KEEPING IT IN FRAME: MONITORING RIBOSOMAL FRAMESHIFTING DURING TRANSLATION

Abstract

Programmed -1 ribosomal frameshifting (-1 PRF) is a molecular mechanism that redirects translating ribosomes into a new reading frame. It is widely used by RNA viruses to conserve genome space while expanding the viral proteome and it can help regulate gene expression in eukaryotic cells. Strict regulation of both programmed and non-programmed frameshift events are essential to translational fidelity. This dissertation explores the -1 PRF element of SARS coronavirus 2 (SARS-CoV-2) and the -1 PRF inhibitor, Shiftless. We comparatively analyzed the structural and functional conservation of -1 PRF elements in SARS-CoV and SARS-CoV-2. Both -1 PRF structure and frameshift efficiency were highly conserved between the two viruses and a small molecule effective against SARS-CoV -1 PRF significantly decreased frameshift efficiency in SARS-CoV-2. This suggests -1 PRF is an attractive antiviral target and could be a useful tool to combat the SARS-CoV-2 pandemic or future outbreaks of similar coronaviruses. The innate immune system targets viral frameshifting using an interferon-stimulated -1 PRF inhibitor called Shiftless (SFL) that binds, arrests, and terminates translation of -1 frameshifted ribosomes. We found that SFL is not only expressed in response to interferon but that it may have a role in general translational fidelity. SFL is constitutively expressed at low levels in human-derived cell lines and its effects are not limited to -1 PRF signals. Disruption of SFL homeostasis results in reciprocal 2-fold changes to recoding efficiencies in a panel of human and viral-derived translational recoding signals, decreases reporter gene expression, and decreases mRNA steady state abundances. Additionally, SFL over or under expression combined with knockdown of prominent ribosome-associated protein quality control (RQC) proteins reveals that SFL is epistatic to RQC. These results suggest that SFL has a role in general translational fidelity monitoring for spontaneously frameshifted ribosomes in addition to its role as a member of the innate immune response.