The Roles of Microbe-Derived Transfer RNA Modifications in Eukaryotic Cell Physiology

Across all kingdoms of life, transfer ribonucleic acid (tRNA) is essential for translation of mRNA into protein. These adaptor molecules, comprising 75-95 nucleotides, decode mRNA codons via base pairing and carry the amino acid dictated by the genetic code to the ribosome. The typical tRNA adopts a cloverleaf secondary structure and an L-shaped tertiary structure. This conserved conformation enables tRNA to engage with both the mRNA codon and the ribosome during translation. In eukaryotic cells, tRNAs are both the most abundant and the most extensively modified cellular RNA species. Indeed, eukaryotic tRNAs carry an average of 13 modifications per cytosolic tRNA and an average of 5 modifications per mitochondrial tRNA. Over 100 different tRNA modifications have been described to date. Though diverse in chemical structure and location, modifications are broadly important in maintaining tRNA stability and fine- tuning translational dynamics. A modification of particular interest is queuosine (Q), a hypermodified guanosine analog found at position 34 in the anticodon of cytosolic and mitochondrial tRNAs for asparagine, aspartic acid, histidine, and tyrosine. While bacteria can synthesize queuosine-tRNA (Q-tRNA) de novo, eukaryotes must import extracellular queuine (the corresponding nucleobase of Q nucleotide) sourced from the environment (i.e., gut microbiome or diet). Q-modification is implicated in modulating decoding accuracy and decoding speed of synonymous NAC/U codons. However, despite being discovered decades ago, the full biological significance of queuosine is still being actively explored. In Chapter 2, we investigate the role of Q-modification on mammalian cell physiology using proliferation experiments and multiplex small RNA-seq (MSR-seq). We report that Q-modification promotes proliferation in both HEK293T cells and murine bone marrow-derived dendritic cells (BMDCs). In both cell types, we identify a novel correlative relationship between the Q and m22G (N2,N2-methylguanosine) modifications. Our mRNA-seq results show a cell-type-specific transcriptomic response to Q- modification levels. Using the same data, we propose a codon-usage-based mechanism underlying the Q-dependent changes in transcription and proliferation. In Chapter 3, we study how preQ1, a metabolic precursor of bacterial Q-tRNA, impacts HEK293T cells and BMDCs. We find that preQ1 generally attenuates cell proliferation in a cell-cycle-independent manner, a phenotype that can be rescued by co-treatment with queuine. By conducting mRNA-seq, we also identify global changes to the BMDC transcriptome in response to preQ1 treatment. In Chapter 4, we focus on BMDCs and their cell-specific functions. Using flow cytometry, we demonstrate that both queuine and preQ1 impact the expression of MHC- II, an immune response mediator. Finally, in Chapter 5, we characterize two photoactivatable preQ1 analog probes in mammalian cell culture. We find that the probes exhibit queuine-like bioactivity and are covalently inserted into Q-modifiable tRNAs. Moreover, we present preliminary RT- qPCR results revealing a potential role for preQ1 and queuine in regulating mammalian histone mRNA levels. Together, this work sheds light on the functions of Q and its derivatives, underscoring their importance in eukaryotic cell physiology.