简介

My laboratory studies a wide variety of experiments that can be grouped into five broad areas:Alternative Splicing Regulatory Networks in Drosophila 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 percent 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 has 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.Alternative Splicing of the Drosophila Dscam Pre-mRNA 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 Zipurskys 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.