Exploring the expression and function of RNA-binding proteins and splicing factors in haematopoiesis

Forrester, Nicole (2023) Exploring the expression and function of RNA-binding proteins and splicing factors in haematopoiesis. Masters thesis, University of East Anglia.

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Alternative splicing (AS) contributes to the generation of functional diversity in higher eukaryotic proteomes by producing multiple isoforms of the same protein from the same gene locus. AS is influential in cell lineage commitment in multiple systems and differentiation processes, including haematopoiesis. RNA-binding motif protein 15 (RBM15) is a known regulator of AS in haematopoietic stem cells, influencing homeostasis in this compartment and particularly regulating megakaryocyte differentiation. This project investigates the regulatory functions of RBM15, and furthermore examines the expression of other splicing factor genes, identifying any lineage-specific patterns within the haematopoietic system. RNA-seq was performed on control and CRISPR-Cas9 edited HAP1 cells to explore differential expression. Editing the RBM15 locus impacted the transcriptome dramatically with 8,710 genes upregulated and 7,941 downregulated, including key haematopoiesis genes such as HCLS1 and PDGFRA. Additionally, an inverse relationship between RBM15 and RBM15B expression was discovered, highlighting that these genes appear closely related. Next, a single-cell short-read dataset from haematopoietic stem cells was explored for patterns of RNA-binding protein and splicing factor (RBP/SF) expression. 83 out of 428 RBP/SF genes were found to be expressed and three displayed lineage-specificity: Carhsp1 and Larp1b were upregulated in erythroid progenitors and Khdrbs3 was upregulated in megakaryocyte progenitors. To investigate the transcriptomic influence of these genes, they were targeted for knockout in HAP1 cells using CRISPR-Cas9 and guides were confirmed to successfully edit two of the three genes. Overall, this research highlights the broad influence of RBM15 on transcriptional regulation and has identified RBP/SFs preferentially acting in specific haematopoietic lineages.

Item Type: Thesis (Masters)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 28 Feb 2024 11:04
Last Modified: 28 Feb 2024 11:04
URI: https://ueaeprints.uea.ac.uk/id/eprint/94371


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