The fragile X mental retardation protein interacts with a distinct mRNA nuclear export factor NXF2

Abstract

Loss of fragile X mental retardation protein, FMRP, causes the fragile X syndrome. Highly expressed in the brain and testis, FMRP has been implicated in the transport and translation of specific mRNAs. Here we show that FMRP and the mRNA nuclear export factor NXF2 co-express in the mouse male germ cells and hippocampal neurons and that FMRP associates with NXF2 but not with its close relative NXF1. We thus hypothesize that FMRP and NXF2 may act in concert to promote the nucleocytoplasmic transport of specific mRNAs in male germ cells and neurons.

FIGURE 1.

(A) Schematic representation of the structures of NXF1 and NXF2 (Herold et al. 2000; Tan et al. 2005). The numbers on top are in amino acids and the protein interaction domains are marked with the bars. NLS, nuclear localization signal; RNP, ribonucleoprotein motif; LRR, leucine-rich region. NTF2-like, nuclear transport factor 2 like; UBA, ubiquitin associated. (B) Antibody specificity. The left panel is a Western blot of protein samples extracted from transfected cells, using the NXF2 antibody. The right panel is a reprobing of the same blot with an anti-flag antibody (Sigma). Molecular weight markers in amino acids are on the left.

FIGURE 2.

(a–g ) Fluorescent images of cryostatic sections of mouse testes. Panels a–f show merged images between DAPI and the indicated protein staining., and panel g is a merged image between e and f. (h–j ) Immunohistochemistry of adult mouse brain paraffin sections using antibodies specific for NXF2 (h), FMRP (i) and NXF1 (j), respectively.

FIGURE 3.

FMRP and NXF2 interact with each other biochemically. The antibody specific for FMRP (αFMRP) or an antibody against an unrelated protein (αMS2) was used to isolate FMRP-containing protein complexes from adult mouse testicular (A) or brain (B) lysates in the IP experiments. Purified protein complexes (Pellet), supernatants (Sup) or inputs (Input) were resolved by SDS-PAGE. Indicated antibodies (marked on the right) were used to probe the membranes in Western blot analyses. (C) In vitro pull-down assays. A mixture of ³⁵S-labeled full-length NXF2 (appears as a doublet) and NXF1 proteins (lane 1) was incubated with GST-FMRP (lane 2) or GST alone (lane 3) pre-bound on glutathione beads as previously described (Huang et al. 2003). Input and proteins selected by the beads were resolved by SDS-PAGE and labeled proteins visualized by autoradiography. (D) Interaction domain mapping. ³⁵S-labeled full-length as well as various domains of NXF2 were incubated with GST alone (lane 3) or with GST-FMRP (lane 4) pre-bound on glutathione beads. Unbound and bound fractions were resolved by SDS-PAGE and labeled proteins visualized by autoradiography. The full-length (1–671), N-terminal (1–194), middle (195–426), and C-terminal regions (427–671) of NXF2 are marked on the left in amino acids.