Analysis of queuosine and 2-thio tRNA modifications by high throughput sequencing

Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas the substrate for Q-modification in tRNA in mammals is queuine, the catabolic product of the Q-base of gut bacteria. This gut microbiome dependent tRNA modification may play pivotal roles in translational regulation in different cellular contexts, but extensive studies of Q-modification biology are hindered by the lack of high throughput sequencing methods for its detection and quantitation. Here, we describe a periodate-treatment method of biological RNA samples that enables single base resolution profiling of Q-modification in tRNAs by Nextgen sequencing. Periodate oxidizes the Q-base, which results in specific deletion signatures in the RNA-seq data. Unexpectedly, we found that periodate-treatment also enables the detection of several 2-thio-modifications including tm5s2U, mcm5s2U, cmnm5s2U, and s2C by sequencing in human and E. coli tRNA. We term this method Periodate-dependent analysis of queuosine and thio modification sequencing (PAQS-seq). We assess Q- and 2-thio-modifications at the tRNA isodecoder level, and 2-thio modification changes in stress response. PAQS-seq should be widely applicable in the biological studies of Q- and 2-thio-modifications in mammalian and microbial tRNAs. Overall design: HEK293T cells were grown with and without queuine in duplicate. Extracted RNA was used to prepare libraries with and without periodate treatment : 8 libraries total. Next, RNA from HEK293T cells with and without queuine was mixed at a ratio of 0% from cells with Q, 10%, 20%... 100% cells with Q. This calibration curve involves 11 libraries in total. Next, RNA extracted from E. coli cells was used to prepare libraries with and without periodate treatment: 2 libraries. Finally, E. coli were grown in duplicate. Each culture was split into 4, and subjected to 1 of 3 stresses or a control. RNA was extracted from these culutures, treated with periodate, and used to prepare libraries: 8 samples. In total this project involves 29 librarires.