This work involves the characterization of two genes using mouse models of gene deficiency and thus has two focuses: Focus I: Innate immune responses are vital for pathogen defence but can result in septic shock when excessive. A key mediator of septic shock is TNFα, which is shed into intercellular spaces after cleavage from the plasma membrane by the protease TACE. Here we report that the rhomboid family member iRhom2 interacts with TACE and regulates TNFα shedding in vitro and in vivo. Compared to controls, gene-targeted iRhom2-deficient mice showed reduced serum TNFα after LPS challenge survived a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to adequately control the replication of Listeria monocytogenes and thus succumbed to even mild infections. Our study has identified iRhom2 as a novel regulator of innate immunity that may be an important target for modulating sepsis and pathogen defence. Focus II: Smg1 is a phosphatidylinositol 3-kinase-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbour premature termination codons (PTCs) due to events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. Using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5 (E8.5). High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from ~9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signalling, membrane dynamics, cell death and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

This work involves the characterization of two genes using mouse models of gene deficiency and thus has two focuses: Focus I: Innate immune responses are vital for pathogen defence but can result in septic shock when excessive. A key mediator of septic shock is TNFα, which is shed into intercellular spaces after cleavage from the plasma membrane by the protease TACE. Here we report that the rhomboid family member iRhom2 interacts with TACE and regulates TNFα shedding in vitro and in vivo. Compared to controls, gene-targeted iRhom2-deficient mice showed reduced serum TNFα after LPS challenge survived a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to adequately control the replication of Listeria monocytogenes and thus succumbed to even mild infections. Our study has identified iRhom2 as a novel regulator of innate immunity that may be an important target for modulating sepsis and pathogen defence. Focus II: Smg1 is a phosphatidylinositol 3-kinase-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbour premature termination codons (PTCs) due to events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. Using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5 (E8.5). High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from ~9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signalling, membrane dynamics, cell death and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

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