Mechanism of Translational Control by the Fragile X Mental Retardation Protein and Creation of the FMRP CTAG Mouse

The Fragile X Mental Retardation Protein (FMRP) is a neuronal RNA-binding protein that is predominantly associated with polyribosomes. Loss of FMRP results in Fragile X Syndrome, characterized by mental retardation, autism and epilepsy. FMRP was recently found to be associated with a specific set of mRNAs with key roles in neuronal function and to physically interact with targeted mRNAs along their entire coding sequences (Darnell 2011). Here, we find that FMRP inhibits translation on these target transcripts by stalling ribosomes, both in vivo and in rabbit reticulocyte lysate programmed with endogenous brain polyribosomes. In these systems, loss of FMRP function resulted in increased ribosome runoff after treatment with puromycin, a drug that acts specifically on translocating ribosomes. In addition to genetic loss-of-function models, FMRPdependent relief of ribosome stalling could be induced by acute biochemical removal of FMRP from polysomes, indicating reversibility of stalling. FMRP was also directly visualized on stalled polysomes by immunoelectron microscopy. Together, these results suggest a model in which FMRP actively regulates translation of target mRNAs by stalling ribosomes. To further advance our understanding of FMRP function, we have created the FMRP cTAG mouse, a knock-in model in which FMRP can be conditionally tagged with AcGFP in a Cre-dependent manner while maintaining wt FMRP expression in all Cre-negative cells. The cTAG mouse will be a valuable tool in the study of cell type-specific FMRP function

Effects of di(n-butyl) phthalate exposure on foetal rat germ-cell number and differentiation: identification of age-specific windows of vulnerability: DBP effects on foetal germ cells

Environmental factors are implicated in increased incidence of human testicular germ‐cell cancer (TGCC). TGCC has foetal origins and may be one component of a testicular dysgenesis syndrome (TDS). Certain phthalates induce TDS in rats, including effects on foetal germ cells (GC). As humans are widely exposed to phthalates, study of the effects of phthalates on foetal rat GC could provide an insight into the vulnerability of foetal GC to disruption by environmental factors, and thus to origins of TGCC. This study has therefore characterized foetal GC development in rats after in utero exposure to di(n‐butyl) phthalate (DBP) with emphasis on GC numbers/proliferation, differentiation and time course for inducing effects. Pregnant rats were treated orally from embryonic day 13.5 (e13.5) with 500 mg/kg/day DBP for varying periods. GC number, proliferation, apoptosis, differentiation (loss of OCT4, DMRT1 expression, DMRT1 re‐expression, GC migration) and aggregation were evaluated at various foetal and postnatal ages. DBP exposure reduced foetal GC number by ∼60% by e15.5 and prolonged GC proliferation, OCT4 and DMRT1 immunoexpression; these effects were induced in the period immediately after testis differentiation (e13.5–e15.5). In contrast, DBP‐induced GC aggregation stemmed from late gestation effects (beyond e19.5). Foetal DBP exposure delayed postnatal resumption of GC proliferation, leading to bigger deficits in numbers, and delayed re‐expression of DMRT1 and radial GC migration. Therefore, DBP differentially affects foetal GC in rats according to stage of gestation, effects that may be relevant to the human because of their nature (OCT4, DMRT1 effects) or because similar effects are demonstrable in vitro on human foetal testes (GC number). Identification of the mechanisms underlying these effects could give a new insight into environment‐sensitive mechanisms in early foetal GC development that could potentially be relevant to TGCC origins