The interaction between the fragile X mental retardation protein (FMRP) and BC1 RNA has been the subject of controversy. We probed the parameters of RNA binding to FMRP in several ways. Nondenaturing agarose gel analysis showed that BC1 RNA transcripts produced by in vitro transcription contain a population of conformers, which can be modulated by preannealing. Accordingly, FMRP differentially binds to the annealed and unannealed conformer populations. Using partial RNase digestion, we demonstrate that annealed BC1 RNA contains a unique conformer that FMRP likely binds. We further demonstrate that this interaction is 100-fold weaker than that the binding of eEF-1A mRNA and FMRP, and that preannealing is not a general requirement for FMRP's interaction with RNA. In addition, binding does not require the N-terminal 204 amino acids of FMRP, methylated arginine residues and can be recapitulated by both fragile X paralogs. Altogether, our data continue to support a model in which BC1 RNA functions independently of FMRP. 1. Introduction Fragile X syndrome is the most common inherited cause of mental impairment accounting for ca. 40% of X-linked mental retardation cases. It is also the most common known cause of autism (reviewed in [1–6]). Other characteristics of the fragile X syndrome include hyperactivity [7], increased susceptibility to seizures [8], increased testicular volume [9], macrocephaly, and large ears [10]. In addition, it has been found that carriers of the fragile X premutation, once thought to be free of the effects of the disease, also suffer from subtle behavioral and physical abnormalities [11–14]. This wide and varied constellation of phenotypic features results from the loss of function of a single gene, FMR1 (summarized in: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part-fragilex). The FMR1 gene encodes the RNA-binding protein FMRP [15], a negative [16–18] and positive [19, 20] translational regulator, and it has been of considerable interest to delineate the cellular RNAs that bind to FMRP [21–25] and the mechanism(s) by which FMRP binds and controls these mRNAs [26–35]. In 2003, Zalfa et al. described a bridging mechanism in which the fragile X mental retardation protein (FMRP) via interaction with the 5′ end of the small noncoding RNA, BC1, and bound and repressed FMRP target mRNAs [36]. This model has been subject to great deal of scrutiny owing to findings that appear to be out of step with other studies. These include differences in the prime localization of FMRP with small repressed mRNPs rather than brain
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