Defects in the function of mRNA-binding proteins underlie a broad spectrum of human pathologies. Fragile Mental Retardation Protein (FMRP) is one of these mRNA-binding proteins, involved in transport, storage and control of mRNA;changes in its biosynthesis cause several developmental and cognitive deficiencies including premature ovarian failure and fragile X syndrome (FXS). FXS is the most common inherited form of intellectual disability and a predominant monogenic cause of autism. The syndrome is associated with transcriptional silencing of the FMRP gene or a mutation in an RNA-binding domain of FMRP. On the molecular level, FXS is likely caused by dysregulation of FMRP-mediated translational control in neurons leading to altered synaptic function and other abnormalities. Despite identification of many FMRP-associated mRNAs by various approaches, the basis for mRNA recognition by FMRP is still not understood. The objective of this proposal is to restore the pilot research project focused on elucidating the principles of mRNA recognition by FMRP. The hypothesis is that FMRP recognizes mRNA targets that bear both sequence and structure-specific binding determinants. To test this hypothesis, it is proposed to identify FMRP binding sites using novel methodology and characterize FMRP-RNA interactions biochemically and biophysically.
Specific Aim 1 is devoted to development of genome-wide RNA foot printing that implements conformation-specific nucleases with complementary specificities to identify FMRP-binding sites and probe their conformation. The mRNA structure will be tested in vitro by nuclease cleavage of total mouse mRNA in the absence and presence of FMRP. The cleaved mRNA fragments will be converted to cDNA and sequenced. FMRP binding sites will be located as losses of nuclease cleavages, and mRNA conformation will be assessed based on specificity of cleavages. These experiments will identify bona fide mRNA binding sites for FMRP and their structural environment.
Specific Aim 2 will characterize mRNA binding sites for FMRP biochemically and structurally. Representative mRNAs will be tested for binding to various domains of FMRP. Identified RNA-protein complexes will be co-crystallized and their structures determined by X-ray crystallography. Together, results will define structural and sequence elements required for interactions with FMRP. Complemented by in silico characterization of the sites, these data will provide 'three-dimensional'RNA binding signatures for FMRP. Thus the proposed study will define the principles of mRNA-FMRP recognition and provide insights on the mechanism of FMRP-dependent translational inhibition. The proposal is relevant to public health since it addresses the molecular basis of the most common cause of mental retardation and related disorders. Understanding FMRP function will advance searches for novel therapeutic interventions against this incurable disease. Thus, the proposed research is relevant to the NIH mission to expand the knowledge base in medical sciences to ensure capability to prevent and cure diseases.
Fragile X syndrome is one of the leading causes of inherited mental retardation and autism that develop in the absence of functional Fragile Mental Retardation Protein (FMRP). Through this Sandy-related RFA, we propose to restore our pilot project identifying and characterizing the interactions between FMRP and its molecular targets using novel genome-wide methodology, X-ray crystallography, and biochemical approaches. These studies will provide insights on the molecular mechanisms of FMRP function and advance searches for novel therapeutic interventions against the currently incurable Fragile X syndrome and related disorders.