Mechanisms of hnRNPM in Alternative Splicing Regulation During EMT

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 occurs in nearly all human genes, there are only a handful of examples suggesting the functional role of alternative splicing in biological processes. The long-term goal of our research is to investigate mechanisms by which alternative splicing is regulated and the role of alternative splicing in modulating critical cellular phenotypes important for normal development and diseases. The developmental process termed epithelial-mesenchymal transition (EMT) represents a program of altering cellular phenotypes, from a tightly packed cobble-stone like epithelial state to a spindle shaped mesenchymal state. When abnormally activated, EMT can cause fibrosis in many tissues including the lung, heart, and kidney, severely affecting the quality of life in patients. Aberrantly activated EMT also results in cancer metastasis, which remains the leading cause of cancer-related mortality. Unfortunately, the precise mechanism that drives cells to undergo EMT has not been fully understood. We recently reported a novel mechanism by which alternative splicing causally controls EMT. These studies relied on analysis of a critical molecule CD44, which encodes a family of cell surface proteins produced by differential 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. Remarkably, when CD44 isoform switching is perturbed, cells can no longer undergo EMT. Importantly, this isoform switching is critical for breast cancer progression in mice and patients. These findings imply that it is crucial to determine the mechanisms by which alternative splicing is regulated in order to better understand the function of alternative splicing in EMT and EMT-associated diseases. 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 regulate CD44 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.