The RNA-binding protein FMR1 has a key role in the neurodevelopmental disorder fragile X syndrome, but the RNAs targeted by the protein were mostly unknown. An analysis reveals thousands of possible targets. See Article p.382
Fragile X syndrome was the first genetic disorder found to link RNA regulation to human cognitive function. Of the forms of hereditary autism or autism spectrum disorder associated with a single gene, this is the most common. It results in a range of intellectual disabilities, from mild to severe, and causes sufferers to exhibit certain physical characteristics. The syndrome is caused by an increase in the number of repeats of a short nucleotide sequence located in the X-chromosome gene Fragile X mental retardation 1 (FMR1), which encodes the RNA-binding protein FMRP. On page 382 of this issue, Ascano et al.1 report the discovery of RNA sequences known as RNA-recognition elements that are targeted by the two independent RNA-binding regions of FMRP — a finding that increases our understanding of the molecular basis of fragile X syndromeFootnote 1. The authors also describe the binding sites in messenger RNAs targeted by the FMRP family of proteins, and their use of a mutant FMRP to further study the protein's role in translation2.
Because FMRP is associated with polyribosomes (clusters of ribosomes, the molecular machines that synthesize proteins) and neuron-specific mRNA, it is thought to have an essential role in the post-transcriptional regulation of gene expression in neurons. Accordingly, FMRP and its associated mRNA targets have been studied intensively for more than a decade. Proteins of the FMRP family have been shown to shuttle between the cell's nucleus and the cytoplasm3, to activate translation4 and, in coordination with the Argonaute 2 protein, to associate with certain elements of tumour-necrosis factor α (a protein that regulates immune cells). In addition, FMRP acts as a translational silencer5, especially of mRNAs that encode proteins in synapses6 (the junctions between neurons). A 'kissing complex' structure in mRNA targets can interact with one of the binding domains in FMRP, and this may account for the protein's role in mRNA translation7. FMRP also has a role8 in preventing ribosomal translocation on mRNA.
Fragile-X research has the potential to be a model of translational neuroscience, but the syndrome cannot be properly understood until the mRNAs targeted by FMRP have been identified9. Until now, no specific RNA-recognition element had been determined for FMRP, and only a handful of the protein's mRNA targets had been confirmed. By using a combination of PAR-CLIP and RIP-Chip — two genomic methods for capturing mRNA–protein complexes and identifying the RNA in them10,11,12,13 — Ascano et al. have shed light on FMRP's target mRNAs and established a correlation between mRNA targets from different cell types. The authors identified hundreds of probable mRNA targets, many of which have previously been implicated in autism spectrum disorder.
Ascano and colleagues' computational analysis of their results revealed two main RNA-recognition elements that are bound by FMRP and found in most of the mRNAs. Further analysis also showed that FMRP regulates the production of two proteins: mTor (which forms part of a signalling pathway related to cell growth and proliferation) and Tsc2, both of which have a role in cancer progression.
Fragile X syndrome is largely considered to be a neurological disorder, but other organs, such as the ovaries, can also be affected; there has been less research into these non-neurological aspects, however. The authors therefore validated their PAR-CLIP and RIP-Chip results by examining the ovaries of mice that had been genetically manipulated to knock out the Fmr1 gene. They observed that the ovaries of these mice are larger and have higher levels of fragile-X-regulated proteins than the ovaries of normal mice. They also found that concentrations of proteins related to fragile X syndrome were higher in post-mortem brain samples from humans with the disorder than from those without it.
“The main strength of this study lies in its unbiased search of the miRNAs, and thus of the total cellular pool of proteins that they could have affected.”
Although Ascano and colleagues' study identifies many of the mRNA targets of FMRP, and points to their probable role in fragile X syndrome and autism spectrum disorder, it is unlikely to be the final word on the matter. Genomic studies of this kind are often criticized, partly because the raw data always suffer from signal-to-noise issues, but also because some researchers question the analytical methods used. Such studies also typically identify hundreds, if not thousands, of mRNAs — it will be a huge task to perform the in-depth analysis required to determine the effects of FMRP binding to each of these targets.
Another potential problem is that Ascano et al. performed their RNA-sequencing experiments in human embryonic cell lines, rather than in neuronal cells. Although there is some genetic overlap between these cell types, it will be important to determine the effects of varying FMRP levels in neurons, and this is technically difficult. The role, if any, of microRNAs — short RNAs that modulate a variety of biological functions — in the regulation of fragile-X signalling pathways is also unknown, because PAR-CLIP and RIP-Chip have a limited ability to identify microRNAs as protein-binding targets. Indeed, FMRP is known to associate with the RNA-induced silencing complex14,15,16 (a protein complex that uses a short RNA strand, such as a microRNA, to target complementary mRNAs for degradation), which implies that microRNA regulation and FMRP function are linked.
Ascano and co-workers' findings advance our understanding of the molecular basis of the defects that lead to fragile X syndrome and autism spectrum disorder. In so doing, they might open up avenues of research for drug discovery targeting these neurological disorders, for which there are presently no effective treatments. But it will probably also fan the flames of controversy surrounding the causes of these conditions.
*This article and the paper under discussion1 were published online on 12 December 2012.
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Jayaseelan, S., Tenenbaum, S. Signalling pathways of fragile X syndrome. Nature 492, 359–360 (2012). https://doi.org/10.1038/nature11764
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