An analysis of the putative role of ROQUIN as a T cell RING-directed ubiquitin ligase

Abstract

An analysis of the putative role of ROQUIN as a T cell RING-directed ubiquitin ligase Abstract: Acquiring long-term protective immunity involves the generation and recall production of high affinity antibodies by B cells in response to foreign antigen. This occurs only with the help of T cells. T follicular helper (Tfh) cells in particular are specialised in homing to B cell-enriched follicles and germinal centres (GCs) of secondary lymphoid organs where they provide selective paracrine signals to support high affinity surface immunoglobulin expression on mutating B cells that differentiate into long-lived plasma cells and memory B cells. Deregulation of Tfh cells leads to a breakdown in the negative selection of pathogenic GC B cell mutants that seed lymphomas or produce disease-causing autoantibodies. It is thus in this context, for the potential to realise biomedical innovations, important to characterise signalling networks that control Tfh cell development and effector function. Here, we sought to map the signalling network of an incompletely understood Tfh cell immunomodulator, ROQUIN, encoded by the Rc3h1 locus. ROQUIN, along with its only mammalian paralogue, ROQUIN2 harbours a unique ROQ domain indispensible in RNA regulation and in preventing unrestricted Tfh cell accumulation that causes systemic autoimmunity. Upstream of the ROQ domain is the ROQUIN amino terminus containing the most evolutionarily conserved sequence and a predicted but uninspected RING domain associated with E3 ubiquitin ligase activity. To determine the putative role of this ROQUIN RING domain in T cell-dependent B cell immunity, we generated a novel mouse model (Tringless), harbouring a T cell-specific genetic deletion of Rc3h1 exon 2, encoding for the RING domain. In vivo analysis of Tringless mice revealed minimal perturbations in ROQUIN ROQ-RNA signalling but an unexpected requirement for T cell ROQUIN RING activity in selectively promoting Tfh cell and GC responses to T-dependent antigens. The development of other effector T cell subsets (Th1, Th2, Th17 and Treg) was not affected. In order to identify potential substrates of the ROQUIN RING domain, we systematically screened for protein partners able to directly bind ROQUIN. Adenosine Monophosphate- activated Protein Kinase (AMPK) was discovered to be targeted by ubiquitin-dependent ROQUIN RING signalling, which was required to limit aberrantly high AMPK activity. Well established as an antagonist to mTOR metabolic signalling and as a central regulator of cellular bioenergetics and proliferation, AMPK activity has not been previously described in Tfh cells. Using an mTOR-deficient mutant mouse strain (chino), we confirmed mTOR as an essential pathway in Tfh cell formation. Consequently, we qualify ROQUIN as a bone fide RING-driven E3 ubiquitin ligase and report a novel ROQUIN-AMPK-mTOR metabolic nexus that our data suggests is important in promoting Tfh cell responses. Show more