MFB: RNA modifications of frameshifting stimulators: cellular platforms to engineer gene expression by computational mutation predictions and functional experiments

In this Molecular Foundations for Biotechnology (MFB) project, Dr. Tamar Schlick from New York University and Dr. Alain Laederach from the University of North Carolina will develop advanced computational tools to predict and control how viral protein synthesis is affected when the cell's machinery (the ribosome) shifts and thus changes how the three-letter code in messenger RNA (mRNA) is read. This frameshifting in translating the mRNA triplet code has been found to be preprogrammed in viruses and human cells to modify the expression of gene products and to regulate biochemical processes. This study aims to computationally predict and experimentally test how introducing mutations to mRNA affects its three-dimensional structure and, consequently, programmed frameshifting in prototypical viral genomes. Revealing the specific structural and sequence requirements for frameshifting in prototype viruses will facilitate the design of novel efficient frameshifting elements, with potential applications to viral packaging of genes. This project will provide interdisciplinary training to students in mathematics, computer science, biology, physics, chemistry, and engineering, with particular emphasis on enhancing minority participation in STEM activities. Public outreach efforts will be included to reach general audiences and highlight the intersection of mathematics, biology, computing, and biotechnologies that have implications in human health. Programmed ribosomal frameshifting (PRF) is a widespread mechanism for modifying the gene expressed by altering the mRNA triplet-nucleotide transcript to generate an alternate gene product. Indispensable to many viruses including HIV and SARS-associated coronaviruses for translating overlapping mRNA reading frames, PRF is also a mechanism in endogenous human, eukaryotic and prokaryotic genes. Because PRF has been shown to dramatically influence viral viability or the biochemical regulation of human processes, the modulation of frameshifting defines a platform for engineering gene expression. However, the complex aspects of frameshifting and the structural plasticity of the RNA frameshifting element (FSE) must be understood before engineering and therapeutic strategies can succeed. In this synergistic biological, chemical, mathematical, and computational research program, graph-theory-based tools will be developed to predict FSE mutations for prototype viral systems aimed at substantially lowering frameshifting efficiency as a novel biotechnological strategy against viral infections and related human diseases associated with PRF. The effect of these mutations will be assessed by Luciferase assay measurements, and the resulting FSE structural landscapes analyzed by techniques suitable for RNAs with multiple conformations. Besides an improved understanding of the mechanisms of frameshifting and computational tools for predicting FSE-landscape-altering mutations, this project will produce new biotechnological, RNA modifying tools as potential therapeutic agents against RNA viruses or applicable to human and other genes that employ frameshifting. Applications to viral packaging/drug delivery also arise, as frameshifting is a compact mechanism to store gene coding information and can be exploited to overcome genomic size limitations. This project is jointly funded by the Division of Chemistry (CHE), the Division of Mathematical Sciences (DMS), and the Division of Physics (PHY) in the Directorate for Mathematical and Physical Sciences (MPS). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.