ORCID

https://orcid.org/0000-0002-3457-7903

Date of Award

2021

Document Type

Dissertation

Degree Name

Philosophy (Ph.D)

Department

Biological Sciences

First Advisor

Matteo Ruggiu

Second Advisor

Ales Vancura

Third Advisor

Ivana Vancurova

Abstract

Gene expression is regulated at multiple levels, including transcription, RNA editing, pre-mRNA splicing, mRNA export, translation, and posttranslational modifications. Alternative splicing is a process by which exons can be included or excluded, giving rise to multiple mRNA isoforms from the same transcript. Alternative splicing is an important mechanism in developmental, tissue- and cell-specific control of gene expression, and it is key for expanding proteomic diversity and complexity from a limited number of genes. Moreover, more than 95% of multiexon genes undergo alternative splicing in humans, and about half of all disease-causing point mutations in humans affect pre-mRNA splicing, including neurological disorders and cancer. The central nervous system comprises the tissues and cells with the highest rate of alternative splicing in the body, and RNA-binding proteins play a major functional role in neurons. However, the regulatory mechanisms of splicing are still poorly understood. This dissertation specifically aims to advance the understanding of regulatory mechanism of pre-mRNA splicing. To this end, we collaboratively performed two projects. In the first project, we investigated how NOVA, a neuron-specific splicing factor, regulates nerve cell-specific alternative splicing of Z+ Agrin — a molecule that is the master architect of nerve-muscle synapses at the neuromuscular junction (NMJ). We cloned the Ciona ortholog of NOVA, which is present as a single copy gene in tunicates, and that of Agrin, and dissected the regulatory mechanism of alternative splicing of Z+ Agrin by Nova. Moreover, we characterized their function and expression pattern during larval development, which we will discuss in detail in Chapter 2 of this dissertation. The second project was a case study where we investigated how mutations in the SLC25A10 gene cause epileptic encephalopathy by disrupting pre-mRNA splicing. SLC25A10 codes for a solute carrier protein and is a part of complex I in mitochondria. The patient inherited 3 mutations: 1 from the mother and 2 from the father. The maternal-derived mutation introduces a stop codon in exon 3. Mutations from the paternal allele are located in exon 9 and intron 10. Although the exonic mutation is a synonymous mutation, the patient had very low levels of SLC25A10 mRNA and lacked protein at detectable levels. Using minigene splicing assay we investigated the molecular mechanism underlying disease pathology in the patient. In Chapter 3 of this dissertation, we will discuss how paternal-derived mutations lead to aberrant splicing.

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