Two microbiome metabolites compete for tRNA modification and mammalian cell proliferation

Microbiome interacts with the eukaryotic host through many metabolites to affect cell physiology. A direct molecular pathway of microbial-host interaction is the incorporation of the microbial metabolite queuine at the wobble anticodon nucleotide of host tRNAs by a host enzyme that regulates host cell translation. Microbes also produce the intermediary metabolite pre-queuosine1 (preQ1) in the queuine pathway, and how preQ1 affects the host cell biology has not been explored. Here we show that preQ1 strongly represses human and mouse cell proliferation, but this effect is suppressed or reversible with queuine and depends on the same host enzyme that installs queuosine tRNA modification. PreQ1 and queuine are present in plasma and mouse tissues and incorporated into tRNA in cells and in mice, and preQ1 reduces mouse xenograft tumor growth. Mechanistically, preQ1 reduces cognate tRNA levels specifically and translation of house-keeping genes in a highly codon dependent manner. Genomic-wide CRISPR screen and validation identify pathways in sterol biosynthesis regulation, lipid metabolism, and Golgi-ER transport that mitigate the preQ1 proliferation effects through alleviation of cognate tRNA levels. Our results show an inter-dependent relationship of two microbial metabolites from the same biosynthesis pathway that compete and regulate host cell proliferation through multiple pathways. Overall design: Study the effect of preQ1 and queuine on tRNA levels in HEK293T cells using control, preQ1, and/or queuine treated total RNA . Study the effect of preQ1 on protein translation in HEK293T cells using control or preQ1 treated input and polysome associated mRNA. Study the queuosine modification in different bacterial classes using human stool RNA samples. Study the effect of preQ1 and queuine on tRNA levels in BMDC cells using control, preQ1, and/or queuine treated total RNA. Study the effect of IRE1 inhibitor on preQ1 effect on tRNA levels. Study the effect of +/- PNK treatment on tRNA fragments levels in sequencing. Study the effect of preQ1 on translation using ribo-seq with input mRNA and ribosome protected fragments (RPF) RNA.