Translational Regulation of the Human Achaete-scute Homologue-1 by Fragile X Mental Retardation Protein

doi:10.1074/jbc.M807354200

Translational Regulation of the Human Achaete-scute Homologue-1 by Fragile X Mental Retardation Protein*

^‡Charité, Universitätsmedizin Berlin, Institut für Vegetative Physiologie, Tucholskystrasse 2, D-10117 Berlin, the ^§Technische Universität Berlin, Institut für Biotechnologie, Gustav-Meyer-Allee 25, D-13355 Berlin, and the ^¶RNA Editing and Hyperexcitability Disorders Helmholtz Group, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany

1 To whom correspondence should be addressed. Tel.: 49-30-450-528-268; Fax: 49-30-450-528-972; E-mail: michael.faehling{at}charite.de.

Abstract

Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5′-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)₁₀ sequence located in the 5′-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation.

Footnotes

↵6 The abbreviations used are: FMRP, fragile X mental retardation protein; FMR1, fragile X mental retardation-1; CMV, cytomegalovirus; UTR, untranslated region; P2, postnatal day 2; HES1, Hairy and Enhancer of Split1; siRNA, small interference RNA.
↵* This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG, Grant FA845/2-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.
↵2 Supported by Sonnenfeld-Siftung.
↵3 Supported by a research grant from Charité, Universitätsmedizin Berlin.
↵4 Supported by the DFG (Grant TH459/5).
↵5 Supported by the Helmholtz Association (Grant VH-NG-246) and the DFG (Grants ME2075/3-1 and SFB TR3/B5).
- Received September 23, 2008.
- Revision received December 17, 2008.
The American Society for Biochemistry and Molecular Biology, Inc.