The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Our goal is to gain insight into the molecular basis of learning and memory by using the fragile X mental retardation protein, FMRP, as a model system. Fragile X syndrome is one of the most common forms of inherited mental retardation, with an incidence of 1 in 4000 live male births. Patients with fragile X syndrome are unable to express the protein FMRP (fragile X mental retardation protein), which is one of a growing number of proteins involved in normal cognitive function.

Although much is known about FMRP—it is an RNA binding protein that shuttles into and out of the nucleus and associates with actively translating polyribosomes—it is still unclear how all of these functions are regulated. Our goal is to understand how the activities of FMRP are modulated, both at the protein and RNA level, and how genetic alterations in this process may lead to instructive phenotypes.

Our work addresses the following questions:

1. How do post-translational modifications modulate FMRP function and what are the modifying enzymes?
2. How does FMRP associate with and transport mRNAs?
3. How is expression of the FMR1 mRNA regulated?

How do post-translational modifications modulate FMRP function and what are the modifying enzymes? Phosphorylation of a highly conserved serine modulates the translational state of FMRP. Unphosphorylated FMRP is associated with actively translating polysomes while phosphorylated FMRP is associated with stalled polysomes, suggesting that phosphorylation is the trigger for regulating expression of associated mRNAs. The key question now is how is this phosphorylation regulated? We will begin by identifying the kinase(s) and the phosphatase(s) that use FMRP as a substrate and will determine how they are regulated. We are also developing an antibody that is specific for the FMR-phosphoserine to examine in which tissues and under what conditions phosphorylation occurs. FMRP is also methylated on the RGG box. We will examine how this post-translational modification modulates RNA binding.

How does FMRP associate with and transport mRNAs? FMRP has a nuclear localization signal (NLS) yet it is unclear how FMRP enters the nucleus and what function it might be performing there. We will examine how FMRP enters the nucleus by performing yeast-2-hybrid to identify the gene product(s) that interact with the FMRP NLS. We will also use a fluorescently-tagged FMRP to characterize both the trafficking and the association of FMRP with RNAs in the nucleus (versus the cytoplasm). Finally, we will examine the requirements for export of the FMRP-mRNA complex from the nucleus.

How is expression of the FMR1 mRNA regulated? Our hypothesis is that elements of the 3'UTR control expression of the FMR1 mRNA. To identify regulatory elements within the mRNA, we are performing deletion mutagenesis on a construct of the 3'UTR attached to a reporter gene and are analyzing the effect on expression. We will also identify proteins that bind to and modulate expression of the FMR1 mRNA. The absence of any of these proteins could result in the mis-regulation of the FMR1 mRNA and perhaps a form of mental retardation in individuals lacking those proteins. Patients with fragile X-like features but with a normal FMR1 gene will be excellent candidates for the absence of these proteins and will be examined for such a loss. We will also directly assess the impact of loss of expression of each of these proteins by generating knockout mice.