Neeves, Jacob; (2022) Examining the role of SFPQ and intron retention in physiology and amyotrophic lateral sclerosis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Intron retention (IR) is a well-conserved form of alternative RNA splicing, though its importance in the regulation of mammalian biological processes has only recently been established. The main accepted role for IR is in fine-tuning host gene expression through nuclear detention and/or transcript degradation. However, increasing breadth of functions are being revealed, including stable cytoplasmic coding and non-coding roles. Recent work has identified aberrant IR in amyotrophic lateral sclerosis (ALS), a rapidly progressive and universally fatal neurodegenerative disease with a life expectancy of 2-5 years from diagnosis, and a paucity of lifespan enhancing therapies. To gain further insight I carried out nuclear-cytoplasmic fractionation at multiple stages throughout human induced pluripotent stem (hiPSC)-derived motor neurogenesis in control and familial ALS VCP-mutant lines. RNA sequencing of the resultant samples enabled characterisation of IR in separate cellular compartments, revealing compartment-specific aberrant accumulation of IR transcripts in ALS. Notably, transcripts increased in the cytoplasm possess sequence-specific attributes and increased predicted binding affinity to RNA binding proteins, including those which exhibit hallmark nuclear-to-cytoplasmic mislocalisation in ALS (TDP43, FUS and SFPQ). This implies a novel role for IR in regulating compartment-specific protein abundance. Candidate-specific interrogation begins to characterise the physiological behaviour of aberrant cytoplasmic IR events through assessment of cytoplasmic stability, translation status and stress-responsivity. Further, I developed tools to specifically modulate one such event, which will address the pathogenic relevance of increased specific IR in ALS, including RBP mislocalisation. Intriguingly, SFPQ, which exhibits significantly increased IR and nuclear-tocytoplasmic protein mislocalisation in ALS, also exhibits dysregulation of another, formerly uncharacterised, alternative RNA transcript (termed altSFPQ) in our VCP-ALS dataset. I demonstrate that altSFPQ is highly unstable and may contribute to autoregulation of SFPQ. Surprisingly, this 4 transcript also exhibits physiological translation potential and novel protein production upon upregulation of endogenous levels. AltSFPQ protein is predicted to be located in the cytoplasm and may, therefore, carry out established cytoplasmic SFPQ functions. Finally, this splicing event is shown to be upregulated in sporadic hiPSC-ALS models, representing the major form of ALS (>90% cases). I conclude that altSFPQ is physiologically translated in a context-specific manner, as well as in ALS which contributes to hallmark SFPQ nuclear-to-cytoplasmic protein mislocalisation.

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