About alternative splicing of FMR1 gene
It is estimated that almost 95% human genes would experience certain level of alternative splicing (AS) and contribute to proteome complexity[14]. AS can produce mRNAs that different in their untranslated regions or coding sequence. The mechanisms of AS mainly include exon skipping, intron intention, the use of alternative splice sites and the choice of mutually exclusive exons. The different spliced isoforms might influence mRNA localization, stability and translation. Moreover, some splicing mRNA variants could alter the reading frame and generate various protein isoforms with diverse localizations and functions. The most common place for AS is in the neural tissue, where various spliced transcripts may function as modulators to synaptic functions. Since the cloning of FMR1 gene as the disease gene of FXS in 1991, a large number of FMR1 mRNAs and FMRP isoforms derived from AS were detected in mouse and human[15]. The distribution of different FMRP isoforms in specific cellular roles and different tissues was also relatively well understood. It is reported that a high expression level of different FMR1 mRNAs was found in brain, testis, placenta and lymphocytes, while a lower level of expression in other organs[15]. Besides, there are at least 4 predicted FMRP isoforms identified in mouse brain, which demonstrates that the dissimilar isoforms of FMRP occur together in the same cell type or separately in distinct cell types[16]. At present, the 24 and more predicted mature transcripts were reported, mainly involving the inclusion or exclusion of exons 12 and 14, and the selection of splice acceptor sites at exons 15 and 17[17]. To our knowledge, our work represents the further study that a new cryptic exon from intron 9 of human FMR1 gene was identified.
About subcellular localization of FMRP
It is believed that the longest isoform of FMRP (Isoform 1) is predominantly cytoplasmic and mainly function as a mRNA-binding protein that can directly or indirectly interact with other proteins, regulating the stability of mRNA and maintaining the balance of shuttling between the cytoplasm and the nucleus. The nuclear localization signal (NLS) and nuclear export signal (NES) of FMRP were also associated with the cytoplasmic localization of FMRP. A patient with a novel R138Q mutation in the NLS had developmental delay[18]. The mechanism of this mutation is not very clear, but it may lead to the different distribution of proteins in cytoplasm and nucleus and may indicate the importance of the domain. Besides, the exon 14 of FMR1 gene were shown to encode a cytoplasmic retention domain. Exclusion of exon 14 altered the downstream reading frame, generated two different C-terminal regions, and finally showed a nuclear localization[7]. Furthermore, FMRP C-terminal is one of the determinant factors of nuclear localization and is the key domains that mediate the kinesin and dendrites transmission[19]. FMRP homologous proteins, FXR1 and FXR2, also shuttle between the nucleus and cytoplasm by producing multiple isoforms with different C-terminal[20]. Correspondingly, FMRP N-terminal is highly conserved in species[21]. Banerjee and his colleagues studied the functional difference of long isoforms and short isoforms FMRP of D. melanogaster, showing that the short isoforms, without the C-terminal region, can easily cause short-term or long-term learning and memories disorders[22].
FMRP can bind key protein cytoplasmic FMRP-interacting protein (CYFIP1), a downstream effector of Rac1 in the cytoplasm, remodeling the cytoskeleton and involving in the formation of the translational initiation complex[23]. However, Previous studies presented that FMRP binds its mRNA targets in the nucleus and facilitates the cargo of nuclear proteins, and the export of FMRP from the nucleus depended on mRNA synthesis. Kim and his colleagues knocked down the mRNA exporter Tap/NXF1, resulting in the increase of FMRP protein in nucleus[24]. It also has been proved that FMRP can combine proteins in nucleus, such as NUFIP1 and 82-FIP (FMR1 interacting protein 1), RISC (RNA-induced silencing complex), AGO2 and Dicer (argonaute 2), and eIF5 (eukaryotic translation initiation factor 5), which are the pivotal molecules that mediate translational repression by inhibiting the initiation of translation and causing polyribosomes stalling[25, 26]. Moreover, the combination process of FMRP and ribosomes was presented in the nucleus, being an important mechanism of translational regulation. FMRP has been regarded as chromatin-associated protein. It can coimmunoprecipitate with chromatin confirmed by Chip sequencing, as well as interacting with nucleolin, affecting the transcription of rRNA and the biosynthesis of ribosomes[27].
Due to the missing of nuclear export signal, our newly identified FMRP isoform with a short C-terminal retains in the nucleus. We speculate that the increasing of FMRP alternatively spliced isoforms in the nucleus could break the balance of shuttling between the nucleus and cytoplasm and then affect the translational repression of FMRP.
About new interactors of FMRP in the FXS-related signaling pathways
With the development of several high-throughput approaches, such as microarray analysis, HITS-CLIP and PAR-CLIP, researches revealed that FMRP can interact with about 5% mRNA targets in brain [4]. FMRP is a translational repressor involving in the regulation of synaptic functions via the activation of NMDA receptors, AMPA receptors and GABA receptors, which contribute to the formation of long-term depression (LTD) and long-term potentiation (LTP), according to mGluR theory[28]. It is widely believed that the regulation of neurological function mainly depends on the mGluR-LTD pathway, which mediates the synaptic plasticity and hinges on the local protein synthesis of dendrities. The mGluR theory of FXS emphasizes that FMRP is downstream of mGluRs and upstream of local protein synthesis. It has been suggested that FMRP represses the translation of dendritically localized mRNAs. With the activation of mGluR, FMRP repression would allow the synthesis of local protein in response to synaptic stimulation, resulting in the AMPAR internalization and LTD [28]. For patients with FXS, the absence of FMRP could constructively increase protein synthesis, leading to the over activation of AMPAR internalization and LTD exaggeration. Both of the extracellular signal–related kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways are required for the regulation of mGluR-LTD [29, 30]. Studies have showed that antagonizing the mGluR pathway can alleviate the phenotypes of FXS [31]. Therefore, mGluR theory provides new avenues for the understanding of pathological mechanisms and therapeutic intervention of FXS.
Interestingly, our findings of RNA microarray analysis revealed GABRB3 is the most significantly down-regulated gene and BEX1 the most significantly up-regulated gene. Altered expression of mRNA and protein for GABA receptors has been reported in FMR1 knockout mice, implying the loss of FMRP can affect GABA receptor subunits expression. Recent publications have also identified the absence of FMRP can cause to the upregulation of mGluR signaling, resulting in the lowered expression of GABRB3 protein, which was consistent with our RNA microarray results[32–34]. Therefore, we indicated that overexpression of truncated FMRP protein has profound effects on FMRP-mGluR-GABRB3 signaling pathway. Besides, it is tempting to speculate that BEX1 gene may participate in mGluR-LTD and mGluR-LTP signaling pathways. BEX1 gene was linked to neurotrophin signaling as a interactor of the Trk tyrosine kinases (TrkA, TrkB and TrkC) or p75 neurotrophin receptor (p75NTR), regulating differentiation, growth, and survival of neuronal and glial cells[35]. Trk receptors can be activated by several canonical pathways, including the phosphatidylinositol-3 kinase (PI3K)/AKT/mTOR and Ras/MAP kinase signaling pathways[36]. Additionally, TrkB can be involved in the LTP signaling pathways and mediate the synaptic plasticity[37]. However, the signaling mechanisms of p75NTR are still poorly understood. It has been reported p75NTR can change the functions of the amygdale[38] and contribute to multiple process of cell responses, such as apoptosis, survival, axonal growth and cell death. When BEX1 protein is overexpressed, it can inhibit the NF-κB activity by Trk receptors and p75NTR, without impacting activation of AKT and Erk1/2 signaling[35], which are critical molecules involved in mGluR-LTD signaling pathways. The high BEX1 level in the connection with the mGluR-LTP or mGluR-LTD signaling gives us a new insight into interactions of FMRP in the FXS-related signaling pathways.
In conclusion, our study identified a new cryptic exon from amid intron 9 of human FMR1 gene with widely expressed in normal healthy individuals. In particular, sequences similar to the new exon can be only found in the genomes of primates and the insertion of it can produce a truncated FMRP protein with altered cellular localization. Our preliminary data from RNA microarray analysis points to the possibility that BEX1 gene may be a new player in the FXS-related mGluR-LTP or mGluR-LTD signaling pathways, although the complicated molecular mechanisms of this new alternative exon-influenced roles of FMRP await for further clarification.