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. 2002 Oct 1;16(19):2497-508.
doi: 10.1101/gad.1022002.

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins

Akira Ishizuka  1 , Mikiko C Siomi, Haruhiko Siomi
Affiliations

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins

Akira Ishizuka et al. Genes Dev. .

Abstract

Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.

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Figures

Figure 1
Figure 1
Protein components of TAP-purified dFMR1 complex from S2 cells. (A) The protein components in the TEV and the final extracts obtained from S2 cells expressing dFMR1-TAP and the parental cells (dFMR1-TAP minus) were resolved on SDS-PAGE and visualized by Coomassie Blue and SYPRO Ruby staining, respectively. Four of the distinct bands observed only in the dFMR1-TAP plus lanes (indicated with an asterisk) were analyzed by mass spectrometry and were found to be AGO2, Dmp68, and ribosomal proteins L5 and L11 as indicated at right. The protein bands indicated with two asterisks correspond to dFMR1-CBP (a converted form of dFMR1-TAP after TEV cleavage) and endogenous dFMR1 (see panel B). (B) Western blot analysis on the TEV and the final extracts using anti-dFMR1 antibody. The bands corresponding to dFMR1-CBP and endogenous dFMR1 are indicated at right.
Figure 2
Figure 2
Confirmation of dFMR1 interactions with AGO2, Dmp68, and ribosomal proteins L5 and L11. (A) Cytoplasmic lysate prepared from S2 cells expressing dFMR1-TAP with either AGO2-His, myc-Dmp68, myc-L5, or myc-L11 was incubated with IgG beads and a Western blot performed on the IgG bound fractions using anti-myc and anti-His antibodies. All proteins found as dFMR1-interacting proteins (Fig. 1A) were detected in the bound fractions. RNase A treatment showed no effect on the binding, indicating that the associations occur through protein–protein interactions. The presence of endogenous dFMR1 in the IgG bound fraction was determined by the Western blot using anti-dFMR1 antibody. (B) 35S-labeled AGO2, Dmp68, and ribosomal proteins L5 and L11 were produced by an in vitro transcription and translation system in the presence of [35S]methionine and incubated with either GST-dFMR1 or GST itself immobilized on glutathione-Sepharose resin. After extensive washing, the bound fractions were resolved on a polyacrylamide gel and the proteins labeled with 35S visualized by autoradiography. All proteins tested were detected only in the bound fractions of GST-dFMR1, demonstrating that they interact with dFMR1 in vitro. RNase A treatment showed no effect on the bindings. Coomassie Blue stainings of GST and GST-dFMR1 used in this experiment are shown at left.
Figure 3
Figure 3
Analysis of dFMR1 interaction with the ribosomal proteins L5 and L11. (A) Delineation of dFMR1 to determine the binding domains with L5 and L11. A fragment, dFLeu, containing the region equivalent to the ribosome-binding domain in hFMR1 interacted with neither L5 nor L11. A delineated fragment, dFC150, interacted with 35S-labeled L5 and L11 as in the case of dFC181, whereas dFC120 did not, demonstrating that dFC150 is the L5 and L11 binding domain in dFMR1. (B) The binding domain to L5 and L11 confers to dFMR1 the activity of interacting with ribosomes. A truncated mutant of dFMR1 lacking the binding domains with L5 and L11 (dFMR1Δ150) was expressed in S2 cells and the cytoplasmic lysate was subjected to sedimentation on a linear density sucrose gradient. Western blots were performed on the fractions using anti-dFMR1 antibody. (C) Northern blot on TEV extracts obtained from cytoplasmic lysates with and without dFMR1-TAP. After protease K treatment of the TEV extracts, RNA molecules were recovered and resolved on a 10% denaturing gel containing 6 M urea. A Northern blot was then performed using a riboprobe specific for 5S rRNA. The total RNA lane contains the total RNA isolated from the parental S2 cells. Mass markers are indicated at left. (D) dFMR1 is able to interact directly with L5 and L11 in vitro. GST pull-down assays were carried out using bacterially expressed His-L5 and His-L11, and Western blots with anti-His antibody were performed. (E) The ternary complex formation of 5S rRNA/L5/dFMR1 in vitro. 5S rRNA was labeled with [32P]UTP. When dFMR1 was incubated with preformed L5/5S rRNA, the RNA band was super-shifted. The migration of free 5S rRNA, 5S rRNA plus BSA, or 5S rRNA plus L5 are shown.
Figure 4
Figure 4
AGO2 and Dmp68, but not dFMR1 alone, are essential for the RNAi pathway. (A) When AGO2 was suppressed by introducing specific dsRNA in S2 cells expressing EGFP, the ability of the cells to silence EGFP by RNAi was profoundly reduced as reported previously (Hammond et al. 2001). In contrast, when dFMR1 expression was suppressed by dFMR1 dsRNA, the EGFP silencing effect was unaffected, indicating that dFMR1 is not essential for the RNAi pathway. Interestingly, when Dmp68 expression was repressed, EGFP silencing by RNAi was completely abolished, indicating that Dmp68 is a novel protein playing an essential role in the RNAi pathway. (B) The amino acid sequence alignment of Dmp68 with human p68. Amino acids identical in Dmp68 and human p68 are indicated with black boxes and residues conserved within DEAD box RNA helicases are underlined.
Figure 5
Figure 5
dFMR1 is associated with RNAi-related complexes. (A) An AGO2 complex formed in S2 cells contains dFMR1. The TEV extracts prepared from S2 cells expressing AGO2-TAP or dFMR1-TAP, and extracts from the parental cells were subjected to Western blots using anti-AGO2 and anti-dFMR1 antibodies. As expected, endogenous dFMR1 was found in both fractions of AGO2-TAP and dFMR1-TAP, confirming that AGO2 interacts with dFMR1 in vivo. Endogenous AGO2 was also copurified with AGO2-TAP. (B) A Dmp68 complex formed in S2 cells contains both dFMR1 and AGO2. The TEV extracts prepared from S2 cells expressing Dmp68-TAP, and extracts from the parental cells were subjected to Western blots using anti-dFMR1 or AGO2 antibodies. Endogenous dFMR1 and AGO2 were found in the Dmp68-associated complex, confirming that Dmp68 interacts with dFMR1 and AGO2 in vivo. (C) Dicer physically interacts with dFMR1. The TEV extracts prepared from S2 cells expressing AGO2-TAP or dFMR1-TAP, and extracts from the parental cells were subjected to Western blot using anti-Dicer antibodies (a kind gift of S. Hammond and G. Hannon; Bernstein et al. 2001). Endogenous Dicer was found in both fractions of AGO2-TAP and dFMR1-TAP, confirming that Dicer interacts with dFMR1 in vivo. The S2 control lane (indicated as total) contains cytoplasmic lysates of parental S2 cells. (D) dFMR1 remains associated with AGO2 during RNAi. AGO2-TAP TEV extracts prepared from S2 cells treated with and without dCKII β dsRNA were subjected to Western blot using anti-dFMR1 antibodies. The amount of dFMR1 bound to AGO2 was not affected by RNAi treatment, suggesting the possibility that dFMR1 is part of RISC. (E) An miRNA, miR-2b, is associated with dFMR1 and AGO2 in vivo. TAP-purifications were performed using cytoplasmic lysates of S2 cells expressing dFMR1-TAP or AGO2-TAP. RNAs were isolated from the purified complexes and analyzed by Northern blot as described (Lagos-Quintana et al. 2001; Lee and Ambros 2001). The blot was probed for the miR-2b (Lagos-Quintana et al. 2001).
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