Identify splicing regulatory cis-elements and trans-factors (compiling the splicing code)
Develop new method to modulate splicing (debugging the splicing code)
Research Synopsis:
The long-term goal of our lab is to understand how splicing is regulated in a systematic level. Our main approach is to collect a ‘part list’ of splicing regulation, and to further determine how they functionally interact to each other so that to assemble a set of general rules of splicing (i.e. the splicing code). Our research is motivated by a major surprise after the sequencing of the human genome, which is the total number of protein coding genes is much lower than previously estimated. This finding indicated that additional genomic complexity might be added at the level of RNA processing. More than 60% of human genes undergo alternative splicing (seefigure 1 for simple forms of alternative splicing), which is tightly regulated in different tissues and developmental stages. Like the transcriptional regulation, the splicing is regulated by protein trans-factors that either enhance or silence the use of adjacent splice sites. These trans-factors are recruited to pre-mRNA through binding to specific RNA sequences known as splicing regulatory cis-elements. Depending on the locations and effects on splicing, these cis-elements are defined as exonic splicing enhancer (ESE), exonic splicing silencer (ESS), intronic splicing enhancer (ISE) and intronic splicing silencer (ISS).
From a part list of splicing regulation to the splicing code
Most splicing regulatory cis-elements and the trans-factors that bind to them are remain to be discovered. We have developed a cell-based splicing reporter system to screen a library of random decamers for ESS (see figure 2 for FAS-ESS) and further examined how they regulate alternative splicing (see figure 3 for ESS roles in regulating splicing). This method allowed us to systematically identify ESS, and can be extended to screen and study all other cis-elements. We are developing high throughput method to identify the trans-factors that bind to these elements, and will determine the in vivo rules for their interaction in the genomic scale. With such information, we can (i) use computational approaches to assemble a set of rules for splicing regulation (i.e. the splicing code), (ii) predict splicing behavior of any transcripts, and (iii) study how splicing regulation reacts to outside stimuli and stresses.
Debugging the splicing code
Given the fact that most human genes contain introns and the majority of genes are alternatively spliced, it is not surprising that disruption of normal splicing pattern can lead to human diseases. A better understanding of splicing regulation can help us to modulate splicing behavior of a gene so that to change the functions of its mature protein. We will design artificial molecules as splicing silencers and enhancers to modulate splicing, and screen for chemicals that can interfere with splicing regulation pathway. We will apply this method to a serious of genes whose alternative splicing is critical to apoptosis. Such molecules will have therapeutic potential for treating splicing disease, and can help us to understand mechanisms of splicing regulation.
Wang Z, Xiao X, Van Nostrand E and Burge CB. (2006) General and specific functions of exonic splicing silencers in splicing control. Mol Cell 23(1): 61-70, (cover article). Abstract
Wang Z, Rolish M, Yeo G, Tung V, Mawson M and Burge CB. (2004) Systematic identification and analysis of exonic splicing silencers. Cell 119 (6): 831-845. Abstract
Drew ME, Morris JC, Wang Z, Wells L, Sanchez M, Landfear SM, and Englund PT. (2003) The adenosine analog tubercidin inhibits glycolysis in trypanosoma brucei as revealed by an RNAi library. J Biol Chem 278(47): 46596-46600. Abstract
Wang Z, Drew ME, Morris JC, and Englund PT. (2002) Asymmetrical division of trypanosome’s kinetoplast DNA network. EMBO J 21(18): 4998-5005. Abstract
Morris JC, Wang Z, Drew ME, and Englund PT. (2002) Glycolysis modulates Trypanosome glycoprotein expression as revealed by an RNAi library. EMBO J 21(17): 4429-4438. Abstract
Wang Z and Englund PT. (2001) RNA interference of a trypanosome topoisomerase II causes progressive loss of mitochondrial DNA. EMBO J 20(17): 4674-83 Abstract
Wang Z, Morris JC, Drew ME, and Englund PT. (2000) Inhibition of Trypanosoma brucei gene expression by RNA Interference: A survey using an integratable vector with opposing T7 Promoters. J Biol Chem 275: 40174-40179. Abstract
