Regulation of protein synthesis by PUS1-mediated mRNA modification in hepatocellular carcinoma - PROJECT SUMMARY The tumor stage and grade of patients with hepatocellular carcinoma (HCC) are significantly and directly correlated with the expression of pseudouridine (Ψ) synthase 1 (PUS1). PUS1 installs the second-most abundant modified RNA nucleotide in human messenger RNA (mRNA), Ψ. Recently, work has described that PUS1 enhances HCC growth in mouse models for HCC and in HCC human cell lines. The work further stated PUS1 enhances Ψ on oncogenic mRNAs in these models. Ψ has been previously implicated in affecting RNA-RNA and RNA-protein interactions, pre-mRNA splicing in introns, mRNA translation in mRNA coding regions, and mRNA half-life. Still, how HCC uses PUS1 and Ψ to progress is unknown. Which Ψ on which mRNAs are sufficient for changes in translation? Are they sufficient to drive the growth observed in cells and mice? How might Ψ mechanistically affect these phenotypes? These are critical knowledge gaps in how PUS1 enhances HCC oncogenesis. I hypothesize that modification of HCC-driving mRNAs with Ψ by PUS1 changes translation selectively by altering intra- and intermolecular interactions these mRNA transcripts to alter HCC growth and viability. I have successfully purified recombinant human PUS1, have successfully installed a library of human mRNAs with Ψ with in vitro PUS1 treatment, and have used Direct Analysis of Ribosome Targeting (DART) in HeLa cell-free extract to assess translation initiation in the context of PUS1 treatment. My preliminary data demonstrate an active role for PUS1 in HCC through the installation of Ψ in the 5 untranslated regions (UTRs) of canonical HCC gene transcripts. Here, PUS1 installs many Ψ residues across mRNA 5 untranslated regions (UTRs). Importantly, PUS1 modifies the 5 UTRs of 188 cancer genes, including the canonical drivers of HCC: Myc, ARID1A, and -catenin, as well as its cofactor TCF7. Further, I demonstrate in that PUS1 treatment of mRNA transcripts enhances the translation initiation of these oncogene transcripts relative to mock-treated controls. These data suggest single Ψ in the 5 UTRs of oncogenes are sufficient to drive translational differences. In Aim 1, I will comprehensively assess how PUS1 and Ψ alter the translation of -catenin, TCF7, MYC, ARID1A, and 26 other common cancer genes by DART in HCC cell-free extracts, by luciferase reporter assays in cells, and by measuring endogenous transcript and Ψ levels in polysome fractions. In Aim 2, I will test how PUS1 perturbation contributes to HCC growth and viability, as well as the downstream genetic and metabolic networks of -catenin, TCF7, MYC, ARID1A. In Aim 3, I will assess which RNA binding proteins and structures are preferred in Ψ-modified -catenin relative to an unmodified control. I will further test the necessity of these interactions to enhanced translation of Ψ-modified -catenin in polysome gradients through genetic depletion and structural mutagenesis. The success of this work will describe mechanistically how PUS1 in HCC drives differences in translation to support oncogenesis.
