Dynamic visualization of polyribosomes

Project Summary/Abstract Fragile X syndrome (FXS) is the leading single-gene cause of autism and the most common form of inherited intellectual disability. Although we understand the genetic mechanism of fragile X mental retardation (FMR1) gene silencing and the loss of function mutations, what is not clear is the molecular function of fragile X mental retardation protein (FMRP). There is biochemical and genetic evidence that FMRP interacts with RNA and polysomes to regulate protein synthesis. However, these approaches are unable to detect the dynamic interactions between ribosomes and FMRP. Our plan is to characterize the polysome-FMRP association using imaging techniques. Light microscopy can provide high resolution map of when and where molecular interactions take place. We will first demonstrate fluorescence resonance energy transfer between ribosome subunits (riboFRET) by fluorescence lifetime imaging microscopy (FLIM). In addition to riboFRET from fluorescent proteins, we will utilize orthogonal self-labeling tags that can be conjugated to organic dyes. The enhanced brightness and photostability of synthetic dyes will improve FLIM-FRET measurements. Next, co-expression of FMRP can show which polysomes within dendrites are bound by FMRP and we allow us to test how synaptic stimulation can alter this interaction. We will perform single-particle tracking (SPT) to monitor individual ribosomal subunits and FMRP assembly or disassembly in response to synaptic activity. Innovations in self-labeling tags, organic dyes and FLIM will be combined to provide robust and high resolution FRET and SPT results in neurons. Our strategy intends to provide a comprehensive understanding of molecular interactions between ribosomes and FMRP.