University of Texas at Austin
School of Biological Sciences The Section of Molecular Cell and Developmental Biology The Institute for Cellular and Molecular Biology School of Biological Sciences / The Section of Molecular Cell and Developmental Biology / The Institute for Cellular and Molecular Biology
Research

Current Research Projects in the Lab:
Mechanisms of mRNA metabolism in cellular morphogenesis.

Diminished Fragile X mental retardation protein (FMRP) activity in humans causes Fragile X Syndrome (FXS), the most common form of heritable mental retardation (O'Donnell and Warren, 2002). Although FMRP has been shown to be an RNA-binding protein that represses mRNA translation in vitro and in vivo (Laggerbauer et al., 2001; Li et al., 2001), its precise mechanism of action is unknown. In fact, some evidence suggests that FMRP might also function as a translational activator of specific transcripts in vivo (Brown et al., 2001; Miyashiro et al., 2003; Monzo et al., 2006; Todd et al., 2003).
Figure 5.

Figure 5. Misexpression of TRAL protein is observed in fixed cellularizing dfmr1 embryos by immunofluorescence. Scale bar represents 10 μm. From Monzo, et al., 2006.

The cognitive symptoms of FXS are thought to stem from the aberrant translation of a large number of specific mRNAs in the post-synaptic dendrites of brain neurons, leading to a deterioration of dendrite morphology and function that interferes with synaptic transmission and plasticity (Grossman et al., 2006; O'Donnell and Warren, 2002; Ule and Darnell, 2006). An important step towards developing effective treatments for FXS is to identify in vivo mRNA targets of FMRP in order to understand how it affects neuronal cell morphology and function. To date few definitive in vivo targets have been identified.

We identified a previously unknown requirement for dFMRP, the single Drosophila ortholog of human FMRP, in cleavage furrow formation and have demonstrated that it functions within cytoplasmic ribonucleoprotein (RNP) bodies during the MZT (Monzo et al., 2006). Through a combination of biochemistry and immunofluorescence analysis we have learned that dFMRP colocalizes with the cytoplasmic RNP body components Trailer Hitch (TRAL)(Wilhelm et al., 2005) and Maternal Expression at 31B (Nakamura et al., 2001) in cleavage stage embryos, but it does not complex with either protein. Rather dFMRP associates with tral mRNA and is required for the expression and localization of TRAL protein (Figure 5)(Monzo et al., 2006).

This discovery is significant because tral mRNA is the first in vivo FMRP target identified that encodes another translation factor, raising the possibility that the cognitive symptoms of FXS could involve FMRP-dependent translational regulation of direct and indirect FMRP target mRNAs. Based on these observations we have initiated quantitative proteomic screens to identify in vivo mRNA targets of dFMRP-dependent translational regulation (Figure 6) and protein binding partners of dFMRP required for its function. These studies should provide valuable insights into the etiology of FXS and the molecular and cellular mechanisms of synaptic plasticity and cleavage furrow formation.

Figure 6.Figure 6.
 
Figure 6. (click for details)
Figure 6. A quantitative proteomics screen using two-dimensional difference in gel eletrophoresis (2D-DIGE) has revealed about 40 proteins that are differentially expressed between WT and dfmr1 cleavage stage embryos. A single-channel fluorescent image of a representative 2D gel shows the array of protein spots observed in dfmr1 embryo extracts (left). Red boxes highlight specific regions of the 2D gel containing protein spots that show four types (I, II, III, and IV) of expression differences between WT and dfmr1 embryos. Boxes are magnified to the right and show the different protein expression profiles observed between WT and dfmr1 embryo extracts in the single 2D gel (arrows). Each protein spot indicated has been identified by mass spectrometry in collaboration with Dr. Jonathan Minden’s lab at Carnegie Mellon University.
 

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