- Regulation code for alternative splicing
- Function of splicing factors in human cancer
- Bioengineering of artificial factors to manipulate control RNA splicing and stability
My lab currently works on two main areas of RNA biology. Our main focus is to study how alternative splicing is regulated in a systematic level and how such regulation responds to cellular signaling pathways during key biological processes. In addition, we developed new approaches to manipulate splicing and other RNA processing pathways by engineering artificial factors that specifically recognize a given RNA target. In summary, my lab is seeking to solve the “splicing code” (i.e. a set of rules for splicing regulation by cis-elements and trans-factors) and to reprogram the code to control gene function. Since alternative splicing is a key regulatory mode for the majority of human genes and the splicing dysregulation is a common cause of various diseases, our research can provide important insight to fundamental biology and human diseases.
1. Systematic study of splicing regulatory elements and factors
We use new cell-based screen systems to unbiasedly identify intronic splicing enhancers and silencers (ISE and ISS) from random sequences. These elements are further used as baits to identify putative trans-acting splicing factors. Our results suggested a complex, overlapping network of protein-RNA interactions between ISS or ISE motifs and their trans-factors. This arrangement may enable individual cis-element to exert different regulatory functions in distinct cellular contexts depending on the spectrum of regulatory factors present.
2. Splicing regulation in cancers
One of molecular hallmark for human cancer is dis-regulation of alternative splicing in many genes on a systematic scale. We identified several splicing factors that function as oncogenes or tumor suppressors. One of such genes is DAZAP1 that function as a general splicing activator by to promote inclusion of weak exons through specific recognition of diverse cis-elements. The C-terminal proline-rich domain of DAZAP1 is phosphorylated by the MEK/Erk pathway, and is sufficient to activate splicing when recruited to pre-mRNA. The phosphorylation by MEK/Erk is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Knockdown or over-expression of DAZAP1 caused a cell proliferation defect that was independent of apoptosis. Taken together, these studies reveal a molecular mechanism that integrates the splicing control into MEK/Erk regulated cell proliferation and migration. We found another splicing factor, RBM4, has tumor suppressor activity and involved in the regulation of cell apoptosis. In addition, we found several splicing regulatory proteins can control DNA damage repair and cell cycle pathway though modulating alternative splicing, providing new links between RNA splicing and cancer.
3. Engineering artificial factors to modulate RNA splicing and stability
Another research area of my lab is to use the synthetic biology approach to engineer novel factors that specifically manipulate RNA metabolism. My lab was the first to engineer “designer” splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 (PUF domains) with functional domains that promote or inhibit splicing. We applied these factors to modulate different types of alternative splicing in various targets including an anticancer target Bcl-X. Our work permitted the creation of artificial factors to target virtually any pre-mRNA, providing a strategy to study splicing regulation and to manipulate disease-associated splicing events. Using a similar strategy, we engineered artificial site-specific RNA endonuclease by combining PUF domain with a general RNA endonuclease domain. The resulting enzyme is analogous to a “restriction enzyme” of RNA in that it can specifically recognize RNA and efficiently cleave near the binding site. Since a PUF domain can be reprogrammed to bind any 8-nt sequence, our artificial enzymes provide a new gene tool that is very useful in studying the function of non-coding RNAs.