Objective Ribonucleic acids (RNA) are involved in many cellular functions. In general, RNA is made up by only four different ribonucleotides. The modifications of RNA (epitranscriptome) can greatly enhance the structural diversity of RNA, which in turn support some of the RNA functions. To determine whether the epitranscriptome of a specific probiotic is associated with its adaptation to the source of energy, Lactobacillus agilis (YZ050) was selected as a model and its epitranscriptome was profiled and compared by using mass spectrometry. Results The L. agilis epitranscriptome (minus rRNA modifications) consists of 17 different RNA modifications. By capturing the L. agilis cells during exponential growth, reproducible profiling was achieved. In a comparative study, the standard source of energy (glucose) in the medium was substituted by a prebiotic inulin, and a downward trend in the L. agilis epitranscriptome was detected. This marks the first report on a system-wide variation of a bacterial epitranscriptome that resulted from adapting to an alternative energy source. No correlation was found between the down-regulated RNA modifications and the expression level of corresponding writer genes. Whereas, the expression level of a specific exonuclease gene, RNase J1, was detected to be higher in cells grown on inulin.

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The molecular structure of both coding and non-coding RNA is frequently altered by the enzymatic activity of either writer or eraser of RNA modifications. Since the RNA functions are dependent on its molecular structure, cellular activities that involve specific RNA molecule(s) can potentially be regulated by the reversible nature of RNA modifications. There are more than 140 known RNA modifications. Mass spectrometry is a proven technique for the identification of RNA modifications. However, due to the lack of ribonucleoside standards and biases in normalizing the level of RNA modifications, it remains a challenge to perform the quantitative analysis of multiple RNA modifications that may co-exist in a biological sample. Our group has recently developed a method for standard-free quantitative epitranscriptomic profiling (SqEP) that allows us using mass spectrometry to accurately determine the abundancy of each detectable RNA modification without using any ribonucleoside standards to calibrate the detection signals. Thus, the SqEP method can be used for the untargeted analysis of RNA modifications. It also allows us to directly compare the level of different RNA modifications that are detectable in an individual cell line. This is an important feature for studying the relationships between various RNA modifications. Using glioblastoma (GBM) as our model, the results from using the SqEP method to determine the level of specific RNA modifications in patient-derived GBM cell lines were shown to be more accurate than the existing methods, and complied with the gene expression data from GBM patients that are available in the Cancer Genome Atlas (TCGA) program. Citation Format: Norman H. Chiu, Jennifer H. Simpson, Renata L. Fleming, Jian Teng, Bakhos A. Tannous. Towards elucidating the role of RNA modifications in cancer by improving the quantitative accuracy of mass spectrometric profiling of RNA modifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB507.