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 the basal transcriptional machinery. GR is expressed in virtually every tissue in the body, however transcriptional regulation of GR is highly promoter and cell type dependent. While transcriptional activation or repression changes absolute levels of mRNA transcripts, an additional layer of complexity is created in the cell by the alternative splicing of the pre-messenger RNA. Much progress has been made in the field of alternative splicing through the study of RNA binding proteins which are important in reading the “splicing code”, relatively little is known about how ligand-dependent transcriptional activation and alternative splicing are controlled in cells. Using a high-throughput mammalian cell co-transfection assay measuring GR-mediated luciferase output we screened a human brain cDNA library and uncovered a highly evolutionarily conserved protein named arginine and glutamate rich 1 (ARGLU1) that strongly potentiated GR transcriptional activation. ARGLU1 had no similarities to already uncovered NR coregulators but was just recently implicated as a co-activator of the estrogen receptor. Using a proteomics approach with a tandem affinity purified ARGLU1 complex we determined that ARGLU1 interacting proteins were primarily ribosomal proteins and RNA splicing factors. Intriguingly, we also found that ARGLU1 associated with JMJD6 (Jumanji domain containing 6), a histone arginine demethylase implicated in chromatin remodeling and alternative gene splicing. The proteomics data suggested that this may be a protein that could function in coupling transcriptional activation to alternative splicing. A number of earlier literature reports have shown that heterologous “mini-genes” driven by NR response elements can be modulated by NR activation. However, the in vivo significance of this was not possible to determine prior to the technological advances in exon sequencing that occurred over the past several years. With the currently available technology we would now be able to identify the endogenous role of these NR binding proteins in alternative splicing. This research program will examine the molecular mechanisms by which members of the NR superfamily help promote transcriptional diversity by coordinating ligand-dependent transcriptional activation with alternative splicing. Because over 95% of human genes are estimated to undergo alternative splicing, understanding how nuclear receptors contribute to the transcriptional diversity at the molecular level is essential for understanding how NRs function in cell and tissue-specific contexts.