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Scientific Report 2005


Protein-Protein and Protein-RNA Interactions in the Nervous System

B.A. Cunningham, A.R. Atkins, S.A. Aschrafi, P.W. Vanderklish, G.M. Edelman

The nervous system is by far the most complex organization in the body. Accordingly, intricate signals regulate the temporal and spatial development of this system, including signals generated by cell-surface molecules such as cell adhesion molecules and by intracellular protein-protein and protein-RNA interactions.

The neural cell adhesion molecule (N-CAM) is expressed early during development, and its expression pattern is developmentally regulated. N-CAM mediates cell-cell adhesion through homophilic interactions: N-CAM on one cell binds to N-CAM on an apposing cell. However, defining the mechanism of interaction between 2 cell-surface glycoproteins has been difficult. We used recombinant proteins in a bead-binding assay and transfected and primary cells to clarify the molecular mechanism of N-CAM homophilic binding.

We found that the entire extracellular region of N-CAM mediated bead aggregation; however, the N-terminal immunoglobulin (Ig) domains, Ig1 and Ig2, were essential. These findings were largely in accord with the results of aggregation experiments with transfected L cells or primary chick brain cells. Additionally, maximal binding depended on the integrity of the intramolecular domain-domain interactions throughout the extracellular region. We propose that these interactions maintain the relative orientation of each domain in an optimal configuration for binding.

Thus, it appears that reciprocal interactions between Ig1 and Ig2 are necessary and sufficient for N-CAM homophilic binding, but that maximal binding requires the quaternary structure of the extracellular region defined by intramolecular domain-domain interactions between all 5 Ig domains and the first fibronectin-like repeat.

Adaptation of cells as a consequence of both intracellular and extracellular cues involves changes in protein expression. Regulation of these changes can occur at many levels, including translational regulation. An example of such adaptation is the induction of selected proteins upon exposure of cells to mild hypothermia. One protein whose level in the cell is increased in response to cold shock is the small RNA-binding motif protein 3 (Rbm3). In conjunction with V. Mauro and
J. Dresios, Department of Neurobiology, we found that the level of expression of Rbm3 affects overall translation in cells. In stably overexpressing neuronal cell lines, the level of translation increased 3-fold. In addition, overexpression of Rbm3 correlated with a reduction in the levels of microRNAs. Furthermore, we discovered a tight association between Rbm3 and the 60S ribosomal subunit. These findings suggest that Rbm3 plays a significant role in regulating translation.

Currently, we are characterizing endogenously expressed Rbm3 in brain, primary neurons, and selected cell lines. Our results indicate that at least 2 isoforms of Rbm3 are expressed in mouse and that these forms arise from alternative splicing. Rbm3 is found in both the nucleus and the cytoplasm of cells; the precise distribution depends on both the Rbm3 isoform and the specific cell type. This subcellular distribution is consistent with a role of Rbm3 in mRNA biogenesis. In addition, we found that Rbm3 undergoes extensive posttranslational modifications. Under basal conditions, a proportion of the protein is phosphorylated. We are identifying the additional modifications observed under basal and stimulated conditions; our goal is to determine how such modifications functionally affect Rbm3.

Additional evidence supporting a role for Rbm3 in regulating translation has come from observation of Rbm3 in granules. Granules are protein-RNA complexes that function as vehicles for the transport of selected mRNAs to designated locations within a cell. Granules are thought to be translationally silent and as such provide the machinery whereby protein expression can be restricted to specific cellular locations, such as synapses. The recent identification of many of the protein and mRNA components of granules has provided insight into the assembly and transport of these complexes. We also established that Rbm3 binds to specific mRNAs in primary neurons. This interaction is a direct association between Rbm3 and the RNA, because we were able to show binding between purified recombinant protein and isolated brain mRNA. Currently, we are trying to establish whether the association of Rbm3 with granules is due to protein-protein interactions, protein-RNA interactions, or a combination of both. Our aim is to provide additional insight into the many levels of translational regulation through fundamental studies on the functions of Rbm3.


Bruce A. Cunningham, Ph.D.