Investigations into the Role of RNA Binding Proteins and Alternative Splicing in the Epithelial-Mesenchymal Transition

Harvey, Samuel

doi:https://doi.org/10.21985/n2-rfqb-kv49

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Investigations into the Role of RNA Binding Proteins and Alternative Splicing in the Epithelial-Mesenchymal Transition

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The critical importance of alternative mRNA splicing and the RNA binding proteins that orchestrate this essential layer of post-transcriptional gene regulation is increasingly recognized in gene regulatory programs. We and others have shown that alternative splicing plays a causal role during the Epithelial-Mesenchymal transition, a cell-developmental program that is hijacked by many cancers to acquire the ability to survive, invade, and metastasize to distant sites. While alternative splicing events may be the ultimate effectors of splicing in regulating cell function, RNA binding proteins play a critical role in regulating EMT and regulating cell-state plasticity in normal and oncogenic environments. This thesis describes four situations where RNA binding proteins and the global splicing regulons they control globally influence changes in cell-state, with a particular focus on the shift from the epithelial-cell state to the mesenchymal-cell state that occurs during EMT. Chapter 1 describes coregulation of alternative splicing by hnRNPM and ESRP1, two RNA binding proteins that play opposite roles in regulating EMT, with hnRNPM necessary for EMT to occur while ESRP1 antagonizes EMT by promoting epithelial-associated alternative splicing. In this chapter, we describe a nuanced overlap of regulation of splicing by hnRNPM and ESRP1 where a small set of skipped exon splicing events controlled discordantly by these two factors is highly associated with EMT-regulated biological processes and predicts breast cancer patient survival. These events are highly enriched in hnRNPM and ESRP1 shared binding sites in intronic sequence downstream of the 5’ splice site, with one splicing event at APLP2 exon 7 showing competitive binding between hnRNPM and ESRP1 to drive APLP2 exon 7 skipping or inclusion in concordance with mesenchymal or epithelial splicing, respectively. Chapter 2 examines cell-state specific regulation of alternative splicing by hnRNPM specifically by integrating epithelial and mesenchymal hnRNPM-depletion datasets from an in vitro model of EMT coupled with crosslinking-associated hnRNPM binding sites across the transcriptome in both cell states. hnRNPM regulates a partially overlapping set of genes and splicing events in these two states, with a shift towards EMT-promotive regulation at both the gene and splicing level in the mesenchymal state. This shift is manifest in increased enrichment of hnRNPM binding sites downstream of skipped exon events specifically in the mesenchymal state. Additionally, hnRNPM’s necessity in the EMT phenotype is clarified through gene set enrichment analysis where hnRNPM depletion upregulates epithelial-associated genes such as CDH1 which are typically suppressed during EMT while playing little role in the gain of mesenchymal expressed genes. Ultimately, this chapter describes how hnRNPM, a ubiquitously expressed RNA binding protein, regulates distinct transcriptional processes in a cell-state specific manner while retaining the ability to suppress epithelial-associated gene regulation in both cell-states. Chapter 3 describes an investigation of the role of RNA secondary structures, specifically RNA G-quadruplexes, during EMT. We previously identified that RNA G-quadruplexes serve as an important binding site for hnRNPF, a splicing factor which this chapter shows promotes epithelial-associated alternative splicing and antagonizes the EMT phenotype. RNA G-quadruplexes are enriched near hnRNPF-dependent splicing events, and hnRNPF shows a high enrichment of G-quadruplex structures near binding sites compared to other hnRNP family members. In addition, this chapter describes the effect of emetine, a small molecule we previously identified as a G-quadruplex destabilizer, and its ability to promote an EMT associated transcriptional signature. The dichotomous roles of hnRNPF, an RNA binding protein that antagonizes EMT through recognition of G-quadruplexes, and emetine, which promotes EMT through G-quadruplex destabilization, highlights the critical role RNA secondary structure plays in regulating alternative splicing and cell phenotypes. Chapter 4 discovers the role of AKAP8, a non-traditional RNA binding protein, as an antagonist of EMT-associated alternative splicing through differential RNA binding topology in epithelial and mesenchymal states. Integration of AKAP8 depletion RNA sequencing datasets in epithelial and mesenchymal cells with AKAP8 crosslinking binding sites shows that AKAP8 specifically antagonizes EMT associated alternative splicing in the epithelial state although AKAP8 expression does not change much during EMT. This antagonism is linked to increased binding of AKAP8 to proximal intronic sequences, which are more highly enriched in splicing-regulatory cis-elements, in the epithelial state compared to the mesenchymal state. Thus, this chapter identifies AKAP8 as another RNA binding protein with cell-state specific binding properties and splicing regulation that polarizes the EMT phenotype towards the epithelial state. Together, these four chapters interrogate a fundamental question of interest in the field of post-transcriptional regulation; how and to what extent does RNA metabolism contribute to cell-phenotype change and cell-state plasticity? Using EMT as a model cell-state transition with clinically relevant importance, especially relating to the underlying mechanism of cancer metastasis, this thesis provides new insight into how RNA binding proteins, through direct and indirect interaction with downstream regulatory targets, play diverse and nuanced roles in bridging genotype to phenotype.

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