Fragile X syndrome – defects in a molecular braking system?
Fragile X is the most common cause of genetically inherited mental disorders – even more common than Down syndrome. Its symptoms range from mild learning disabilities to severe mental retardation and include autistic behaviors, seizures and physical abnormalities (The Fragile X Foundation). Although scientists knew that fragile X is due to mutations in the fragile X mental retardation 1 (FMR1) gene, which encodes the fragile X mental retardation protein (FMRP), FMRP's role was a mystery. That mystery is slowly being unraveled. A team of scientists led by Drs. Robert and Jennifer Darnell of the Rockefeller University, New York, recently reported that FMRP stalls translation, preventing the over-expression of proteins that appear to cause fragile X and other neurological disorders (Darnell et al. 2011). The team's research may lead to novel therapies for these disorders.
Before the Darnell team's findings, some broad mRNA translation-regulating mechanisms were known, but the specific proteins that regulate the translation of the transcripts involved in synaptic remodeling were not. Whatever those regulators were, their roles were likely pretty important because the synthesis of new neuronal proteins is critical to synaptic plasticity– a phenomenon thought to play a key role in forming and maintaining memory. Because FMRP is expressed in neuronal cells and binds to mRNA that is being translated on polyribosomes, it was a likely candidate. Moreover, exogenous FMRP had been shown to repress the translation of a variety of mRNA transcripts in vitro.
To better understand FMRP's functions, its RNA targets needed to be identified. Previous attempts to identify them had met with limited success. The Darnell team was undaunted. They developed an ultraviolet radiation-mediated technique called "crosslinking IP" (CLIP), which, when combined with high-throughput sequencing (HITS-CLIP), can identify specific mRNA-protein interactions. They used this and other biochemical assays to compare mRNA-FMRP interactions in the brains of two Fmr1-deficient mouse models – the Fmr1 knockout mouse FVB.129P2-Fmr1tm1Cgr/J (004624) and an I304N point mutant knockin mouse B6.129-Fmr1tm1Rbd/J (010504) – to mRNA-FMRP interactions in the brains of FVB/NJ (001800) and C57BL/6J (000664) wild-type littermate controls. Following are their key findings:
- In vivo, FMRP binds to at least 842 neuronal mRNA target transcripts.
- Those transcripts encode proteins from many gene families, most of which function in synaptic signaling.
- A large percentage of both pre- and post-synaptic proteins are targets of FMRP regulation.
- 28 of the 842 FMRP target transcripts are implicated in autism spectrum disorders.
- Whereas ribosomes are stalled in an FMRP-dependent manner in the brains of wild-type mice, they are not stalled in the brains of Fmr1- knockout mice.
- FMRP appears to stall translation by physically associating and forming large complexes with polyribosomes and target mRNA transcripts.
Significantly, the results obtained by the Darnell team are consistent in the two different fragile X mouse models – two different Fmr1 mutations on two different genetic backgrounds – indicating that genetic background effects did not influence their results. The mRNAs bound to FMRP indicated that it directly regulates translation of synaptic proteins and synaptic plasticity. The fact that many FMRP target transcripts are linked to autism spectrum disorders may explain why people with fragile X exhibit autistic behaviors. That FMRP binds to and stalls the translation of synaptic protein-encoding mRNA transcripts suggests that neurological disorders such as fragile X are caused by the over-expression of genes normally repressed by FMRP. This revelation may lead to the development of novel therapies for fragile X and related disorders.
"It is significant that we used two independent fragile X mouse models to validate our findings. In addition to the Fmr1 knockout, we used the I304N point mutant knockin mouse that we made (Zang et al. 2009) and bred into two genetic backgrounds (FVB and C57BL/6) to match the conventional Fmr1 KO. I think the use of both models adds significance to the findings, and I'd love to see more people use multiple models, especially given the occasional problems inherent in genetically engineered models. For example, the conventional Fmr1 KO still has the neo cassette, and there can be neighboring gene effects from genes originating from the 129 strain of ES cells still present near the engineered gene, even after multiple generations of backcrossing into C57 or FVB." -- Dr. Jennifer Darnell