Their studies, published in the April 15th issue of Genes & Development, provide a new direction for efforts to understand how the loss of FMRP function leads to the complex behavioral and cognitive defects characteristic of Fragile X syndrome.
While the importance of identifying a function for FMRP has been clear for some time, what this function actually is has continued to evade researchers. FMRP is a protein characterized by the presence of three RNA binding domains: two tandem KH-type RNA binding domains and an RGG box. Scientists have focused on the identification of FMRP RNA ligands in an effort to understand FMRP function. This effort is particularly meaningful since FMRP is believed to regulate mRNA translation in the brain, and identifying the mRNA targets of this regulation would be a huge step in understanding how loss of this protein results in the varied and complex phenotypes of Fragile X syndrome.
In most Fragile X patients, loss of FMRP is due to silencing of FMR1 resulting from the unusual amplification of a CGG repeat (over 200 copies in affected patients versus less than 60 copies in unaffected individuals) that leads to hypermethylation of FMR1 and shut down of transcription of the gene. However, Fragile X patients expressing mutations or deletions within the FMR1 gene have also been described, including a severely affected patient harboring a missense mutation that resulted in a one amino acid change, isoleucine at position 304 for asparagine, in one of the KH domains of FMRP, KH2.
Dr. Darnell and colleagues focused on understanding how this specific mutation leads to loss of FMRP function. They first screened an RNA library to identify what RNA motif is recognized by the KH2 domain. They found that the KH2 domain of FMRP recognizes a loop-loop pseudoknot, or "kissing complex" structure in the RNA, and that this recognition is abrogated by the isoleucine to asparagine mutation. Notably, they show that the association of FMRP with the translation machinery (in brain polyribosomes) can be competed out with kissing complex RNA, an important finding since previous biochemical studies have reported altered polyribosome distribution of mRNAs in Fragile X patients.
These findings will redirect the search for the RNA targets of FMRP whose misregulation is responsible for the disease, to those containing kissing complex motifs.
Though much remains to be understood in the biology leading to Fragile X syndrome and the function of FMRP, Dr. Darnell is confident that "these findings may provide a crucial link between the association of FMRP in brain polyribosomes, its proposed role in regulation mRNA translation, and neurologic dysfunction in the Fragile X syndrome".
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