Guramrit Singh
Control of post-transcriptional gene expression by dynamic ribonucleoprotein (RNP) complexes
Research Interests
Be it a run-of-the-mill epithelial cell or an activated T-cell hot on the trail of an invading pathogen, 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. The overall vision of our research is to gain knowledge of the inner workings of molecular machineries that operate at a post-transcriptional level to ensure optimal gene expression, and understand how their dysfunction may cause disease.
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 gain deeper insights into fidelity and accuracy of gene expression. Utilizing experimental tools ranging from cutting-edge RNA-Seq based high-throughput methods to more traditional yet ever-powerful biochemical and molecular approaches, we are investigating the following post-transcriptional phenomena in cultured mammalian cell lines:
Connectivity between seemingly distant steps in post-transcriptional gene expression
In comparison to transcription that is confined to euchromatic territories in the nucleus, post-transcriptional events (i.e. splicing, nuclear export, mRNA transport, translation and degradation) are spread over a much more vast cellular landscape. It is now well established that, although seemingly unlinked, many of these post-transcriptional steps are interconnected. Coupling between different steps of gene expression can increase efficiency and ensure tight quality control during each step, but the underlying mechanisms remain poorly understood.
One of the best-known molecular links between post-transcriptional steps is the Exon Junction Complex (EJC), 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 the DEAD-box protein eIF4AIII, which along with its co-factors, Y14 and Magoh, 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 localization, translation and degradation of messages in the cytoplasm. Intriguingly, mutations in genes encoding EJC core and peripheral proteins lead to several neurological and developmental disorders. Recently, we uncovered the in vivo EJC interactome – its transcriptome-wide RNA binding sites and its proteome-wide interactions (Figure 1; Singh et al., Cell 2012). This work revealed a new connection between EJC and SR proteins, another key class of mRNP constituents. Moreover, a function for EJC in mRNP compaction and packaging was uncovered (Figure 2). We aim to build on this work to further understand how a multitude of peripheral proteins work together with the EJC core to control and connect several steps in post-transcriptional gene expression.
