Phosphorylation of FMRP inhibits association with Dicer - PubMed (original) (raw)

Phosphorylation of FMRP inhibits association with Dicer

Anne Cheever et al. RNA. 2009 Mar.

Abstract

Fragile X syndrome is caused by an absence of the protein product of the fragile X mental retardation gene (FMR1). The fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates translation of associated mRNAs; however, the mechanism for this regulation remains unknown. Constitutively, phosphorylated FMRP (P-FMRP) is found associated with stalled untranslating polyribosomes, and translation of at least one mRNA is down-regulated when FMRP is phosphorylated. Based on our hypothesis that translational regulation by P-FMRP is accomplished through association with the microRNA (miRNA) pathway, we developed a phospho-specific antibody to P-FMRP and showed that P-FMRP associates with increased amounts of precursor miRNAs (pre-miRNA) compared with total FMRP. Furthermore, P-FMRP does not associate with Dicer or Dicer-containing complexes in coimmunoprecipitation experiments or in an in vitro capture assay using a P-FMRP peptide sequence bound to agarose beads. These data show that Dicer-containing complexes bind FMRP at amino acids 496-503 and that phosphorylation disrupts this association with a consequent increase in association with pre-miRNAs. In sum, we propose that in addition to regulating translation, phosphorylation of FMRP regulates its association with the miRNA pathway by modulating association with Dicer.

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Figures

FIGURE 1.

FIGURE 1.

PSER antibody is specific for phosphorylated FMRP. (A) Schematic representation of the FMRP. NLS-nuclear localization sequence, NES-nuclear export sequence, phospho-serines 496, 499, and 503 (adapted from data in Ceman et al. 2003). (B) Western blot of whole-cell lysates from VC, WT, and S499A FMRP expressing cells were immunoblotted (ib) with the indicated antibody: PSER, 1a (anti-FMRP 1a-1C3) (Devys et al. 1993), or eIF5. (C) Anti-Flag immunoprecipitations (IP) of Flag-FMRP expressing L-M[TK-] cells were split and mock-treated or phosphatase (P-tase)-treated (Ceman et al. 2003) before probing with PSER or 1a (anti-FMRP). Ig-immunoprecipitating antibody alone. (D) Flag-FMRP expressing L-M[TK-] cells immunoprecipitated with NP antibody and probed with 1a (anti-FMRP), FXR1, or FXR2 antibodies. WCL-L-M[TK-] whole-cell lysate. Ig-NP indicates antibody alone.

FIGURE 2.

FIGURE 2.

P-FMRP associates with increased amount of precursor miRNAs. (A) We immunoprecipitated 2 × 109 Flag-FMRP cells with pre-immune serum (pre), NP, or PSER. RNA was extracted and end-labeled with P32 as described (Duan and Jin 2006), resolved on a 15% acrylamide TBE/urea gel, and visualized using autoradiography (8 h). The longer exposure of the gel (16 h, right) shows associated miRNAs. Decade marker system (Ambion) indicates RNA size in nucleotides. (B) Equivalent amounts of FMRP were immunoprecipitated by PSER and NP antibodies in A.

FIGURE 3.

FIGURE 3.

P-FMRP is unable to associate with Dicer. (A) Lysate from 2 × 107 HeLa cells was immunoprecipitated with NP or PSER antibodies and immunoblotted (ib) with Dicer antibody or FMRP (1a) antibody. (WCL)-50 μg HeLa whole-cell lysate; IP, immunoprecipitations; and Ig, immunoprecipitating antibody alone. (B) Lysate from 109 Flag-FMRP-expressing cells was immunoprecipitated with anti-FMRP antibody 7G1 (Brown et al. 2001), split, and incubated with either RNase (+) or mock-treated (−) and immunoblotted (ib) with the Dicer antibody. (WCL)-50 μg whole-cell lysate. (C) Lysate from 107 HeLa cells was incubated with matrix-coupled peptide (NH2-SNA[pS]ETES-CONH2) (P-FMRP), (NH2-SNASETES-CONH2) (FMRP), or random peptide (RP) (KETAAAKFERQHMDS); resolved on a 6% SDS-PAGE gel; and immunoblotted with Dicer antibody.

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