A New Bacterial Adenosine‐Derived Nucleoside as an Example of RNA Modification Damage

Abstract

The fields of RNA modification and RNA damage both exhibit a plethora of non‐canonical nucleoside structures. While RNA modifications have evolved to improve RNA function, the term RNA damage implies detrimental effects. Based on stable isotope labelling and mass spectrometry, we report the identification and characterisation of 2‐methylthio‐1,N6‐ethenoadenosine (ms2ϵA), which is related to 1,N6‐ethenoadenine, a lesion resulting from exposure of nucleic acids to alkylating chemicals in vivo. In contrast, a sophisticated isoprene labelling scheme revealed that ms2ϵA biogenesis involves cleavage of a prenyl moiety in the known transfer RNA (tRNA) modification 2‐methylthio‐N6‐isopentenyladenosine (ms2i6A). The relative abundance of ms2ϵA in tRNAs from translating ribosomes suggests reduced function in comparison to its parent RNA modification, establishing the nature of the new structure in a newly perceived overlap of the two previously separate fields, namely an RNA modification damage.

Reversible oxidative dimerization of 4-thiouridines in tRNA isolates

“…We have scrutinised the nucleoside content of tRNA isolates from E. coli for novel structures using a specifically designed LC–MS regimen termed neutral loss scan (NLS, vide infra ), and previously reported on two newly identified modification types, along with three dozen candidates of yet unidentified structures. 11,33,34 Here we report on the pursuit of a group of four unusual LC–MS signals, which we found to originate from what was initially a putatively new RNA modification. Its structure turned out to be an oxidatively damaged dimeric form of s 4 U, linked via a disulfide.…”

Chemical Synthesis of Modified RNA

“…In general, the thiocarbonyl groups of nucleobases can be problematic in ammonia or methylamine solutions (as required for RNA deprotection), leading to amination/imination [84] . They are also sensitive towards oxygen, leading to oxidative dimerization [85] …”

Pitfalls in RNA Modification Quantification Using Nucleoside Mass Spectrometry

“…Several approaches for the acquisition of SILISs and their applications have been presented by us and others in the last years and are not further discussed. ,,, If a SILIS is not available, isotope-labeled (deoxy)­ribonucleosides or chemically similar modifications are often used as SILISs. , Here, the analyst must be highly confident in the sample preparation as small differences in, e.g., salt load can lead to deviations in the MS detection efficiency. If an online UV detector is available, the MS signals can be normalized to UV signals of canonical nucleosides. ,, Step 5: For absolute quantification, the analyte of interest must be available in weighable quantities to produce calibrant solutions. For external calibration, 5–12 calibrant solutions of varying analyte concentration are prepared to ensure that the analyte signal of the sample is within the calibrated range.…”

“…Several approaches for the acquisition of SILISs and their applications have been presented by us and others in the last years and are not further discussed. ,,, If a SILIS is not available, isotope-labeled (deoxy)­ribonucleosides or chemically similar modifications are often used as SILISs. , Here, the analyst must be highly confident in the sample preparation as small differences in, e.g., salt load can lead to deviations in the MS detection efficiency. If an online UV detector is available, the MS signals can be normalized to UV signals of canonical nucleosides. ,, …”

“…However, chemical modification of RNA is necessary, as the four so-called canonical nucleosides (adenosine, guanosine, cytidine, and uridine) are not sufficient to fulfill the myriad roles RNA plays within a cell’s biology. Thus, a large chemical variety of RNA nucleosides has evolved: So far, over 150 different nucleosides have been discovered in RNA across all domains of life, and the number is ever-growing. − RNA modifications have been reported to be present in most types of RNA in various stoichiometries, with transfer RNA (tRNA) being the most extensively modified. For ribosomal RNA (rRNA), some positions are reported to be fully modified, while other positions are only partially modified .…”