Research Overview
Our research is focused on the regulation of alternative splicing and small RNA mediated gene regulation which are fascinating and important mechanisms by which genes can be regulated. Our long-term goals are to understand how these processes are regulated at a mechanistic level and the logic of these processes in significant biological settings. To achieve these goals, we use a wide variety of approaches that include biochemistry, genetics, imaging, deep sequencing, large-scale RNAi screening and bioinformatics.
ENCODE: The goal of the ENCODE project is to identify all sequence-based functional elements in the human genome. In collaboration with Chris Burge, Xiang-Dong Fu, and Gene Yeo we are working to identify the RNA sequence elements bound by 250 RNA binding proteins (RBPs) in two human cell lines and to perform functional assays to assign biological roles to the binding sites we identify. The end product of this project will be a comprehensive catalog of functional RNA sequence elements encoded in the human genome. To do this, we are performing CLIP-Seq assays to identify the RNA elements recognized by 250 individual RBPs in each cell line. These experiments will be supplemented with sequence-based in vitro binding assays to independently validate the consensus binding sites identified for each RBP. We will perform ChIP-seq on a subset of nuclear RBPs, to obtain an integrated view on RNA-protein interactions in relationship to genomic DNA. We will also deplete each RBP and perform RNA-seq on RNA isolated from these cells. Together, these assays, coupled with informatics analysis, will allow us to ascribe the RNA binding sites with functions in splicing, RNA stability, and RNA editing.
Dscam Alternative Splicing: The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene is one of the most interesting genes in any organism. Dscam is remarkable in that it has the potential to encode over 38,000 isoforms by virtue of extensive alternative splicing. Dscam contains 115 exons, 95 of which are alternatively spliced. The alternative exons are organized into four distinct clusters – the exon 4, 6, 9, and 17 clusters – that contain 12, 48, 33, and 2 variable exons each, respectively. Importantly, the exons within each cluster are alternatively spliced in a mutually exclusive manner. DSCAM functions a both an axon guidance receptor and an immune receptor. In the nervous system, the collection of isoforms that are expressed each neuron a unique identity and determines its wiring pattern. In the immune system, DSCAM functions in a matter analogous to vertebrate antibodies where different isoforms recognize different pathogens. Thus, understanding the regulation and mechanisms of Dscam alternative splicing will provide tremendous insight into how both the nervous and immune systems function. We are working to determine the expression patterns of individual isoforms at single cell resolution, determine the mechanisms involved in mutually exclusive splicing, and identify RNA binding proteins that regulate Dscam alternative splicing and the mechanisms by which they act.
Trans-Splicing: In some eukaryotes, including nematodes, trypanosomes and planarians, splicing can occur in trans. In these cases, a specialized spliced leader RNA is spliced to the 5' end of protein coding RNAs. Interestingly, there are a few cases where a distinct type of trans-splicing has been shown to occur, namely, the splicing of exons from protein coding genes. The two best characterized examples are the mod(mdg4) and lola genes from Drosophila. We have used genomic approaches to identify additional trans-spliced genes in Drosophila. We are currently working to determine the mechanisms by which trans-splicing occurs and the function of trans-spliced isoforms. This work will provide insight into the mechanisms of trans-splicing, a completely understudied yet important process. Given the potential utility of trans-splicing in treating human diseases and that trans-spliced mRNAs in humans have recently been linked to cancer, it is likely the discoveries we make will be of direct relevance to human health.
Collaborative work: In addition to our modENCODE and ENCODE work, which by its nature is very collaborative, we have many collaborations with other labs to explore fascinating areas of biology. We have a long-standing collaboration with the Terns lab to study the CRISPR systems in various bacteria and archaea. We have collaborated with Trisha Wittkopp to study various aspects of gene expression using interspecies Drosophila hybrids. We also are collaborating with Jeff Coller to use genomic approaches to study RNA turnover.
