Fragile X Mental Retardation Protein (FMRP) Binds Specifically to the Brain Cytoplasmic RNAs BC1/BC200 via a Novel RNA-binding Motif

Abstract

Fragile X mental retardation protein (FMRP), the protein responsible for the fragile X syndrome, is an RNA-binding protein involved in localization and translation of neuronal mRNAs. One of the RNAs known to interact with FMRP is the dendritic non-translatable brain cytoplasmic RNA 1 BC1 RNA that works as an adaptor molecule linking FMRP and some of its regulated mRNAs. Here, we showed that the N terminus of FMRP binds strongly and specifically to BC1 and to its potential human analog BC200. This region does not contain a motif known to specifically recognize RNA and thus constitutes a new RNA-binding motif. We further demonstrated that FMRP recognition involves the 5 stem loop of BC1 and that this is the region that exhibits complementarity to FMRP target mRNAs, raising the possibility that FMRP plays a direct role in BC1/ mRNA annealing.The fragile X mental retardation protein (FMRP) 3 is the protein involved in the fragile X syndrome, the most common cause of inherited mental retardation. FMRP is highly expressed in neurons, where it is involved in mRNP transport and translation, two processes required for synaptic plasticity (1, 2). Thus, FMRP acts as a translational repressor both in vitro and in vivo (3)(4)(5)(6)(7)(8), and its effect is more pronounced at the synapses (7). The specific mechanism(s) through which FMRP regulates translation still remains to be understood; in particular, it is not clear whether the regulation occurs at the level of translation initiation (3), during the translation elongation phase (according to a "stalling polysomes" hypothesis) (8, 9), or both, depending on the different stages of development. With respect to mRNP transport, FMRP has both a nuclear localization signal (NLS) and a nuclear export signal (NES) and is capable of shuttling between the nucleus and the cytoplasm (10); it therefore seems likely that FMRP accompanies specific mRNAs from the nucleus to the cytoplasm. Furthermore, granules containing FMRP are transported to locations throughout the dendrite, where translation is regulated by synaptic activation (11), reminiscent of the granules in which mRNPs are thought to be transported. Indeed, mass spectrometric analysis of RNase-sensitive mRNP transport granules also identified, among several proteins involved in transport along the cytoskeleton, FMRP (12).As a protein involved in mRNP transport and regulation of translation, FMRP is expected to bind selectively to a subset of the mRNAs. Quite a variety of mRNAs have been identified in vitro and in vivo as potential targets of the entire FMRP (13-16), and it is still not clear how the mRNAs are recognized. There are at least three RNA elements that can direct FMRP binding (for a recent review, see Ref. 17). The first is a G-rich RNA structure called the G quartet (14,18,19), and the second consists of U-rich stretches (16). Thirdly, we have demonstrated that FMRP binds specifically to the non-coding RNA BC1, which in turn exhibits significant complementarity to and anneals with some mRNAs regulated by FMRP (7)...

On BC1 RNA and the fragile X mental retardation protein

“…4B). The data show that FMRP associates with target mRNAs in the absence of BC1 RNA, a result incompatible with a requirement for BC1 RNA to provide a physical link between FMRP and its target mRNAs, as postulated by Zalfa and colleagues (16,17). Does BC1 RNA associate with FMRP in vivo?…”

“…We next turned to the question of whether BC1 RNA physically interacts with mRNAs that are targets of FMRP regulation. Zalfa and colleagues (16,17) proposed that the 5Ј BC1 domain directly interacts, via base-pairing, with such FMRP target mRNAs and that BC1 RNA thus bridges these mRNAs and FMRP. These authors probed BC1-mRNA interactions by annealing biotinylated BC1 RNA with total RNA from mouse brain and by subsequently amplifying RNAs that were recovered from streptavidin beads (17).…”

“…Various RNAs are captured and amplified in this procedure because of (i) lack of specificity of the procedure per se and (ii) the selection of U-rich RNAs by an A-rich segment in BC1 RNA. The model proposed by Zalfa and colleagues (16,17) specifically predicted that BC1 RNA is required as an adaptor to link FMRP with its target mRNAs. If so, FMRP should be unable to interact with such target mRNAs in the absence of BC1 RNA.…”

“…Consensus has developed over recent years that FMRP is, like BC1 RNA, a translational repressor that is active in postsynaptic microdomains (7, 9, 10). However, in contrast to BC1 RNA, FMRP is associated with polysomes (11-15), indicating that BC1 RNA and FMRP operate at different levels in the translation pathway.In an alternative model, it has been proposed that BC1 RNA and FMRP interact directly with each other (16,17). In this scenario, BC1 RNA (i) physically binds to FMRP, (ii) directly interacts, by base-pairing of its 5Ј domain, with select mRNAs that are FMRP targets, and (iii) acts as a bridge between FMRP and such target mRNAs, thus serving as a requisite adaptor (16, 17).…”

“…In this scenario, BC1 RNA (i) physically binds to FMRP, (ii) directly interacts, by base-pairing of its 5Ј domain, with select mRNAs that are FMRP targets, and (iii) acts as a bridge between FMRP and such target mRNAs, thus serving as a requisite adaptor (16,17).…”