Every cell relies on precise and efficient gene expression. Multiple layers of controls operate at transcriptional (DNA to RNA) and post-transcriptional (RNA to protein) levels to govern faithful expression of genes into proteins. Much of the post-transcriptional fate of an mRNA rests in the hands of proteins that complex with RNA to form ribonucleoproteins (mRNPs). mRNP assembly begins as early as the precursor-to-mRNA (pre-mRNA) is transcribed, and proceeds as pre-mRNA is sculpted into mRNA during several processing steps. mRNPs continue to evolve throughout their lifetime, shedding proteins and acquiring others as they move from one cellular compartment to another and/or as they are acted upon by numerous macromolecular machines (e.g. the nuclear pore, the translating ribosome). How these dynamic RNA-protein machineries assemble and function to control mRNA fate remains under intense investigation to fully understand the fidelity and accuracy of gene expression. Utilizing experimental tools ranging from cutting-edge RNA-Seq based methods to more traditional yet ever-powerful biochemical and molecular approaches, we are investigating the following post-transcriptional phenomena in mammalian cell culture and whole animal models:
I. Control of post-transcriptional gene expression by the Exon Junction Complex
The Exon Junction Complex (EJC) is an extremely conserved multi-protein complex deposited ~24 nucleotides upstream of most mRNA exon-exon junctions during pre-mRNA splicing. The EJC is anchored on the RNA by eIF4AIII, which along with its co-factors, Y14, Magoh and MLN51, forms the EJC core. This core provides a platform for assembly of other peripheral EJC proteins that participate in pre-mRNA splicing and mRNA export from the nucleus, and in mRNA localization, translation and degradation in the cytoplasm. We previously uncovered the in vivo EJC interactome – its transcriptome-wide RNA binding sites and its proteome-wide interactions (Singh et al., Cell 2012). This work revealed that the EJC is arguably the most prominent mRNP constituent as it occupies the majority of exon junctions of human mRNAs. We have also discovered that in addition to the canonical exon junction sites, EJCs are also associated with non-canonical sites away from exon junctions. This connection is likely forged via EJC’s interactions with RNA binding proteins such as SR and SR-like proteins. We are now working to further understand the composition of the EJC at each assembly site at a transcriptome-wide scale, factors that influence EJC composition and how EJC composition impacts its function during post-transcriptional gene expression. We are also investigating the function of EJC and SR proteins in overall mRNP compaction, packaging and structure.
II. Developmental roles of the Exon Junction Complex in a vertebrate model (in collaboration with the Amacher lab)
The extraordinarily conserved EJC proteins eIF4AIII, Y14, and Magoh play critical roles during animal development. Mutations in these proteins cause several human disorders: reduction in Y14 levels causes thrombocytopenia-absent radius syndrome, eIF4AIII mutation results in an autosomal recessive disorder with cleft mandible and limb anomalies, and copy number variation of EJC core proteins underlies X-linked mental retardation syndrome and intellectual disability. Additionally, Magoh haploinsufficiency in the mouse causes microcephaly and pigmentation defects. These observations hint at shared and non-overlapping roles for EJC core proteins and/or EJC core regulation by cell- or tissue-specific regulators. However, the precise role of the EJC in post-transcriptional gene regulation during development and differentiation is largely unknown. We are studying EJC core functions during embryonic development using zebrafish, a vertebrate model amenable to genetics, cell biology, and high throughput molecular biology and biochemistry. We are creating and characterizing zebrafish EJC null mutants to reveal whether these ubiquitously expressed proteins have broad or more tissue specific functions during development and/or if they play common or non-overlapping roles. Intriguingly, despite extreme conservation, the EJC is thought to function in NMD only in mammals; we are also testing whether the zebrafish EJC core is required for NMD. Alternative non-NMD EJC functions that may regulate animal development are pre-mRNA splicing, mRNA export, localization, decay and translational regulation. Our RNA-Seq based molecular characterization of EJC core mutant embryos will assess some of these functions.