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Faculty
 Brenton
R. Graveley
Associate Professor of Genetics and Developmental Biology
graveley@neuron.uchc.edu
Areas of Interest:
My laboratory studies a wide variety of experiments that can be grouped
into five broad areas:
Alternative Splicing Regulatory Networks in Drosophila
- Alternative Splicing in Human Embryonic Stem Cells
- Alternative Splicing of the Drosophila Dscam Pre-mRNA
- The Drosophila modENCODE Project
- The Role of microRNAs in Planarian Regeneration
Alternative Splicing Regulatory Networks
In eukaryotes, genes are organized into segments referred to as exons
and introns. When genes are transcribed into messenger RNA (mRNA), the
exons are joined together and the introns are spliced out. The majority
of eukaryotic genes contain multiple exons and introns. In such cases,
the exons can be joined together in different patterns in a process
called alternative splicing to generate multiple mRNAs from a single
gene, each of which can encode a protein with a distinct function.
Alternative splicing is a common means by which eukaryotes regulate gene
expression and is the primary means of enhancing the diversity of
proteins encoded in the genome. In addition, defects in alternative
splicing can result in the onset of many human diseases including
cancer, Alzheimer’s disease, and myotonic dystrophy. Thus, understanding
the mechanisms by which alternative splicing is regulated is of
tremendous importance to human health.
My laboratory is interested in all aspects of the regulation of
alternative splicing in Drosophila melanogaster. Drosophila is an
excellent system in which to study alternative splicing due to the
ability to use cutting-edge genetic, biochemical, tissue culture, and
genomic technologies. In addition, the majority of the genes involved in
the regulation of alternative splicing are shared between humans and
flies. Thus, the principals we learn by studying this process in
Drosophila are directly applicable to the regulation of alternative
splicing in humans.
Alternative Splicing in Human Embryonic Stem Cells
Cells and organisms function based on the expression patterns,
actions, and interactions of thousands of genes and their products. A
tremendous amount of work has gone into dissecting the transcriptional
regulatory and protein interaction networks that drive cell function.
However, one important aspect of gene regulation is often overlooked in
these studies - alternative splicing. Alternative splicing is the
process by which exons can be joined together in different patterns to
generate multiple mRNAs from a single gene. Alternative splicing is a
tremendously important mechanism by which eukaryotes regulate gene
expression and is the primary means of enhancing the diversity of
proteins encoded by the genome. It is currently estimated that as many
as 75% of human genes encode pre-mRNAs that are alternative spliced to
generate multiple mRNAs, each of which can potentially encode a protein
with a distinct function. Thus, just like transcription regulation,
alternative splicing can function as a developmental switch.
A number of microarray studies have been conducted to identify a set of
“stemness” genes - genes that are expressed in and define the core gene
regulatory program for all types of stem cells. While these studies have
led to the identification of a number of genes that play important roles
in controlling various aspects of stem cell biology, they have not
examined alternative splicing in stem cells. Thus, a crucial aspect of
the gene regulatory programs of all types of human stem cells have been
overlooked.
The goal of our work is to fill this gap by performing expression
profiling experiments of hES cells in their undifferentiated state and
as they differentiate down different lineage pathways using microarrays
that can monitor alternative splicing and to elucidate the role of
specific RNA binding proteins in controlling alternative splicing in hES
cells. These experiments should lead to a more complete understanding of
the gene expression programs of hES cells which is critical to our
ability to guide stem cells down different lineage pathways and to
realizing the full therapeutic potential of hES cells.
Dscam Alternative Splicing
A major project in the laboratory is to determine the mechanisms
involved in controlling alternative splicing of the Drosophila Down
Syndrome Cell Adhesion Molecule (Dscam) gene. The Dscam gene, which was
discovered in Larry Zipursky’s lab, encodes an axon guidance receptor
that is most similar to one of the human genes implicated in causing
Down Syndrome. Perhaps the most interesting aspect of this gene is that
it is the most extensively alternatively spliced gene that we know of in
any organism. This single gene can generate over 38,000 different
isoforms by virtue of extensive alternative splicing. In fact, the
number of proteins generated by this gene is two to three times the
number of genes in the entire Drosophila genome! It is thought that the
diversity of Dscam isoforms contributes to the specificity of neuronal
wiring. We have found that the alternative splicing of Dscam transcripts
is regulated throughout development and in a tissue-specific manner.
Moreover, this regulated alternative splicing is evolutionarily
conserved. We are now using RNAi, genetics, evolutionary, and
biochemical approaches to identify trans-acting factors and cis-acting
RNA sequences that participate in controlling this extraordinarily
complex alternative splicing event.
By performing an RNAi screen in which we depleted hundreds of RNA
binding proteins in the Drosophila genome, we have identified about 40
different proteins that regulate the splicing of various Dscam exons. We
have also identified several splicing regulatory elements that are
required for the inclusion of any alternative exons and we are currently
working to identify the mechanisms by which these elements act. In
addition, we are using a variety of techniques to determine the role of
the different Dscam isoforms in specifying axon guidance in the fly.
Although there are differences in the properties of the human and
Drosophila Dscam proteins, these studies may lead to insights regarding
the role of this gene in the development of Down syndrome in humans.
The Drosophila modENCODE project
The goal of this project is to generate a comprehensive list of all
the sequence-based functional elements in the Drosophila genome. This
will be done by generating a set of developmentally staged and tissue-
and cell-specific RNAs for expression profiling using high-density
genome tiling microarrays and 454 pyrosequencing of small RNAs. These
expression data will be used for sophisticated transcript modeling that
integrates extant EST and cDNA sequence and comparative data from the 12
sequenced Drosophila genomes. These gene models will be experimentally
validated and functionally analyzed in RNAi assays. The final product of
these efforts will include comprehensive annotations of transcription
start sites, exon/intron structures, polyadenylation sites and the cis-elements
required for splicing.
The role of microRNAs in Planarian Regeneration
Planarians are free-living flat worms that are best known for their
regenerative capacity. For example, after surgical bisection of the
animal in either the vertical or horizontal plane each half of the
animal will regenerate the missing structures. In fact, a fragment as
small as 1/279th the size of the original individual has the capacity to
regenerate into an entire animal. The key to the amazing regenerative
prowess of these creatures is a population of cells known as neoblasts
that are distributed throughout the body of the animal. Neoblasts are
totipotent cells that are the only dividing cells in the animal. The
division progeny of neoblasts replace cells lost during normal cellular
turnover in the animal. After injury, however, neoblasts migrate to the
wound site, divide, and their progeny eventually replace the missing
structures. Thus, neoblasts are the planarian equivalent of stem cells
making these unique organisms an excellent model system for studying
stem cell biology. We have recently identified over 70 microRNAs - small
RNA molecules that regulate gene expression post-transcriptionally -
from the planarian Schmidtea mediterranea. We are currently working to
determine the expression patterns of each miRNA, identify mRNA targets
that are regulated by each miRNA, and to identify the biological
functions of each miRNA. It is likely that understanding how
regeneration in planarians is regulated will provide insight into the
biology of stem cells in other organisms, including humans.
Lab Rotation Projects:
A wide variety of potential rotation projects in the general areas of
post-transcriptional gene regulation (i.e., the RNA world) are available
to highly motivated students. Please contact me to discuss potential
projects or even better, to suggest a project of your own.
Selected Publications:
Graveley, B.R. 2008. The Haplo-Spliceo-Transcriptome: Splicing
Variations in the Human Population. Trends in Genetics, in press.
Olson, S. Blanchette, M., Park, J.W., Savva, Y., Yeo, G.W., Yeakley,
J.M., Rio, D.C. and Graveley, B.R. 2007. A Regulator of Dscam Mutually
Exclusive Splicing Fidelity. Nature Structural and Molecular Biology, in
press.
“Alternative Splicing in the Postgenomic Era” 2007. Edited by
Blencowe, B.J. and Graveley, B.R. Landes Bioscience, in press.
Park, J.W., and Graveley, B.R. 2007. Complex Alternative Splicing. in
“Alternative Splicing in the Postgenomic Era”, ed. B.J. Blencowe and B.R.
Graveley. in press.
Yang, L., Park, J.W., and Graveley, B.R. 2007. Splicing from the
Outside In. Molecular Cell 27:861-862.
Palakodeti, D., Smielewska, M., and Graveley, B.R. 2006. MicroRNAs
from the Planarian Schmidtea mediterranea: A model system for stem cell
biology. RNA, 12:1640-1649. Epub 2006 Jul 18.
Black, D.L. and B.R. Graveley. 2006. Splicing Bioinformatics to
Biology. Genome Biology, 7:317-320.
Park, J.W. and B.R. Graveley. 2005. The Use of RNA Interference to
Dissect the Roles of trans-Acting Factors in Alternative Pre-mRNA
Splicing, Methods, 37:341-344.
Graveley, B.R. 2005. Coordinated control of splicing and translation.
Nature Structural & Molecular Biology, 12:1022-1023.
Kreahling, J.M. and B.R. Graveley. 2005. The iStem: A long-range RNA
secondary structure element required for efficient exon inclusion in the
Drosophila Dscam pre-mRNA. Molecular and Cellular Biology,
25:10251-10260.
Graveley, B.R. 2005. Mutually Exclusive Splicing of the Insect Dscam
Pre-mRNA Directed by Competing Intronic RNA Secondary Structures. Cell,
123:65-73. See accompanying Preview article by Chris Smith (Cell,
123:1-7).
Kreahling, J.M., B.J. Lam, K.J. Hertel, and B.R. Graveley 2005. The
female-specific 5' splice site of the Drosophila fruitless pre-mRNA
is silenced by a splicing repressor recognized by PSI. Submitted.
Graveley, B.R. 2005. Alternative Splicing: One Gene, many products.
in "The Implicit Genome". Edited by Lynn Caporale. Oxford University
Press. In press.
Celotto, A.M., J.W. Lee, and B.R. Graveley 2005. Exon-specific RNA
Interference: A tool to determine the functional relevance of
proteins encoded by alternatively spliced mRNAs. Methods in Molecular
Biology 309:273-282.
Hertel, K.J. and B.R. Graveley. 2005. RS domains contact the pre-mRNA
throughout spliceosome assembly. Trends in Biochemical Sciences
30:115-118.
Graveley, B.R. 2005. Small molecule control of pre-mRNA splicing.
RNA, 11:355-358.
Philipps, D.L., J.W. Park, and B.R.Graveley. 2004. A computational
and experimental approach towards a priori identification of
alternative exons. RNA, 10:1838-1844.
Park, J. K. Parisky, A.M. Celotto, R. Reenan, and B.R. Graveley.
2004. Identification of Alternative Splicing Regulators by RNA
Interfernce in Drosophila. PNAS, 101:15974-15979.
Graveley, B.R., A. Karu, D. Gunning, S.L. Zipursky, L. Rowen, and J.
C. Clemens. 2004. The Organization and Evolution of the Dipteran and
Hymenopteran Dscam genes. RNA, 10:1499-1506.
Graveley, B.R. 2004. A protein interaction domain that contacts RNA
in the pre-spliceosome. Molecular Cell, 13:302-304.
Celotto, A.M. and B.R. Graveley. 2004. Using Single-Strand
Conformational Polymorphism Gel Electrophoresis to Analyze Mutually
Exclusive Alternative Splicing. Methods in Molecular Biology 257:65-73.
Celotto, A.M. and B.R. Graveley. 2004. RNA Interference of mRNA
Processing Factors in Drosophila S2 Cells. Methods in Molecular
Biology 257:245-254.
Kreahling, J. and B.R. Graveley. 2004. The origins and implications
of Aluternative splicing. Trends in Genetics, 20:1-4.
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