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The Skaggs Institute
for Chemical Biology


Scientific Report 2008




Fundamental Processes in Neural Development

G.M. Edelman, A. Atkins, D.C.Y. Koh, D. Matsuda, 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 cold-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.

Julie Pilotte has observed that RBM3 is highly expressed in proliferative zones and plastic regions of the brain in rats and is developmentally regulated. Within mature neurons, RBM3 is present in nuclei and in dendrites. Dr. Pilotte's studies in neurons and other cells have shown that RBM3 localizes to leading edges of processes and to structures containing translation machinery that presage the formation of mature adhesion plaques. Functional studies indicate that RBM3 has a profound effect on cell motility and morphology, an effect that appears to extend to neurite outgrowth. These effects of RBM3 likely involve the potent regulatory influence of RBM3 on mRNA translation. Our previous work established that RBM3 greatly enhances mRNA translation rates, potentially by altering the production of microRNAs. Dr. Pilotte has conducted a comprehensive analysis of the relationship between RBM3 expression levels and the production of microRNAs. The data suggest several possible mechanisms underlying the effects of RBM3 on cell morphology and motility that involve microRNAs that target components of the translation machinery and cytoskeleton. These studies are ongoing. Overall, RBM3 appears to have a variety of functions critical for cell morphology, migration, and maturation that may be involved in the brain during neural development and in plasticity in adults.

In the cytoplasm, RBM3 plays important roles in regulating mRNA transport and translation. In many cells, however, the protein is more strongly expressed in the nucleus, where its function is just beginning to be defined. Annette Atkins used immunoprecipitation of RBM3 from nuclear extracts and proteomic analyses in collaboration with L. Liao and J.R. Yates, Scripps Research, to show that the protein colocalizes with a subset of proteins from the splicing machinery. Her further studies have shown that the protein can influence the splicing of specific mRNAs. Moreover, she obtained convincing evidence that RBM3 regulates its own expression by splicing an exon with a premature termination codon from its message, preventing degradation of the mRNA via a process known as nonsense-mediated decay. These results add an important new dimension to the role of RBM3 and related RNA-binding proteins in regulating protein synthesis at multiple levels.

Vincent Mauro and his colleagues have been studying basic mechanisms of translation initiation in eukaryotes. Earlier studies by this group showed that a 9-nucleotide segment from the 5′ leader of the Gtx homeodomain mRNA facilitated translation initiation by base pairing to 18S rRNA, the RNA component of 40S ribosomal subunits. Although the Gtx element was tested in isolation in earlier studies, the results indicated that eukaryotic mRNAs could initiate translation much as the Shine-Dalgarno interaction does in bacteria. Studies by Panagiotis Panopoulos have now shown the physiologic relevance of this element in the context of 2 natural mRNAs that contain this sequence in their 5′ leaders: Gtx itself and fibroblast growth factor 2. For these studies, he used modified RNA oligonucleotides to block mRNA-rRNA base pairing by targeting complementary sequences in either the 18S rRNA or mRNAs and by mutating the Gtx element in the context of the natural mRNA sequences.

Dora Koh has investigated the translation of the β-site amyloid precursor protein–cleaving enzyme 1 (BACE1) mRNA. The increased translation of this mRNA has been implicated in the etiology of Alzheimer's disease. Her studies resolved an apparent discrepancy between various published studies by showing that the various results could be explained by the use of different expression systems and differences in interpretation. She showed that the translation of the BACE1 mRNA was affected by the expression system and that it occurred by a ribosomal shunting mechanism when the mRNA was expressed in the nucleus via RNA polymerase II. In other studies, she has probed the RNA conformation of 2 endogenous RNAs in living cells: RNase P, which was probed as a proof of concept, and BACE1. These studies revealed a strong correlation between nucleotide accessibility and the site of translation initiation in the BACE1 mRNA, supporting the tethering/clustering model of translation initiation that was previously postulated by Dr. Mauro's group. Daiki Matsuda is using synthetic mRNAs with multiple potential initiation codons and various obstacles designed to block individual codons to further investigate the notion that the accessibility of the initiation codon is a key factor affecting its use.

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

 

Gerald M. Edelman, M.D., Ph.D.
Professor
Chairman, Department of Neurobiology

Department of Neurobiology