ARS2 is a member of the nuclear cap-binding complex (CBC) that is critical for a number of RNA processing pathways. The emerging model is that ARS2 acts as a master regulator of RNAPII transcript maturation by bringing capped RNA substrates together with the appropriate processing machinery. ARS2 is essential for early mammalian development but it remains unclear precisely how ARS2 functions in stem and progenitor cell maintenance and differentiation. The purpose of this study was to answer basic questions about the localization and function of ARS2 in muscle progenitor cells. Here I describe the localization of ARS2 in proliferating myoblasts and post-mitotic differentiating myotubes and show that disruption of ARS2 expression levels by knockdown or overexpression results in impaired myogenic differentiation. I also discovered a new isoform of ARS2 that is localized exclusively in the cytoplasm and found preliminary evidence that ARS2 is required for nonsense-mediated decay (NMD). This study includes the first evidence that an ARS2 isoform is expressed in the cytoplasm and opens the door for the discovery of new ARS2 functions beyond its reported roles in the nucleus. / Graduate

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The aim of this thesis was to identify genetic factors involved in frontotemporal lobar degeneration (FTLD), a neurodegenerative disorder clinically characterised by a progressive change in personality, behaviour and language. FTLD is a genetically complex disorder and a positive family history is found in up to 40% of the cases. In 10-20% of the familial cases the disease can be explained by mutations in the gene encoding the microtubule associated protein tau (MAPT). In the first study we describe the clinical and neuropathological features of a Finnish family with FTLD caused by a mutation in MAPT. We also provide evidence that the pathogenic mechanism of this mutation is through altered splicing of MAPT transcripts. Recently, mutations in the gene encoding progranulin (PGRN) were identified as a major cause of FTLD. In the second study we describe a Swedish family with FTLD caused by a frameshift mutation in PGRN. We provide a clinical and neuropathological description of the family, as well as evidence that the pathogenicity of this mutation is through nonsense-mediated decay of the mutant mRNA transcripts and PGRN haploinsufficiency. In the third study we describe a novel PGRN splice site mutation and a previously described PGRN frameshift mutation, found in a mutation screen of 51 FTLD patients. We describe the clinical and neuropathological characteristics of the mutation carriers and demonstrate that haploinsufficiency is the pathogenic mechanism of the two mutations. In the fourth study we investigate the prevalence of PGRN and MAPT gene dosage alterations in 39 patients with FTLD. No gene dosage alterations were identified, indicating that variations in copy number of the PGRN and MAPT genes are not a common cause of disease, at least not in this FTLD patient collection.

Nonsense mediated mRNA decay (NMD) is a surveillance mechanism that targets transcripts containing premature stop codons (PTCs) for degradation, and that also regulates up to 10% of the whole transcriptome. During the course of my PhD I set out to identify novel NMD factors by performing a genome-wide RNA interference (RNAi) screen in a transgenic strain of Caenorhabditis elegans carrying an NMD reporter. I identified five novel proteins that are putative NMD factors in worms: NGP-1, NPP-20, AEX-6, PBS-2 and NOAH-2. Knock-down of these proteins led to severe developmental defects: worms were either arrested during various larval stages or died prematurely. The only exception was AEX-6, the knockdown of which led to a milder phenotype. Homology analysis of the novel C. elegans NMD factors showed that these proteins are conserved in human, with the exception of NOAH-2, which only has a homologue in Drosophila melanogaster, NOMPA. By performing an NMD assay in human cells, I demonstrated that GNL2 (NGP-1) and SEC13 (NPP-20) are functionally conserved NMD factors in human. Analysis of the consequences of depletion of GNL2, SEC13, UPF1 or UPF2 on the transcriptome of HeLa cells revealed that these four proteins co-regulate a subset of endogenous NMD targets, whilst also independently regulating the expression of other sets of transcripts. The findings presented in this thesis further our knowledge of the biology of NMD in both nematodes and humans. They demonstrate the existence of further regulators of this surveillance pathway, and add a layer of complexity to this fine-tuned biological process.

The aim of this thesis was to identify genetic factors involved in frontotemporal lobar degeneration (FTLD), a neurodegenerative disorder clinically characterised by a progressive change in personality, behaviour and language. FTLD is a genetically complex disorder and a positive family history is found in up to 40% of the cases. In 10-20% of the familial cases the disease can be explained by mutations in the gene encoding the microtubule associated protein tau (MAPT). In the first study we describe the clinical and neuropathological features of a Finnish family with FTLD caused by a mutation in MAPT. We also provide evidence that the pathogenic mechanism of this mutation is through altered splicing of MAPT transcripts. Recently, mutations in the gene encoding progranulin (PGRN) were identified as a major cause of FTLD. In the second study we describe a Swedish family with FTLD caused by a frameshift mutation in PGRN. We provide a clinical and neuropathological description of the family, as well as evidence that the pathogenicity of this mutation is through nonsense-mediated decay of the mutant mRNA transcripts and PGRN haploinsufficiency. In the third study we describe a novel PGRN splice site mutation and a previously described PGRN frameshift mutation, found in a mutation screen of 51 FTLD patients. We describe the clinical and neuropathological characteristics of the mutation carriers and demonstrate that haploinsufficiency is the pathogenic mechanism of the two mutations. In the fourth study we investigate the prevalence of PGRN and MAPT gene dosage alterations in 39 patients with FTLD. No gene dosage alterations were identified, indicating that variations in copy number of the PGRN and MAPT genes are not a common cause of disease, at least not in this FTLD patient collection.

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The life of RNA polymerase II (RNAPII) transcripts is shaped by the dynamic formation of mutually exclusive ribonucleoprotein complexes (RNPs) that direct transcript biogenesis and turnover. A key regulator of RNA metabolism in the nucleus is the scaffold protein ARS2 (arsenic resistance protein 2), that binds to the cap binding complex (CBC) and regulates processing, degradation, and export of RNAPII transcripts. We report here that alternative splicing of ARS2’s intron 5, generates cytoplasmic isoforms that lack 270 amino acids from the N-terminal of the protein and are functionally distinct from nuclear ARS2. ARS2 isoforms distinctive roles are evidenced under physiological conditions and stress. Under physiological conditions, ARS2 isoforms differentially regulate transcript degradation through nonsense mediated decay (NMD). Switching of ARS2 isoforms within the CBC in the cytoplasm has dramatic functional consequences, changing ARS2 from a NMD inhibitor to a NMD promoter that enhances the binding of UPF1 to CBP80 and ERF1, favouring SURF complex formation, SMG7 recruitment and transcript degradation. ARS2 isoform exchange is also relevant during arsenic stress. Cytoplasmic ARS2 is specifically induced during arsenic exposure. It is crucial for arsenic sensitivity, and promotes a global response to arsenic in a CBC independent manner. We propose that ARS2 isoform switching promotes the proper recruitment of RNP complexes during NMD and the cellular response to arsenic stress. The existence of non-redundant ARS2 isoforms is relevant for cell homeostasis, stress response and cancer treatment. / Graduate / 2023-04-14

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