Temporal control of spliceosome activity to modulate splicing switches during T cell activation and serum stimulation.
Precursor messenger RNA (pre-mRNA) splicing is mediated by a large and dynamic molecular RNA-protein (RNP) machine, the spliceosome. Pre-mRNAs of higher eukaryotes are typically alternatively spliced, leading to the production of multiple mature mRNAs from a single protein-coding gene. Upon differentiation or stimu- lation, cells undergo dramatic changes in gene expression, which induce functional changes and the develop- ment of effector cell phenotypes. Some of these changes in gene expression are the result of switches in alternative splicing programs. Project A15 investigates the molecular mechanisms underlying such functional splicing switches. Specifically, we focus on the roles of signaling cascades/post-translational modifications in installing and regulating splicing switches. In the past funding period, we have delineated and characterized a concerted “immediate early” splicing switch that is implemented shortly after T cell activation via RAF/MEK/ERK pathway-dependent phosphorylation of the key splicing regulator, hnRNPC. This splicing switch leads to increased intron retention in many pre-mRNAs encoding ribosomal proteins and translation- associated factors. It may thereby modulate the translation capacity of T cells in a temporal manner shortly after stimulation. Furthermore, we provided proof-of-principle that an “anchor-away” strategy targeting splicing factors, based on reversible chemical dimerizers, may enable a precise temporal control of splicing. We have also further investigated the inner workings of a key spliceosomal NTPase and the structural basis and func- tional consequences of a higher eukaryote-specific splicing factor composition, which may be involved in the implementation or modulation of splicing switches. In the coming funding period, we strive to further charac- terize the identified immediate early splicing switch and its link to a switch in translation, delineate structural principles underlying this splicing switch and further refine and apply strategies to temporally control splicing by anchor-away and targeted protein degradation strategies.