A Preliminary Survey of Transfer RNA Modifications and Modifying Enzymes of the Tropical Plant Cocos nucifera L.

The coconut (Cocos nucifera L.) is a commercial crop widely distributed among coastal tropical regions. It provides millions of farmers with food, fuel, cosmetics, folk medicine, and building materials. Among these, oil and palm sugar are representative extracts. However, this unique living species of Cocos has only been preliminarily studied at molecular levels. Benefiting from the genomic sequence data published in 2017 and 2021, we investigated the transfer RNA (tRNA) modifications and modifying enzymes of the coconut in this survey. An extraction method for the tRNA pool from coconut flesh was built. In total, 33 species of modified nucleosides and 66 homologous genes of modifying enzymes were confirmed using a nucleoside analysis using high-performance liquid chromatography combined with high-resolution mass spectrometry (HPLC-HRMS) and homologous protein sequence alignment. The positions of tRNA modifications, including pseudouridines, were preliminarily mapped using a oligonucleotide analysis, and the features of their modifying enzymes were summarized. Interestingly, we found that the gene encoding the modifying enzyme of 2'-O-ribosyladenosine at the 64th position of tRNA (Ar(p)64) was uniquely overexpressed under high-salinity stress. In contrast, most other tRNA-modifying enzymes were downregulated with mining transcriptomic sequencing data. According to previous physiological studies of Ar(p)64, the coconut appears to enhance the quality control of the translation process when subjected to high-salinity stress. We hope this survey can help advance research on tRNA modification and scientific studies of the coconut, as well as thinking of the safety and nutritional value of naturally modified nucleosides.

Potential Misidentification of Natural Isomers and Mass-Analogs of Modified Nucleosides by Liquid Chromatography-Triple Quadrupole Mass Spectrometry.

Triple quadrupole mass spectrometry coupled to liquid chromatography (LC-TQ-MS) can detect and quantify modified nucleosides present in various types of RNA, and is being used increasingly in epitranscriptomics. However, due to the low resolution of TQ-MS and the structural complexity of the many naturally modified nucleosides identified to date (>160), the discrimination of isomers and mass-analogs can be problematic and is often overlooked. This study analyzes 17 nucleoside standards by LC-TQ-MS with separation on three different analytical columns and discusses, with examples, three major causes of analyte misidentification: structural isomers, mass-analogs, and isotopic crosstalk. It is hoped that this overview and practical examples will help to strengthen the accuracy of the identification of modified nucleosides by LC-TQ-MS.

Micro-flow hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry detects modified nucleosides in the transfer RNA pool of cyanobacteria.

Post-transcriptional modification of nucleosides is observed in almost all elements of RNA. Modified nucleosides finely tune the structure of RNA molecules and affect vital functions, such as the modified wobble position 34 of transfer RNAs expanding the reading preference of anticodons to codons. Recent investigations have revealed that the modification species and their frequencies in an RNA element are not stable but vary with specific cellular factors including metabolites and particular proteins (writers, readers, and erasers). To understand the link between dynamic RNA modifications and biological processes, sensitive and reliable methods for determining modified nucleosides are required. In this study, micro-flow (8 µL/min) hydrophilic interaction liquid chromatography was coupled with triple quadrupole mass spectrometry for the simultaneous determination of adenosine, uridine, cytidine, guanosine, and 20 modified nucleosides. The method was calibrated using 0.1-1000 nM standards (∼0.03-300 ng/mL) and successfully applied to the determination of transfer RNA modifications in the model cyanobacterium Synechococcus elongatus PCC 7942. A protocol for the isolation of a clean transfer RNA pool was optimized, requiring only 25 ng for the identification and quantification of transfer RNA modifications. This micro-flow liquid chromatography-tandem mass spectrometry method constitutes the first step toward monitoring dynamic ribonucleoside modifications in a limited RNA sample.