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.
Highlights
The fragile X mental retardation-1 (FMR1) gene encodes the fragile X mental retardation protein (FMRP),6 an RNA-binding protein, which is expressed among various tissues with the highest levels in neurons of the developing brain and in spermatogonia in adult testis [1, 2]
We identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1, 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
FMRP target mRNAs with a role in neuronal development we selected all genes belonging to the gene ontology class “nervous system development” and searched within their mRNA untranslated regions (UTRs) for poly-U stretches (U[Ն8]) and G-quartet motifs according to the consensus [DWGG(N0–2)
Summary
The FMR1 gene encodes the fragile X mental retardation protein (FMRP), an RNA-binding protein, which is expressed among various tissues with the highest levels in neurons of the developing brain and in spermatogonia in adult testis [1, 2]. FMRP has been suggested to play a role in synaptic development and plasticity through regulating mRNA transport and local protein synthesis at synapses (10 –12). FMRP was found to gate the translation of a large set of mRNAs in dendrites that are involved in synaptic plasticity. Some of these transcripts are transported in mRNA granules together with FMRP. Alternative approaches consisted in the use of random oligonucleotides linked to anti-FMRP antibody to reveal putative FMRP targets [28] and in differential display analysis [29, 30]. The goal of this study was to identify potential new FMRP targets, which play a pivotal role in gene regulation during neuronal development. Because hASH1 is crucial in generating neuronal diversity by regulating neuronal subtype specification and differentiation [33], our findings offer a new view of how FMRP influences neuronal development
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