Profiling and functional interactomics of circular RNAs across human stem cell differentiation

Author: Stuart Webb

Webb, Stuart, 2022 Profiling and functional interactomics of circular RNAs across human stem cell differentiation, Flinders University, College of Medicine and Public Health

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Human embryonic stem cells (hESC) are a powerful tool to examine many aspects of cell development, differentiation and disease. Directed differentiation of ESCs into specific lineages can be achieved with high fidelity and reproducibility using specific growth factors invoking dramatic phenotypic and transcriptomic responses. Each of these differentiation processes incorporate a key process called epithelial-mesenchymal transition (EMT), which has a significant prognostic impact in cancer. EMT is the conversion of stationary cells into cells with greater motility and is indispensable for processes including cellular differentiation, wound repair and organ development. Therefore, as EMT is required for cellular differentiation, transcriptomic profiling was performed to investigate circular RNA (circRNA) expression changes and molecular functions during hESC differentiation into cell types of the three germ cell lineages.

Alternative Splicing (AS) is a regulated process that generates a variety of RNA molecules from the same pre-mRNA transcript. AS is observed in ~95% of multi-exonic human genes resulting in unique mRNA transcripts that may function synonymously, in opposition to, or in an unrelated manner. Importantly, AS is regulated during cell differentiation leading to changes in both coding and non-coding RNAs.

CircRNAs are a highly abundant class of RNA, ubiquitous among eukaryotes and usually located within protein coding genes. Circular RNAs are produced during a non-canonical AS event, termed back-spicing, and therefore changes to AS have a significant impact on circRNA biosynthesis which leads to the production of differentiation and EMT-associated circRNAs. Currently, over 400,000 circRNAs have been identified and despite a growing repertoire of functions being reported, this is only for comparatively few circRNAs. Therefore, the vast proportion (>99%) remain functionally enigmatic.

Performing high-throughput RNA-sequencing of H9 hESCs differentiated into 5 different cell types, lead to the identification of 27 candidate circRNAs that are either (i) commonly regulated during each cell-specific differentiation event, or (ii) showed lineage-specific changes in expression. Furthermore, unique characteristics of individual circRNAs, including, differential splicing of exons and co-regulation with their parental gene’s mRNA transcripts provides insight into additional regulation of circRNAs. To study their effect on differentiation, and associated programs including EMT, circRNA abundance was manipulated through knockdown (KD) experiments. Reduction to circACVR2A levels was found to induce consistent and rapid neuronal differentiation in hESCs and partially block the cell cycle in A549 cells. Undertaking a circACVR2A-protein interactome analysis, Cyclin H was identified as a binding partner. Cyclin H, together with CDK7 and MAT1, forms the CDK-activating kinase (CAK) trimeric complex, necessary for regulating proliferation and cell cycle progression. Altering circACVR2A levels altered CDK activity and phosphorylation of targets, providing a functional explanation for altered passage through the cell cycle and for cellular phenotypes observed in both cell lines, altering cell fate and resulting in neuronal differentiation of hESCs.

The outcome of this project identified that specific circRNAs play critical functional roles within the context of regulating parental mRNA levels, cell cycle control, human stem cell differentiation and EMT.

Keywords: Stem cell differentiation, circular RNAs, circRNAs, EMT, Neuron

Subject: Medical Science thesis

Thesis type: Doctor of Philosophy
Completed: 2022
School: College of Medicine and Public Health
Supervisor: Professor Simon Conn