How does variation in gene expression contribute to phenotypic diversity and disease? Gene expression involves transcription of DNA into mRNA, alternative splicing of mRNA, translation of mRNA into proteins, and regulation of mRNA and protein levels through turnover pathways. Research in the McManus lab focuses on understanding mechanisms that regulate mRNA translation, and how variation in RNA sequences and structures affects protein production. Our lab develops and employs novel high-throughput assay systems to identify RNA cis-regulatory elements and structures, and then quantitate their impact on mRNA translation using massively parallel reporter systems. We computationally mine the resulting data to distill features and develop models that predict the functions of mRNA transcript leaders. Our work addresses two major questions regarding translational control.

1. How do uORFs control translation? Upstream Open Reading Frames (uORFs) are short open reading frames found in mRNA transcript leaders that regulate mRNA translation and stability in eukaryotes. These fascinating regulatory elements control Intriguingly, many uORFs initiate at non-AUG (“near-cognate”) start codons (e.g. UUG). We use quantitative analysis of ribosome profiling to identify candidate uORFs in the yeast Saccharomyces cerevisiae and its sister species. This footprinting technique identifies the locations on ribosomes on mRNA genome wide. By computationally assessing the resulting profiles of ribosome occupancy, we have predicted thousands of candidate uORFs in three Saccharomyces species. To test their functions, we have developed massively parallel reporter assays that quantitate the regulatory impacts of thousands of uORFs simultaneously. We use the resulting data to build computational models that predict uORF functions.

2. How is translation regulated by a fungal pathogen during infection of a mammalian host? Candida albicans is a deadly fungal pathogen that causes thousands of deaths annually. During infection, the fungal pathogen C. albicans resists numerous stresses imposed by the host immune system, including restriction of heavy metals and exposure to oxidative stress. mRNA translation is highly regulated in response to stress conditions. However, little is known regarding how translational control contributes to virulence. In collaboration with the Mitchell (CMU) and Filler (UCLA) labs, we have developed novel approaches to assay genome-wide changes in mRNA translation during infection in a mouse host. By characterizing the dynamic translatome during infection, we expect to identify novel virulence mechanisms and potential drug targets.