Studies have shown that the loss of fragile X mental retardation protein (FMRP) causes a mental retardation condition known as fragile X syndrome. The protein contains two RNA binding domains known as K homology domains (KH domains). The specific function and target of these RNA binding domains are not known nor is it known why the loss of FMRP leads to mental retardation. Therefore to understand the molecular function of the FMRP, one needs to first study these domains. It is the aim of this proposal to identify RNA targets and RNA binding characteristics of the first KH domain in FMRP. The SELEX procedure (Systematic Evolution of Ligands by Exponential Enrichment) will be used to identify RNA targets. SELEX is an in vitro selection procedure that identifies high affinity nucleic acid ligands for a given protein. The sequences of the ligands obtained from SELEX are not necessary that of the in vivo target but they can be used as templates in searching genome sequences for possible targets. Once a high affinity target is identified, phage display will be used to investigate the binding of the KH domain to the target. By displaying mutant proteins on the coat of the phage, phage display enables efficient screening and selection of a large pool of mutants. The purpose of the mutation analysis is to identify the residues that participate in RNA binding. Identifying potential RNA targets and regions of the KH domain responsible for RNA binding may help to elucidate the physiological function of the FMR protein.
