Costello Heaven, Neve Elizabeth; (2025) The role of nuclear speckles in protein-RNA homeostatic regulation and its perturbation in neurodegeneration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The concentration of proteins containing intrinsically disordered regions (IDRs) must be tightly controlled to maintain cellular homeostasis and prevent disease, yet no mechanism for such regulation has been identified. Through my PhD research, I have contributed to the elucidation of a novel mechanism termed interstasis; a homeostatic feedback pathway that senses the concentration of co-condensing proteins and controls their gene expression through mRNA-mediated negative feedback. I focus on the mechanism that exploits the multivalency of GA-rich mRNAs that encode arginine-enriched mixed-charge domains (R-MCDs), enriched in nuclear speckle condensates. Accumulation of R-MCD proteins increases cohesion of nuclear speckles, recruiting TRA2B proteins that strongly bind purinergic mRNAs and selectively retain them in speckles. Cdc-like kinase (CLK) activity controls the localisation of TRA2B to speckles, modulating the setpoint of interstasis. Employing transcriptomics and high-resolution imaging, I demonstrate that interstasis is a collective feedback mechanism that responds to the accumulation of condensation-prone speckle proteins and sequesters their encoding mRNAs to promote their mutual homeostasis. Next I begin to explore potential roles of nuclear speckle-mediated protein-RNA homeostatic regulation in neurodegeneration and ageing. Using mouse hippocampal-derived neural stem cells and single-molecule RNA imaging, I examine the potential contribution of interstasis to the protein-RNA uncoupling observed in quiescence, demonstrating slight nuclear retention of specific GA-rich transcripts in quiescent cells. Additionally, I assess the possible contribution of interstasis to proteostatic perturbations by aggregation-prone mutants of tau. Given that tau reportedly colocalises with and mislocalises nuclear speckle protein SRRM2, I quantify changes in subcellular mRNA abundance in iPSCs with changing nucleocytoplasmic tau aggregate localisation. Finally, employing iCLIP I explore tau-RNA interactions, uncovering binding between tau and ribosomal RNAs in close proximity to the polypeptide ribosome exit channel. Together, my work has assessed the roles of nuclear speckles in cellular homeostasis and how their deregulation may contribute to neurodegenerative processes.

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