Student Theses and Dissertations

Date of Award

2013

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Darnell Robert Laboratory

Abstract

The Fragile X Mental Retardation Protein (FMRP) is a neuronal RNA-binding protein that is predominantly associated with polyribosomes. Loss of FMRP results in Fragile X Syndrome, characterized by mental retardation, autism and epilepsy. FMRP was recently found to be associated with a specific set of mRNAs with key roles in neuronal function and to physically interact with targeted mRNAs along their entire coding sequences (Darnell 2011). Here, we find that FMRP inhibits translation on these target transcripts by stalling ribosomes, both in vivo and in rabbit reticulocyte lysate programmed with endogenous brain polyribosomes. In these systems, loss of FMRP function resulted in increased ribosome runoff after treatment with puromycin, a drug that acts specifically on translocating ribosomes. In addition to genetic loss-of-function models, FMRPdependent relief of ribosome stalling could be induced by acute biochemical removal of FMRP from polysomes, indicating reversibility of stalling. FMRP was also directly visualized on stalled polysomes by immunoelectron microscopy. Together, these results suggest a model in which FMRP actively regulates translation of target mRNAs by stalling ribosomes. To further advance our understanding of FMRP function, we have created the FMRP cTAG mouse, a knock-in model in which FMRP can be conditionally tagged with AcGFP in a Cre-dependent manner while maintaining wt FMRP expression in all Cre-negative cells. The cTAG mouse will be a valuable tool in the study of cell type-specific FMRP function.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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