Alternative splicing of pre-mRNA is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. Despite the fact that alternative splicing occur in nearly all human genes, our understanding of molecular mechanisms regulating alternative splicing and the role of alternative splicing in normal and disease states remains incomplete and fragmented. In this project we propose to investigate the molecular mechanisms by which alternative splicing is regulated during a developmental process, termed epithelial-mesenchymal transition (EMT). EMT involves the change from a tightly packed cobble-stone like epithelial state to a motile and spindle shaped mesenchymal state. When abnormally activated, EMT can cause fibrosis in many tissues including the lung, heart, and kidney, severely affecting health. Aberrantly activated EMT also results in cancer metastasis, the leading cause of cancer-related mortality. Unfortunately, the precise mechanism that drives cells to undergo EMT has not been fully understood. Through the study of the CD44 gene, we recently demonstrated that alternative splicing regulation causally controls EMT. CD44 encodes a family of cell surface proteins produced by alternative splicing. Inclusion of one or more of the variable exons generates CD44 variant (CD44v), whereas skipping all of the variable exons produces CD44 standard (CD44s). We discovered that CD44 alternative splicing is differentially regulated during EMT, resulting in a switch in expression from CD44v in epithelial cells to CD44s in mesenchymal cells. Importantly, when CD44 isoform switching is perturbed, cells can no longer undergo EMT. These findings imply that it is crucial to determine the mechanisms regulating alternative splicing in order to better understand the function of alternative splicing in EMT and EMT-associated diseases. Towards this goal, we have recently identified hnRNPM as a critical splicing factor that stimulates the production of CD44s, promoting an EMT phenotype. In the proposed studies, we will test our hypothesis that hnRNPM and other splicing regulators compete or cooperate to regulate alternative splicing of critical genes including CD44 during EMT. To test this hypothesis, we have developed the following Specific Aims:
Aim 1. Define trans-acting factors and cis-acting elements that govern cell-type dependent alternative splicing during EMT.
Aim 2. Characterize the molecular mechanism by which hnRNPM promotes CD44 exon skipping.
Aim 3. Define hnRNPM-regulated splicing events that are critical for EMT. Understanding the molecular mechanisms of alternative splicing regulation that controls EMT could be important for the development of therapeutic strategies that perturb EMT-reactivated diseases such as tissue fibrosis and cancer metastasis. Our newly- defined alternatively spliced genes critical for EMT may also offer new ideas regarding the pathogenesis and treatment of EMT-associated human diseases.
Alternative splicing of pre-mRNA is a fundamental mechanism for gene regulation in mammals. We have demonstrated that alternative splicing regulation causally controls epithelial-mesenchymal transition (EMT), a developmental process that is abnormally activated in fibrosis and tumor metastasis. A better understanding on the molecular mechanisms of alternative splicing regulation in EMT may facilitate the development of novel strategies for the treatment of EMT-associated diseases.
|Xu, Yilin; Gao, Xin D; Lee, Jae-Hyung et al. (2014) Cell type-restricted activity of hnRNPM promotes breast cancer metastasis via regulating alternative splicing. Genes Dev 28:1191-203|