Molecular Regulation of Alternative Splicing by Nuclear Receptors and Coregulators

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The nuclear hormone receptors comprise a superfamily of ligand activated transcription factors that regulate diverse physiological processes by coordinating patterns of gene expression. Transcriptional activation by a nuclear hormone receptor (NR) depends strongly on its interactions with coactivators and corepressors that either directly or indirectly modify the surrounding chromatin configuration to allow recruitment of basal transcriptional machinery. My laboratory is studying the molecular mechanisms contributing to changes in alternative splicing in response to NR hormone signaling. We recently discovered that the glucocorticoid receptor (GR) ligand Dexamethasone (Dex) can alter genome-wide patterns of alternative splicing (in addition to gene expression) in neuronal N2a cells within 4 hours of administration. Intriguingly, genes whose levels of mRNA expression were altered by Dex were largely distinct from those whose alternative splicing (AS) was affected. Thus, AS represents a new layer of gene regulation not previously appreciated for nuclear receptors. Recently, we discovered a new NR coregulator called arginine and glutamate rich 1 (ARGLU1) that plays a role in coactivating transcription as well as modulating AS, both basally and in response to glucocorticoid signaling. Using a proteomics approach, ARGLU1 was shown to interact primarily with RNA splicing factors. Interestingly, we also found that ARGLU1 associated with RBM39 (a member of the U2AF65 family that was previously shown to play a role in steroid-hormone mediated transcription and AS) and JMJD6 (Jumanji domain containing 6), a lysyl hydroxylase and arginine demethylase implicated in chromatin remodeling and AS. Prior studies found that JMJD6 can post-translationally modify splicing factors and impact their function. We will use a variety of gain and loss of function models incorporating the use of neural cells and in vivo inducible neuron-specific ARGLU1 knockout mice to analyze changes in gene expression/AS in response to GC treatment by RNAseq and correlate these to behavioural measures. To directly assess the genome-wide set of RNAs bound to ARGLU1 we will perform HITS-CLIP and compare the identity of the bound RNAs with those shown to be alternatively spliced and transcriptionally altered from RNAseq. We will use biochemical, molecular and proteomic methods to assess the importance of the interaction of ARGLU1 and GR with the chromatin modifier JMJD6 for mediating each of its distinct functions and gain a better understanding of how GR is contributing to AS. The study of RNA binding proteins has yielded significant progress in our understanding of the "splicing code", however, relatively little is known about how hormone-dependent AS is controlled in cells or tissues. This research program will examine the molecular mechanisms by which members of the nuclear receptor superfamily, including GR, help promote transcriptional diversity by coordinating ligand-dependent AS.