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. 2021:2353:137-154.
doi: 10.1007/978-1-0716-1605-5_8.

tRNA Modifications as a Readout of S and Fe-S Metabolism

Ashley M Edwards  1 , Maame A Addo  1 , Patricia C Dos Santos  2
Affiliations

tRNA Modifications as a Readout of S and Fe-S Metabolism

Ashley M Edwards et al. Methods Mol Biol. 2021.

Abstract

Iron-Sulfur (Fe-S) clusters function as core prosthetic groups known to modulate the activity of metalloenzymes, act as trafficking vehicles for biological iron and sulfur, and participate in several intersecting metabolic pathways. The formation of these clusters is initiated by a class of enzymes called cysteine desulfurases, whose primary function is to shuttle sulfur from the amino acid L-cysteine to a variety of sulfur transfer proteins involved in Fe-S cluster synthesis as well as in the synthesis of other thiocofactors. Thus, sulfur and Fe-S cluster metabolism are connected through shared enzyme intermediates, and defects in their associated pathways cause a myriad of pleiotropic phenotypes, which are difficult to dissect. Post-transcriptionally modified transfer RNA (tRNA) represents a large class of analytes whose synthesis often requires the coordinated participation of sulfur transfer and Fe-S enzymes. Therefore, these molecules can be used as biologically relevant readouts for cellular Fe and S status. Methods employing LC-MS technology provide a valuable experimental tool to determine the relative levels of tRNA modification in biological samples and, consequently, to assess genetic, nutritional, and environmental factors modulating reactions dependent on Fe-S clusters. Herein, we describe a robust method for extracting RNA and analytically evaluating the degree of Fe-S-dependent and -independent tRNA modifications via an LC-MS platform.

Keywords: Bacteria; Iron-sulfur cluster; LC-MS; RNA extraction; Sulfur metabolism; Thionucleosides; tRNA; tRNA modification.

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Figures

Fig. 1
Fig. 1
Structures of modified bacterial tRNA and derivatives. (a) Structures of modified tRNAs found at positions 32, 34, and 37 whose synthesis is dependent on Fe-S proteins, and (b) structures of modified tRNAs found at positions 17a, 34, 37, and 55b whose synthesis is independent of Fe-S proteins are shown with their corresponding modified/hypermodified moieties indicated in red. The numbers “1” and “9” found within each base denote the β-glycosidic bond orientation found in the pyrimidine and purine bases, with their adjacent riboses in tRNA. (a) Pseudouridine is also found at positions 32, 38, 39, 40 in B. subtilis and E. coli, and additionally at positions 13 and 35 in E. coli. (b) Dihydrouridine is also found at positions 20 and 20a in B. subtilis and E. coli, at position 47 in B. subtilis only, and at position 16 in E. coli only
Fig. 2
Fig. 2
The layout of MestreNova application displaying a total ion chromatograph of a 70-min LC-MS run. Arrows indicate the application commands for selecting the mass browser (a), TIC panel (b), new chromatogram (c), and a new window (d)
Fig. 3
Fig. 3
Layout of MestreNova application when mass browsing to produce an extracted ion chromatogram (EIC). Arrows indicate commands for selecting type of chromatogram (a) and tolerance range (b)
Fig. 4
Fig. 4
Extracted ion chromatogram of 382.1544 m/z and the corresponding retention times of all analytes that fall within 5 PPM of this m/z of interest. Arrows indicate commands for selecting the range (a) and zoom in (b)
Fig. 5
Fig. 5
Mass spectrum of the 43.7- to 44.9-min retention time, the m/z values found within this time range, and the spectral count associated with the ms2i6A37 modified nucleoside
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