The Djuranovic lab is interested in developing methods to evaluate the cause and mechanism of translational repression by miRNAs and RBPs (Cottrell et al., 2016, 2017, 2018). We have developed methods for fast RNA pull-downs using previously engineered RNA recognition motifs (RRM, Cottrell et al., 2016) and established that translational efficiency is a major determinant of the magnitude of miRNA-mediated translational repression (Cottrell et al. 2017). Using an unbiased approach with Massively Parallel Reporter Gene Assay (MPRA), we further established that miRNAs repress translation at the step of translation initiation that leads to the reduction in translation efficiency (Cottrell et al 2018). Besides providing further evidence for the mechanisms of post-transcriptional regulation by miRNAs, Pumilio and AU-Rich elements (AREs), we also established PTRE-seq as a valuable tool for studying the effects of multiple cis-acting elements on RNA stability and translational control.
We are continuing our pursuit towards understanding molecular mechanisms of miRNA- and RBP-mediated translation regulation using in vitro kinetics and thermodynamics as well as in vivo reporter or global gene expression approaches. We have invested our knowledge in gene attenuation techniques to create sets of cell lines where certain RBPs are knocked-out or expressed at hypomorphic levels. We are now following the impact of these proteins on miRNA-mediated translational repression as well as on overall cellular homeostasis.
In addition, using our PTRE-Seq assay, we have started to assay the impact of 5’ and 3’ UTR mutations in tumor-suppressor genes and oncogenes found in cancer patients (tissue cultures) and in collaboration with Dougherty Lab at Department of Genetics, mutations found in autism-spectra disorder patients using brain specific delivery of libraries in mouse models.
In yet another approach we are analyzing the impact of other RNA regulatory elements such as upstream open reading frames (uORFs) and/or internal ribosome entry sites (IRESs) in combination with specific mRNAs to define the earliest steps in translation initiation that are targeted by miRNAs. These studies are complemented by genome-wide approaches aimed at investigating the influence of specific miRNAs across all endogenous gene targets. Since these approaches are not restricted to miRNA-mediated gene silencing processes, we further plan on using similar techniques to more deeply explore the mechanisms of post-transcriptional gene regulation caused by stress (selective translation), by the inhibitory/stimulatory effects of uORFs, by eIF4E inhibitory proteins and by other specific mRNA elements.
