The Skaggs Institute
for Chemical Biology
Scientific Report 2005
Fundamental Processes in Neural Development
G.M. Edelman, S. Aschrafi,
A. Atkins, S. Chappell, J. Dresios, O. Harismendy, D.C.Y. Koh, G.W. Rogers, T. Stevens, M.
Tsatmali, W. Zhou
to focus on primary cellular processes and their influence on the development of the vertebrate
nervous system. Much of this effort has emphasized the regulation of translation of mRNA into protein,
including basic mechanisms of translation and the specific regulation of translation at synapses
during synaptic plasticity. Equally important have been studies of the nature and differentiation
of neural stem cells and the basic factors that regulate gene transcription.
In examining the mRNAs expressed in response
to neuronal adhesion, Vince Mauro noted that many of them had sequences that matched or were complementary
to ribosomal RNA. He and his colleagues, Stephen Chappell, Wei Zhou, George Rogers, Jr., John Dresios,
and Dora Koh, are working on several mRNAs to characterize a mechanism of translation enhancement
that uses base pairing of mRNAs to ribosomal mRNA sequences. Dr. Zhou developed an important positive
feedback vector to identify translational enhancers. In collaboration with Drs. Zhou and Chappell,
Dr. Dresios used a related system in yeast to show definitively that base pairing was the underlying
mechanism by which a 9-nucelotide sequence from the mouse Gtx homeodomain mRNA enhanced translation.
This study was the first to confirm such a mechanism in eukaryotic translation, similar to the Shine-Dalgarno
sequence in prokaryotes.
Drs. Rogers and Koh investigated a mechanism
that regulates the translation efficiency of the BACE1 mRNA, which encodes the enzyme
enzyme is overexpressed in Alzheimers disease without a corresponding increase in BACE1
mRNA levels, indicating that the translation efficiency of this mRNA is increased. Earlier studies
suggested that the translation of this mRNA is regulated by factors that affect the accessibility
of the initiation codon. Dr. Koh is testing this notion by adapting a method that was used to probe
RNA secondary structures in bacteria.
Translation of dendritic mRNAs is involved
in many functions of the nervous system. For example, several forms of changes in efficacy occur
in a brain region called the hippocampus, and translation of dendritically localized mRNAs is
required for each change to persist beyond a few hours. Peter Vanderklish has been studying how
translation is regulated by receptor-mediated signaling that occurs during the induction of
changes in synaptic efficacy.
In collaborative studies, Bruce Cunningham,
Armaz Aschrafi, Dr. Dresios, Ann Atkins, and Dr. Vanderklish have examined 2 RNA-binding proteins
important in synaptic function: RNA-binding motif protein 3 (Rbm3) and the fragile X mental retardation
protein (FMRP). The protein Rbm3 is associated with a subset of large granules containing specific
proteins, mRNAs, and much of the translation machinery. Rbm3 binds ribosomes and alters microRNA
levels to enhance translation globally. FMRP also occurs in large mRNA granules, where it acts
as a translational suppressor. Loss of FMRP results in enhanced translation of granule mRNAs in
response to metabotropic glutamate receptor signaling.
Transcription of genes into messenger
RNAs is a highly regulated process that is essential for normal development and when altered can
result in disease states. Transcription requires several factors that maintain the DNA in an appropriate
conformation and activate the ability of DNA polymerase to synthesize mRNA. Robyn Meech and colleagues
are studying 2 such transcription factors: Barx2 and the neural restrictive silencer factor (NRSF).
Barx2, discovered in our laboratory, is
critical for the development of muscle and cartilage, and Tracy Stevens showed that it regulates
the expression of the gene for estrogen receptor α
in breast cancer cells. She showed that Barx2 regulates the expression of 2 estrogen receptor α
mRNAs that differentially affect gene expression and cell growth. In addition, overexpression
of Barx2 promoted anchorage-independent cell growth and induced cellular invasion. These mechanisms
coordinately regulate cell growth, invasion, and gene expression that are critical in normal
development of the mammary gland and in progression of breast cancers.
NRSF is a transcription factor that represses
the expression of neuron-specific genes in nonneuronal cells. Oliver Harismendy is developing
a novel chromatin immunoprecipitation-based technology for genome-wide analysis of transcription
factor binding sites. This method, serial analysis of genomic sites, should increase the efficiency
of the identification of active targets of transcription factors such as NRSF in various cells
and phases of development. When used in conjunction with bioinformatics, the method can provide
unique and essential information about the use of DNA-binding sites in particular contexts.
Understanding the factors that stimulate
the conversion of stem cells to neurons and that sustain neuronal survival are critical issues
for understanding neuronal development. Kathryn Crossin and her colleagues have shown that newborn
neurons have higher levels of reactive oxygen species (ROS) than their progenitors do, a surprising
finding because high levels of ROS are usually a reflection of dying cells. More recently, Marina
Tsatmali pursued the hypothesis that elevated ROS levels play an important role in neuronal differentiation.
She perturbed the expression of ROS and found that this manipulation dramatically alters neuronal
morphology, although not neuronal fate. She also examined the physiologic properties of neurons
in brain slices with high or low levels of ROS and showed that the cells with high levels of ROS have
normal physiologic properties in situ.
The aim of all of these activities is the
study of the molecular and cellular events that define and regulate the development of the nervous
system. We are focusing on fundamental processes rather than on specific diseases. This strategy
is based on the notion that understanding even a single primary process can provide the necessary
framework for defining the mechanisms that underlie not just one but a variety of diseases.
Aschrafi, A., Cunningham, B.A.,
Edelman, G.M, Vanderklish, P.W. The fragile X mental retardation
protein and group I metabotropic glutamate receptors regulate levels of mRNA granules in brain.
Proc. Natl. Acad. Sci. U. S. A. 102:2180, 2005.
Atkins, A.R., Gallin, W.J., Owens,
G.C., Edelman, G.M., Cunningham, B.A. Neural cell adhesion
molecule (N-CAM) homophilic binding mediated by the two N-terminal Ig domains is influenced by
intramolecular domain-domain interactions. J. Biol. Chem. 279:49633, 2004.
Dresios, J., Aschrafi, A., Owens,
G.C., Vanderklish, P.W., Edelman, G.M., Mauro, V.P. Cold
stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances
global protein synthesis. Proc. Natl. Acad. Sci. U. S. A. 102:1865, 2005.
Meech, R., Edelman, D.B., Jones,
F.S., Makarenkova, H.P. The homeobox transcription factor
Barx2 regulates chondrogenesis during limb development. Development 132:2135, 2005.
Tsatmali, M., Walcott, E.C., Crossin,
K.L. Newborn neurons acquire high levels of reactive oxygen
species and increased mitochondrial proteins upon differentiation from progenitors. Brain
Res. 1040:137, 2005.
Vanderklish, P.W., Edelman, G.M.
Differential translation and fragile X syndrome. Genes Brain Behav. 4:360, 2005.
Zhou, W., Edelman, G.M., Mauro,
V.P. A positive feedback vector for identification of nucleotide
sequences that enhance translation. Proc. Natl. Acad. Sci. U. S. A. 102:6273, 2005.