Saturday, May 4, 2024
5/4/2024
ABSTRACT The exon junction complex (EJC) is deposited by the spliceosome upstream of exon–exon junctions during the process of pre-mRNA splicing. If an exon–exon is sufficiently downstream of either a normal termination codon or a premature termination codon (the latter of which can be generated by splicing errors or frameshift or nonsense mutations), then EJC-dependent nonsense-mediated mRNA decay (NMD) is triggered. NMD destroys aberrant mRNAs and regulates the normal gene expression of ~5-10% of unmutated mRNAs. I recently discovered that AKT, a central cell signaling kinase, is a constituent of an alternative EJC containing CASC3 (the AKT EJC) but not UPF2 or RNPS1 (in the canonical UPF2 EJC). AKT functionally replaces UPF2 in UPF2- independent NMD by activating the key NMD factor UPF1. AKT does so by phosphorylating UPF1; UPF2 does so by binding UPF1. I found that insulin-mediated activation of AKT signaling increases AKT EJCs at the expense of UPF2 EJCs, which is the first example of cell-signaling mediated regulation of EJC composition. These findings are important because AKT signaling and NMD, which converge at the EJC, are both dysregulated in some cancers, Fragile X syndrome, and type 1 and 2 diabetes. Despite its importance, there are many remaining uncertainties about how EJCs are formed, including: when and how the commitment to either the AKT EJC or UPF2 EJC is made; the mechanism by which insulin increases AKT EJC formation; if insulin increases and/or alters EJC binding at exon-exon junctions; if spliceosomes are involved in alternative EJC formation; the choreography of steps involved in core EJC formation, presumably on the spliceosome; and how EJCs are transferred to upstream of exon-exon junctions on newly spliced mRNA, to name a few. In this proposal, I will start addressing some of these uncertainties while receiving training in concepts and techniques from my mentoring team so as to continue pursuing these questions in my own lab. In Aim 1 (K99), I will test (i) the precursor-product relationship between UPF2 EJCs and AKT EJCs (while learning how to generate and use HEK293T cells engineered for auxin-induced degradation of EJC proteins), (ii) if the insulin-promoted increase in AKT EJCs and decrease in UPF2 EJCs requires splicing, and (iii) if insulin signaling increases and/or alters the number of EJC-bound exon–exon junctions (while learning RIP-Seq). In Aim 2 (K99), I will identify and define any insulin-promoted post-translational modifications (PTMs) on EJC proteins that promote the increase in AKT EJCs and decrease in UPF2 EJCs (while learning mass spectrometry (MS)-based proteomics and PTM profiling). In Aim 3 (R00), I will apply my background in mechanistic biochemistry and training in new techniques to build an independent research program aimed at elucidating yet-undetermined molecular mechanisms in EJC biology, starting by defining which splicing factors are needed for core EJC assembly, which splicing factors, if any, are involved in alternative EJC formation, and for the latter, whether PTMs of splicing factors are involved.
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