The Skaggs Institute
for Chemical Biology

Scientific Report 2007

Fundamental Processes in Neural Development

G.M. Edelman, A. Atkins, S. Chappell, J. Dresios, O. Harismendy, K.N. Gonzalez, D.C.Y. Koh, P. Panopoulos, J. Pilotte

Scientists in the Department of Neurobiology focus on the features of primary cellular processes that regulate the development of the vertebrate nervous system. In the past year, the emphasis has been on factors that control the translation of mRNA into protein, including the specific regulation of local translation at synapses. Equally important have been studies of RNA-binding proteins such as the cell-induced RNA-binding motif protein 3 (RBM3) and its influence on the differentiation and function of neurons at the level of both transcription and translation.

A major effort in the department is to define the fundamental mechanisms of translation initiation in eukaryotes. Stephen Chappell and John Dresios have investigated the notion that ribosomal subunits do not scan from the site of recruitment to the initiation codon, but rather reach the initiation codon by mechanisms that involve tethering of ribosomal subunits to the mRNA or clustering of ribosomal subunits at binding sites in mRNAs. Earlier studies by this group indicated that one mechanism used by eukaryotic mRNAs to recruit ribosomal subunits involves Watson-Crick base pairing between complementary segments of mRNA and ribosomal RNA. Panagiotis Panopoulos is further investigating this base-pairing mechanism of ribosomal recruitment by assessing the extent to which several putative mRNA-binding sites in ribosomes affect the translation of natural mRNAs. In addition, Dora Koh has developed a new method to monitor nucleotide accessibility in natural mRNAs of living cells. She probed the RNase P RNA as a proof of concept and the β-secretase mRNA to test a key prediction of ribosomal tethering/clustering, which is that the accessibility of an AUG codon affects the use of the codon.

In other studies, we are defining the structure and function of a neuronal member of a family of mRNA-binding proteins that are upregulated during cold shock and other forms of stress. Annette Atkins and Julie Pillote have found that RBM3 is highly expressed throughout much of the brain in early development, particularly in the cerebellum and olfactory bulb. High expression in these regions, and in proliferative zones and differentiating cell fields, persists into adulthood. Subcellularly, RBM3 appears in both the cytoplasm and the nucleus, and evidence suggests an early postnatal shift between these compartments. In cytoplasm, RBM3 is found in granules that transport mRNA and other components of the translation machinery into dendrites, where the components can provide the basis for local translation of specific mRNAs in response to synaptic activity. The mRNA encoding RBM3 is also present in dendrites, suggesting that this mRNA may itself be locally translated. In the nucleus, RBM3 occurs in defined structures resembling speckles that contain elements of the RNA splicing machinery. Proteomic and other data indicate that RBM3 influences alternative splicing of its own and other mRNAs. Overall, RBM3 appears to have a variety of functions critical for neuronal development, maturation, and activity.

Vertebrate development depends on both temporal and spatial regulation of gene expression. Katie Gonzalez and Olivier Harismendy have examined 2 developmental regulators that are exemplars of the homeobox and zinc finger transcription factor families, respectively. Development of skeletal muscle is regulated by the coordinate activities of basic helix-loop-helix, MADS domain, and homeobox transcription factors. Dr. Gonzalez has shown that the Barx2 homeobox protein is important for muscle development in vivo. Barx2 is expressed in a muscle progenitor population called satellite cells that are required for muscle growth and repair. Recently, she established primary cultures of satellite cells from wild-type mice and from mice lacking the gene for Barx2 and showed that the cells from the mutant mice proliferate and differentiate more slowly. This result supports her earlier findings that mice lacking Barx2 have diminished muscle growth and repair. She is currently investigating the regulation of muscle-specific gene expression by Barx2 and the role of direct interactions between Barx2 and the muscle-expressed basic helix-loop-helix and MADS domain proteins known as myogenic differentiation protein and serum response factor.

Brain development involves the precise orchestration of a neural-specific gene expression program. A zinc finger transcription factor called the neural restrictive silencer factor (NRSF) restricts this genetic program to the nervous system by repressing neuron-specific genes in nonneuronal cells. To investigate the regulatory mechanisms used by NRSF, Dr. Harismendy is using a novel technology for genome-wide analysis of transcription factor binding sites that takes advantage of new advances in high-throughput multiparallel sequencing. In addition to identifying NRSF binding sites, the method generates genomic footprinting information. Analysis of these footprints revealed 2 different categories of NRSF target genes: those with extended genomic DNA footprints and those without. Currently, Dr. Harismendy is investigating the regulatory mechanisms that distinguish these different categories of targets.

All of these efforts are designed to elucidate the molecular and cellular events that define and regulate the development and function of the nervous system. Our focus on fundamental processes rather than on specific diseases is based on the belief that understanding these processes can provide the necessary framework for defining the mechanisms that underlie not just one but a variety of diseases. Indeed, our studies have already provided insights into aspects of several diseases, including Alzheimer's disease, breast cancer, and mental retardation syndromes.

Publications

Liao, L., Pilotte, J., Xu, T., Wong, C.C., Edelman, G.M., Vanderklish, P.W., Yates, J.R. III. BDNF induces widespread changes in synaptic protein content and up-regulates components of the translation machinery: an analysis using high-throughput proteomics. J. Proteome Res. 6:1059, 2007.

Mauro, V.P., Chappell, S.A., Dresios, J. Analysis of ribosomal shunting during translation initiation in eukaryotic mRNAs. Methods Enzymol. 429:323, 2007.

Mauro, V.P., Edelman, G.M. The ribosome filter redux. Cell Cycle 6:2246, 2007.

Smart, F., Aschrafi, A., Atkins, A., Owens, G.C., Pilotte, J., Cunningham, B.A., Vanderklish, P.W. Two isoforms of the cold-inducible mRNA-binding protein RBM3 localize to dendrites and promote translation. J. Neurochem. 101:1367, 2007.