{"id":{"repo_id":"cork","oai_identifier":"oai:cora.ucc.ie:10468/18898"},"canonical_url":"https://search.dev.ndltd.org/etd/cork/oai:cora.ucc.ie:10468/18898","repository":{"repo_id":"cork","name":"University College Cork","base_url":"https://cora.ucc.ie/server/oai/request"},"display":{"title":"Analysis of SMAUG1 protein motifs and interacting partners - linking regulation of phase separation to human disease","abstract":"RNA-binding proteins (RBPs) are central regulators of gene expression, influencing RNA processing, localisation, translation, and decay. A growing body of evidence shows that many RBPs exert their functions through the formation of dynamic, membrane-less organelles (MLOs) via liquid–liquid phase separation (LLPS). Dysregulation of these MLOs or “biomolecular condensates” is increasingly linked to human disease, including cancer and neurological disorders. Human SMAUG1 (SAMD4A) is a conserved translational repressor and condensate-forming protein implicated in neuronal function, metabolism, antiviral defence, and cancer. However, little is known regarding its condensate formation, localisation, and whole cell interactome. The aim of this PhD project was to define the molecular determinants of SMAUG1 phase separation, identify regulatory mechanisms controlling its condensates, and establish how condensate formation influences SMAUG1 localisation, protein interactome and RNA regulatory functions in the context of human health and disease. In Chapter 2, we defined the protein regions and molecular interactions that control SMAUG1 condensate formation in cells. Using protein domain and short linear motif (SLiMs) deletion mutants, we demonstrated that SMAUG1 phase separation is driven by its structured ‘Smaug similarity region 1’ (SSR1) dimerization domain and a novel intrinsically disordered prion-like domain. In contrast, the RNA-binding ‘sterile alpha motif’ (SAM) domain and other disordered regions were dispensable for condensation. We further identified members of the 14-3-3 protein family as potent negative regulators of SMAUG1 condensation. Dimeric 14-3-3 proteins that bind multiple phosphorylated motifs across SMAUG1 caused condensate dissolution. This work establishes a signalling dependent mechanism for dynamic control of SMAUG1 phase behaviour and with findings published as a first-author manuscript. In Chapter 3, we tested SMAUG1 condensation in vitro and defined its intrinsic phase separation behaviour. Recombinant SMAUG1 formed liquid-like condensates at low micromolar concentrations (0.1-2μM) under physiological salt (0-200 mM NaCl) and pH conditions (pH 7.5), in the absence of RNA or molecular crowders. SMAUG1 condensates exhibited hallmark liquid properties including fusion, surface wetting and ageing. Addition of purified 14-3-3γ dissolved SMAUG1 condensates in vitro, confirming that 14-3-3 proteins regulate SMAUG1 phase behaviour through direct interactions. In Chapter 4, we investigated how SMAUG1 phase separation influences its subcellular localisation and RNA interactome. Using motif prediction, in silico protein complex modelling, cell-based assays, and in vitro phenylalanine/glycine (FG) phase separation assays, we showed that SMAUG1 undergoes nucleocytoplasmic shuttling and identified a functional C-terminal nuclear export signal that promotes rapid export to the cytoplasm via exportin-1. Crucially, investigation of SMAUG1’s RNA targets in and out of condensates revealed a potential nuclear role for SMAUG1 in splicing through binding of intronic and exonic transcript regions. These findings expand SMAUG1’s functionality beyond translational repression and implicate it as a regulator of splicing, with the ability to bind more transcripts in its disperse state. Top RNA targets revealed further roles for SMAUG1 in nervous system development and degeneration, cell structure integrity, cell motility and guanosine triphosphatase (GTPase) enzyme regulation. Collectively, this thesis establishes SMAUG1 as a dynamically regulated phase-separating RBP that integrates intrinsic sequence features and interactions with 14-3-3 proteins to modulate phase behaviour. Meanwhile, phase state dictates SMAUG1’s RNA interactome in the nucleus and cytoplasm. These findings provide a mechanistic framework linking SMAUG1 condensate biology to processes associated with neurodevelopment, neurodegeneration and cancer, and highlight SMAUG1 as a potential target for future therapeutic intervention.","abstract_html":"RNA-binding proteins (RBPs) are central regulators of gene expression, influencing RNA processing, localisation, translation, and decay. A growing body of evidence shows that many RBPs exert their functions through the formation of dynamic, membrane-less organelles (MLOs) via liquid–liquid phase separation (LLPS). Dysregulation of these MLOs or “biomolecular condensates” is increasingly linked to human disease, including cancer and neurological disorders. Human SMAUG1 (SAMD4A) is a conserved translational repressor and condensate-forming protein implicated in neuronal function, metabolism, antiviral defence, and cancer. However, little is known regarding its condensate formation, localisation, and whole cell interactome. The aim of this PhD project was to define the molecular determinants of SMAUG1 phase separation, identify regulatory mechanisms controlling its condensates, and establish how condensate formation influences SMAUG1 localisation, protein interactome and RNA regulatory functions in the context of human health and disease. In Chapter 2, we defined the protein regions and molecular interactions that control SMAUG1 condensate formation in cells. Using protein domain and short linear motif (SLiMs) deletion mutants, we demonstrated that SMAUG1 phase separation is driven by its structured ‘Smaug similarity region 1’ (SSR1) dimerization domain and a novel intrinsically disordered prion-like domain. In contrast, the RNA-binding ‘sterile alpha motif’ (SAM) domain and other disordered regions were dispensable for condensation. We further identified members of the 14-3-3 protein family as potent negative regulators of SMAUG1 condensation. Dimeric 14-3-3 proteins that bind multiple phosphorylated motifs across SMAUG1 caused condensate dissolution. This work establishes a signalling dependent mechanism for dynamic control of SMAUG1 phase behaviour and with findings published as a first-author manuscript. In Chapter 3, we tested SMAUG1 condensation in vitro and defined its intrinsic phase separation behaviour. Recombinant SMAUG1 formed liquid-like condensates at low micromolar concentrations (0.1-2μM) under physiological salt (0-200 mM NaCl) and pH conditions (pH 7.5), in the absence of RNA or molecular crowders. SMAUG1 condensates exhibited hallmark liquid properties including fusion, surface wetting and ageing. Addition of purified 14-3-3γ dissolved SMAUG1 condensates in vitro, confirming that 14-3-3 proteins regulate SMAUG1 phase behaviour through direct interactions. In Chapter 4, we investigated how SMAUG1 phase separation influences its subcellular localisation and RNA interactome. Using motif prediction, in silico protein complex modelling, cell-based assays, and in vitro phenylalanine/glycine (FG) phase separation assays, we showed that SMAUG1 undergoes nucleocytoplasmic shuttling and identified a functional C-terminal nuclear export signal that promotes rapid export to the cytoplasm via exportin-1. Crucially, investigation of SMAUG1’s RNA targets in and out of condensates revealed a potential nuclear role for SMAUG1 in splicing through binding of intronic and exonic transcript regions. These findings expand SMAUG1’s functionality beyond translational repression and implicate it as a regulator of splicing, with the ability to bind more transcripts in its disperse state. Top RNA targets revealed further roles for SMAUG1 in nervous system development and degeneration, cell structure integrity, cell motility and guanosine triphosphatase (GTPase) enzyme regulation. Collectively, this thesis establishes SMAUG1 as a dynamically regulated phase-separating RBP that integrates intrinsic sequence features and interactions with 14-3-3 proteins to modulate phase behaviour. Meanwhile, phase state dictates SMAUG1’s RNA interactome in the nucleus and cytoplasm. These findings provide a mechanistic framework linking SMAUG1 condensate biology to processes associated with neurodevelopment, neurodegeneration and cancer, and highlight SMAUG1 as a potential target for future therapeutic intervention.","abstract_has_math":false,"creators":["Carey, Olivia"],"institution":"University College Cork","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Dean, Kellie"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-12-31","date_published":"2025-12-31","updated_at":"2026-07-24T01:48:42Z","subjects":["Liquid–liquid phase separation","Short linear motifs","SMAUG1","14-3-3","RNA-binding protein","RNA regulation"],"languages":["en"],"rights":["© 2025, Olivia Carey."],"rights_urls":["https://creativecommons.org/licenses/by-nc/4.0/"],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/10468/18898","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Dean, Kellie"]},{"key":"dc:creator","label":"Author","values":["Carey, Olivia"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2026-05-26T14:52:47Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2026-05-26T14:52:47Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-12-31"]},{"key":"dc:publisher","label":"Institution","values":["University College Cork"]},{"key":"dc:type","label":"Dc Type","values":["Doctoral thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["PhD - Doctor of Philosophy"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Liquid–liquid phase separation","Short linear motifs","SMAUG1","14-3-3","RNA-binding protein","RNA regulation"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["© 2025, Olivia Carey."]},{"key":"dc:rights.uri","label":"Rights URI","values":["https://creativecommons.org/licenses/by-nc/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10468/18898"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["RNA-binding proteins (RBPs) are central regulators of gene expression, influencing RNA processing, localisation, translation, and decay. A growing body of evidence shows that many RBPs exert their functions through the formation of dynamic, membrane-less organelles (MLOs) via liquid–liquid phase separation (LLPS). Dysregulation of these MLOs or “biomolecular condensates” is increasingly linked to human disease, including cancer and neurological disorders. Human SMAUG1 (SAMD4A) is a conserved translational repressor and condensate-forming protein implicated in neuronal function, metabolism, antiviral defence, and cancer. However, little is known regarding its condensate formation, localisation, and whole cell interactome. The aim of this PhD project was to define the molecular determinants of SMAUG1 phase separation, identify regulatory mechanisms controlling its condensates, and establish how condensate formation influences SMAUG1 localisation, protein interactome and RNA regulatory functions in the context of human health and disease. In Chapter 2, we defined the protein regions and molecular interactions that control SMAUG1 condensate formation in cells. Using protein domain and short linear motif (SLiMs) deletion mutants, we demonstrated that SMAUG1 phase separation is driven by its structured ‘Smaug similarity region 1’ (SSR1) dimerization domain and a novel intrinsically disordered prion-like domain. In contrast, the RNA-binding ‘sterile alpha motif’ (SAM) domain and other disordered regions were dispensable for condensation. We further identified members of the 14-3-3 protein family as potent negative regulators of SMAUG1 condensation. Dimeric 14-3-3 proteins that bind multiple phosphorylated motifs across SMAUG1 caused condensate dissolution. This work establishes a signalling dependent mechanism for dynamic control of SMAUG1 phase behaviour and with findings published as a first-author manuscript. In Chapter 3, we tested SMAUG1 condensation in vitro and defined its intrinsic phase separation behaviour. Recombinant SMAUG1 formed liquid-like condensates at low micromolar concentrations (0.1-2μM) under physiological salt (0-200 mM NaCl) and pH conditions (pH 7.5), in the absence of RNA or molecular crowders. SMAUG1 condensates exhibited hallmark liquid properties including fusion, surface wetting and ageing. Addition of purified 14-3-3γ dissolved SMAUG1 condensates in vitro, confirming that 14-3-3 proteins regulate SMAUG1 phase behaviour through direct interactions. In Chapter 4, we investigated how SMAUG1 phase separation influences its subcellular localisation and RNA interactome. Using motif prediction, in silico protein complex modelling, cell-based assays, and in vitro phenylalanine/glycine (FG) phase separation assays, we showed that SMAUG1 undergoes nucleocytoplasmic shuttling and identified a functional C-terminal nuclear export signal that promotes rapid export to the cytoplasm via exportin-1. Crucially, investigation of SMAUG1’s RNA targets in and out of condensates revealed a potential nuclear role for SMAUG1 in splicing through binding of intronic and exonic transcript regions. These findings expand SMAUG1’s functionality beyond translational repression and implicate it as a regulator of splicing, with the ability to bind more transcripts in its disperse state. Top RNA targets revealed further roles for SMAUG1 in nervous system development and degeneration, cell structure integrity, cell motility and guanosine triphosphatase (GTPase) enzyme regulation. Collectively, this thesis establishes SMAUG1 as a dynamically regulated phase-separating RBP that integrates intrinsic sequence features and interactions with 14-3-3 proteins to modulate phase behaviour. Meanwhile, phase state dictates SMAUG1’s RNA interactome in the nucleus and cytoplasm. These findings provide a mechanistic framework linking SMAUG1 condensate biology to processes associated with neurodevelopment, neurodegeneration and cancer, and highlight SMAUG1 as a potential target for future therapeutic intervention."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Analysis of SMAUG1 protein motifs and interacting partners - linking regulation of phase separation to human disease"]}]}],"canonical_facts":{"dc:contributor.advisor":["Dean, Kellie"],"dc:creator":["Carey, Olivia"],"dc:date.accessioned":["2026-05-26T14:52:47Z"],"dc:date.available":["2026-05-26T14:52:47Z"],"dc:date.issued":["2025-12-31"],"dc:description.abstract":["RNA-binding proteins (RBPs) are central regulators of gene expression, influencing RNA processing, localisation, translation, and decay. A growing body of evidence shows that many RBPs exert their functions through the formation of dynamic, membrane-less organelles (MLOs) via liquid–liquid phase separation (LLPS). Dysregulation of these MLOs or “biomolecular condensates” is increasingly linked to human disease, including cancer and neurological disorders. Human SMAUG1 (SAMD4A) is a conserved translational repressor and condensate-forming protein implicated in neuronal function, metabolism, antiviral defence, and cancer. However, little is known regarding its condensate formation, localisation, and whole cell interactome. The aim of this PhD project was to define the molecular determinants of SMAUG1 phase separation, identify regulatory mechanisms controlling its condensates, and establish how condensate formation influences SMAUG1 localisation, protein interactome and RNA regulatory functions in the context of human health and disease. In Chapter 2, we defined the protein regions and molecular interactions that control SMAUG1 condensate formation in cells. Using protein domain and short linear motif (SLiMs) deletion mutants, we demonstrated that SMAUG1 phase separation is driven by its structured ‘Smaug similarity region 1’ (SSR1) dimerization domain and a novel intrinsically disordered prion-like domain. In contrast, the RNA-binding ‘sterile alpha motif’ (SAM) domain and other disordered regions were dispensable for condensation. We further identified members of the 14-3-3 protein family as potent negative regulators of SMAUG1 condensation. Dimeric 14-3-3 proteins that bind multiple phosphorylated motifs across SMAUG1 caused condensate dissolution. This work establishes a signalling dependent mechanism for dynamic control of SMAUG1 phase behaviour and with findings published as a first-author manuscript. In Chapter 3, we tested SMAUG1 condensation in vitro and defined its intrinsic phase separation behaviour. Recombinant SMAUG1 formed liquid-like condensates at low micromolar concentrations (0.1-2μM) under physiological salt (0-200 mM NaCl) and pH conditions (pH 7.5), in the absence of RNA or molecular crowders. SMAUG1 condensates exhibited hallmark liquid properties including fusion, surface wetting and ageing. Addition of purified 14-3-3γ dissolved SMAUG1 condensates in vitro, confirming that 14-3-3 proteins regulate SMAUG1 phase behaviour through direct interactions. In Chapter 4, we investigated how SMAUG1 phase separation influences its subcellular localisation and RNA interactome. Using motif prediction, in silico protein complex modelling, cell-based assays, and in vitro phenylalanine/glycine (FG) phase separation assays, we showed that SMAUG1 undergoes nucleocytoplasmic shuttling and identified a functional C-terminal nuclear export signal that promotes rapid export to the cytoplasm via exportin-1. Crucially, investigation of SMAUG1’s RNA targets in and out of condensates revealed a potential nuclear role for SMAUG1 in splicing through binding of intronic and exonic transcript regions. These findings expand SMAUG1’s functionality beyond translational repression and implicate it as a regulator of splicing, with the ability to bind more transcripts in its disperse state. Top RNA targets revealed further roles for SMAUG1 in nervous system development and degeneration, cell structure integrity, cell motility and guanosine triphosphatase (GTPase) enzyme regulation. Collectively, this thesis establishes SMAUG1 as a dynamically regulated phase-separating RBP that integrates intrinsic sequence features and interactions with 14-3-3 proteins to modulate phase behaviour. Meanwhile, phase state dictates SMAUG1’s RNA interactome in the nucleus and cytoplasm. These findings provide a mechanistic framework linking SMAUG1 condensate biology to processes associated with neurodevelopment, neurodegeneration and cancer, and highlight SMAUG1 as a potential target for future therapeutic intervention."],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["https://hdl.handle.net/10468/18898"],"dc:language.iso":["en"],"dc:publisher":["University College Cork"],"dc:rights":["© 2025, Olivia Carey."],"dc:rights.uri":["https://creativecommons.org/licenses/by-nc/4.0/"],"dc:subject":["Liquid–liquid phase separation","Short linear motifs","SMAUG1","14-3-3","RNA-binding protein","RNA regulation"],"dc:title":["Analysis of SMAUG1 protein motifs and interacting partners - linking regulation of phase separation to human disease"],"dc:type":["Doctoral thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["PhD - Doctor of Philosophy"]},"updated_at":"2026-07-24T01:48:42Z"}