Viruses have evolved mechanisms to use 5? untranslated regions (UTRs) to control viral protein synthesis during infection. For example, many RNA viruses use internal ribosome entry sites in the 5? UTRs. By contrast, poxviruses, such as Vaccinia virus, encode polyadenosine (polyA) repeats in the 5? UTR of post-replicative mRNA that enhance the translation of poxviral transcripts. The functionality of the polyA enhancer effect in human cells is dependent on poxvirus-mediated phosphorylation of an extended loop of the small subunit protein receptor of activated C kinase 1 (RACK1). Although we know ribosome specification plays a key role in selectively regulating poxviral protein synthesis, it is unclear how negative charge in the loop regulates mRNA selectivity of poxvirus-customized ribosomes and how this selectivity is influenced by global changes in cellular transcriptional and translational activity during infection. Using structure modeling and biochemical approaches, we have previously shown that charge in the RACK1 loop increases repulsive electrostatic interactions with the negatively charged backbone of 18S rRNA near the mRNA exit channel. Therefore, we hypothesize that introduction of negative charge remodels the loop or mRNA exit channel to more broadly regulate translation by altering recognition of specific 5? UTR elements, such as polyA leaders. We will address this hypothesis by developing two complementary cell systems that we will use to study RACK1-loop mediated mRNA selectivity under different conditions that mimic aspects of poxvirus infection. We will use polysome profiling and RNA-Seq to analyze global changes in translational efficiency to gain a broader understanding of transcript-specific translation modulated by the RACK1 loop. The findings from the proposed research will lay a solid foundation for our understanding of how poxviruses control translation, as well as future studies of ribosome-centric modes of translation regulation in diverse biological and pathological contexts.
Growing evidence suggests that ribosome specification enables selective translation through recognition of specific 5?UTR elements in a given transcript. We recently revealed how Vaccinia virus customizes ribosomes through phosphorylation of RACK1 to enhance translation of viral mRNAs that contain unusual 5? polyA leaders. The proposed research aims to determine how vaccinia virus-modified RACK1 regulates transcript specificity during cellular reprogramming, with broader relevance to our understanding of ribosome function in diverse pathological contexts.