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
Lee, C.*, Kim, N., Roy, M., and Graveley, B.R.* 2009. Massive
Expansions of Dscam Splicing Diversity via Staggered Homologous
Recombination During Arthropod Evolution PLoS Biology,
Submitted.
Hale, C.R., Zhao, P., Olson, S., Duff, M.O., Graveley, B.R.,
Wells, L., Terns, R.M., and Terns, M.P.* 2009. RNA-Guided RNA
Cleavage by a CRISPR RNA-Cas Protein Complex. Cell, in press.
Lu, Y.C., Smielewska, M., Palakodeki, D., Lovci, M.T., Aigner,
S., Yeo, G.W., and Graveley, B.R.* 2009. Deep sequencing
identifies new and regulated microRNAs in Schmidtea mediterranea.
RNA, doi:10.1261/rna.1702009.
Susan E. Celniker, Laura A. L. Dillon, Mark B. Gerstein, Kristin
C. Gunsalus, Steven Henikoff, Gary H. Karpen, Manolis Kellis,
Eric C. Lai, Jason D. Lieb, David M. MacAlpine, Gos Micklem,
Fabio Piano, Michael Snyder, Lincoln Stein, Kevin P. White,
Robert H. Waterston & modENCODE Consortium. 2009. Unlocking the
secrets of the genome. Nature 459:927-930.
Graveley, B.R.* 2009. Regulation without Regulators. Nature
Structural & Molecular Biology, 16:13-15.
McManus, C.J. and Graveley, B.R.* 2008. Getting the Message Out.
Molecular Cell, 31:4-6.
Graveley, B.R.* 2008. Molecular Biology: Power Sequencing.
Nature, 453:1197-1198.
Palakodeki, D., Smielewska, M., Lu, Y.C., Yeo, G.W., and
Graveley, B.R.* 2008. The PIWI Proteins SMEDWI-2 and SMEDWI-3
are Required for Stem Cell Function and piRNA Synthesis in
Planarians. RNA, 14:1174-1186.
Graveley, B.R.* 2008. The Haplo-Spliceo-Transcriptome: Splicing
Variations in the Human Population. Trends in Genetics, 24:5-7.
“Alternative Splicing in the Postgenomic Era” 2007. Edited by
Blencowe, B.J. and Graveley, B.R. Landes Bioscience and Springer
Science+Business Media, LLC, Springer series: Advances in
Experimental Medicine and Biology, Volume 623.
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. 623:50-63.
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
& Molecular Biology, 14:1134-1140.
Drosophila 12 Genomes Consortium. 2007. Evolution of genes and
genomes on the Drosophila phylogeny. Nature 450:203-218.
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.
Graveley, B.R.* 2006. Alternative Splicing: One Gene, Many
Products. in “The Implicit Genome”, ed. Lynn Caporale. Oxford
University Press, USA.
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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