFXR protein sequence. The KH (hnRNP K homology) domains are boxed in black and the RGG box is indicated in blue (accession no. dFXR NP_611645). (C) Peptides obtained from microsequencing are indicated in red on the VIG protein sequence; the RGG box is indicated in blue. At right, a comparison of the domain structure of VIG to the human homolog PAI-RBP-1 (accession nos. VIG NP_523572 and PAI-RBP-1 NP_056455).

Figure 2

dFXR [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)] and VIG associate with RISC. (A) S2 cells were transfected with a combination of luciferase double-stranded RNA (dsRNA), his-tagged VIG, and T7-tagged dFXR1. Ribosomal extracts were size-fractionated on Superose 6 and Western blotted. (B) Western blots of Argonaute-2 (Ago-2) and tagged dFXR in cytoplasmic and high-salt ribosomal extract from cells expressing the indicated tagged constructs. (C) In a separate experiment, Flag-tagged dFXR-1 was transfected, extracted, and size-fractionated as in A, and fractions containing dFXR were further fractionated on Source S. Fractions were Western blotted for Ago-2 and Flag-dFXR. (D,E) Drosophila S2 cells were transfected with dsRNA and either a control vector or vectors directing the expression of either T7-VIG (D) or Flag-dFXR (E). Proteins were recovered by immunoprecipitation and analyzed by Western blot using a polyclonal antibody raised against the Ago-2 protein. (F) Drosophila S2 cells were transfected with radiolabeled luciferase dsRNA and the indicated control or T7-tagged expression constructs. Proteins were immunoprecipitated with anti-T7 affinity resin, and associated RNA was separated on denaturing 15% polyacrylamide gel.

Figure 3

dFXR [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)] and VIG are required for efficient RNA interference in Drosophila S2 cells. Drosophila S2 cells were transfected with double-stranded RNAs (dsRNAs) corresponding to the indicated cDNAs. Three days later, cells were transfected with dsRNAs directed against luciferase (or GFP for controls) and with expression vectors for both firefly and renilla luciferases. Values shown represent the expression of luciferase in luciferase dsRNA-transfected cells relative to luciferase expression from cells transfected with control dsRNA.

Figure 4

dFXR [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)] immunoprecipitates contain RISC activity. Drosophila S2 cells were transfected with expression vectors for the indicated T7-tagged cDNAs and double-stranded RNAs corresponding to luciferase. Protein was immunoprecipitated with anti-T7 tag affinity resin, and this resin was incubated for the indicated times with radiolabeled single-stranded RNA (ssRNA) for target (luciferase), or control (GFP). RISC activity was indicated by loss of targeted RNA.

Figure 5

dFXR- [the Drosophila homolog of the Fragile X Mental Retardation Protein (FMRP)] and VIG-based RISC complexes contain microRNAs (miRNAs). (A) S2 cells were lysed hypotonically, and the nuclei were removed by centrifugation. The supernatant was pelleted at low salt, producing a S100 and P100. The P100 was extracted with high salt and centrifuged again to yield a high-salt P100 and a high-salt S100. RNA was extracted from equivalent portions of each and analyzed by Northern blotting with an oligonucleotide complementary to the Drosophila miRNA 13a. (B) S2 cells were soaked in luciferase double-stranded RNA, and extracts were prepared. Ribosomes were pelleted, salt extracted, and precipitated under low-salt conditions. The precipitates were dissolved again at high salt and size-fractionated on Superose 6. Column fractions were analyzed by Western blotting for Argonaute-1 (Ago-1) and Argonaute-2 (Ago-2) and by Northern blotting for miR2b, mir13a, and luciferase RNA. (C) S2 cells were transfected with T7-tagged FXR and his-tagged Vig expression constructs and size-fractionated. Fractions corresponding to the Ago-2 peak were immunoprecipitated using anti-T7 agarose beads and analyzed by Northern blotting for miR2b. Control immunoprecipitations were from similarly fractionated untransfected cells. (D) Drosophila S2 cells were transfected with the indicated tagged expression constructs or a GFP control. Proteins were collected by immunoprecipitation using anti-tag agarose beads. The beads were extracted with Trizol to prepare RNA and Northern blotted for miR2b.