Funktionelle Konsequenzen von alternativem Spleißen in T-Zellen
Final Report Abstract
In the post genome era, it has become evident that in higher eukaryotes mechanisms other than regulated transcription play a fundamental role in controlling gene expression. Alternative splicing multiplies the number of possible proteins generated from a single pre-mRNA and therefore is one of the most abundant mechanisms to control protein expression and function post-transcriptionally. Splicing defects are associated with many human diseases, underlining the central importance of splicing for proper cellular function. The aim of the Emmy-Noether-Project was to investigate the mechanism and functionality of alternative splicing events during T cell activation. Naive and activated T cells have well defined functional differences and therefore represent a well-suited model system to investigate the contribution of alternative splicing to dynamic changes in cellular functionality. The specific aims of the present project were 1) to address the functionality of selected splicing events in a model T cell line, 2) to confirm functionality in physiological settings such a primary T cells or mouse models, and 3) to investigate the mechanistic basis for activation-induced alternative splicing of functionally important targets. For one alternative splicing events, exclusion of Traf3 exon 8 in activated T cells, we have addressed all three aims. Traf3 exon 8 skipping induces an NFkB signaling pathway in activated T cells that we suggest to be important for the function of T helper cells. This has been shown in a model T cell line and in primary human T cells (aims 1 and 2). This splicing switch is regulated through a protein called CELF2 that is preferentially expressed in activated T cells and directly binds to an intronic splicing silencer upstream of Traf 3 exon 8 (aim 3). In addition, we have analyzed the functionality of Sec16 alternative splicing in controlling an early step in protein export, ER-to-Golgi transport. Although activated T cells show a strong increase in secretory cargo, an adaptation of the early secretory pathway, mediated by COPII-coated vesicles, to this differential requirement had not been described. We were able to show that activated T cells indeed show increased ER-export efficiency and that this adaptation depends on Sec16 alternative splicing (aims 1 and 2). This is the first connection of alternative splicing with the early secretory pathway and one of the few examples, how a single alternative splicing events controls a fundamental cell biological process. While investigating the role of a splicing factor, U2AF26, in activated T cells, we noticed that U2AF26 is itself alternatively spliced and that U2AF26 alternative splicing is controlled in a rhythmic, time of the day dependent manner. This finding led us to start investigating alternative splicing in the context of the mammalian circadian clock, following the same aims as described above for T cell activation: what is the functionality and how is it regulated. We were able to show that rhythmic U2AF26 alternative splicing controls the molecular clock work through altering protein stability (aim 1); in addition, mice lacking U2AF26 show faster adaptation in jet-lag experiments, providing evidence for in vivo functionality of this rhythmic alternative splicing event (aim 2). We were then able to show that rhythmic U2AF26 alternative splicing is controlled by circadian changes in body temperature (aim 3). This is not only true for U2AF26, as we have identified a whole group of exons in functionally related genes, whose splicing pattern is controlled by body temperature cycles. This work provides the first evidence that alternative splicing contributes to the regulation of the mammalian circadian clock and shows for the first time that subtle changes in body temperature are sufficient to control a concerted alternative splicing switch. The contribution of alternative splicing to cellular function and identity remains enigmatic and represent one of the big and largely unsolved questions in RNA biology. By providing the first functional connection between alternative splicing and two fundamental processes, protein export and biological timekeeping by the circadian clock, the project has made a major contribution to this research field. In addition, the finding that very subtle changes in body temperature are sufficient to control alternative splicing, e.g. in a rhythmic manner, provides a new paradigm for splicing regulation in mammals and opens many new research directions.
Publications
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Alternative splicing controlled by heterogeneous nuclear ribonucleoprotein L regulates development, proliferation, and migration of thymic pre-T cells. J Immunol. 2012 Jun;188(11):5377–88
Gaudreau M-C, Heyd F, Bastien R, Wilhelm B, Moroy T
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Alternative splicing networks regulated by signaling in human T cells. RNA. 2012 May;18(5):1029–40
Martinez NM, Pan Q, Cole BS, Yarosh CA, Babcock GA, Heyd F, Zhu W, Ajith S, Blencowe BJ, Lynch KW
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Activation-induced tumor necrosis factor receptor-associated factor 3 (Traf3) alternative splicing controls the noncanonical nuclear factor kappaB pathway and chemokine expression in human T cells. J Biol Chem. 2014 May;289(19):13651–60
Michel M, Wilhelmi I, Schultz A-S, Preussner M, Heyd F
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Alternative splicing - principles, functional consequences and therapeutic implications. Dtsch Med Wochenschr. 2014 Feb;139(7):339–42
Heyd F
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Rhythmic U2af26 alternative splicing controls PERIOD1 stability and the circadian clock in mice. Mol Cell. 2014 May;54(4):651–62
Preussner M, Wilhelmi I, Schultz A-S, Finkernagel F, Michel M, Moroy T, Heyd F
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Alternative splicing of MALT1 controls signalling and activation of CD4(+) T cells. Nat Commun. 2016 Apr;7:11292
Meininger I, Griesbach RA, Hu D, Gehring T, Seeholzer T, Bertossi A, Kranich J, Oeckinghaus A, Eitelhuber AC, Greczmiel U, Gewies A, Schmidt-Supprian M, Ruland J, Brocker T, Heissmeyer V, Heyd F, Krappmann D
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Heterogeneous Nuclear Ribonucleoprotein L is required for the survival and functional integrity of murine hematopoietic stem cells. Sci Rep. 2016 Jun;6:27379
Gaudreau M-C, Grapton D, Helness A, Vadnais C, Fraszczak J, Shooshtarizadeh P, Wilhelm B, Robert F, Heyd F, Moroy T
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Sec16 alternative splicing dynamically controls COPII transport efficiency. Nat Commun. 2016 Aug;7:12347
Wilhelmi I, Kanski R, Neumann A, Herdt O, Hoff F, Jacob R, Preussner M, Heyd F
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Activation-Dependent TRAF3 Exon 8 Alternative Splicing Is Controlled by CELF2 and hnRNP C Binding to an Upstream Intronic Element. Mol Cell Biol. 2017 Apr;37(7)
Schultz A-S, Preussner M, Bunse M, Karni R, Heyd F
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Body Temperature Cycles Control Rhythmic Alternative Splicing in Mammals. Mol Cell. 2017 Aug;67(3):433–446.e4
Preussner M, Goldammer G, Neumann A, Haltenhof T, Rautenstrauch P, Muller-McNicoll M, Heyd F
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Post-transcriptional control of the mammalian circadian clock: implications for health and disease. Pflugers Arch. 2016 Jun;468(6):983–91
Preussner M, Heyd F
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The cancer-associated U2AF35 470A>G (Q157R) mutation creates an in-frame alternative 5’ splice site that impacts splicing regulation in Q157R patients. RNA. 2017;23:1796–806
Herdt O, Neumann A, Timmermann B, Heyd F
