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

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 (ms2 ϵA), 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 ms2 ϵA biogenesis involves cleavage of a prenyl moiety in the known transfer RNA (tRNA) modification 2-methylthio-N6-isopentenyladenosine (ms2 i6 A). The relative abundance of ms2 ϵA 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.

A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure.

Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC-MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high-resolution mass spectrometry. The structure of msms2 i6 A (2-methylthiomethylenethio-N6-isopentenyl-adenosine) was confirmed by methione-D3 feeding experiments and by synthesis of the nucleobase. The msms2 i6 A contains a thioacetal, shown in vitro to be biosynthetically derived from ms2 i6 A by the radical-SAM enzyme MiaB. This enzyme performs thiomethylation, forming ms2 i6 A from i6 A in a first turnover. The new thioacetal is formed by a second turnover. Along with the pool of 36 new modifications, this work describes a new layer of RNA modification chemistry.

One precursor, three apolipoproteins: the relationship between two crustacean lipoproteins, the large discoidal lipoprotein and the high density lipoprotein/ß-glucan binding protein.

The novel discoidal lipoprotein (dLp) recently detected in the crayfish, differs from other crustacean lipoproteins in its large size, apoprotein composition and high lipid binding capacity, We identified the dLp sequence by transcriptome analyses of the hepatopancreas and mass spectrometry. Further de novo assembly of the NGS data followed by BLAST searches using the sequence of the high density lipoprotein/1-glucan binding protein (HDL-BGBP) of Astacus leptodactylus as query revealed a putative precursor molecule with an open reading frame of 14.7 kb and a deduced primary structure of 4889 amino acids. The presence of an N-terminal lipid bind- ing domain and a DUF 1943 domain suggests the relationship with the large lipid transfer proteins. Two-putative dibasic furin cleavage sites were identified bordering the sequence of the HDL-BGBP. When subjected to mass spectroscopic analyses, tryptic peptides of the large apoprotein of dLp matched the N-terminal part of the precursor, while the peptides obtained for its small apoprotein matched the C-terminal part. Repeating the analysis in the prawn Macrobrachium rosenbergii revealed a similar protein with identical domain architecture suggesting that our findings do not represent an isolated instance. Our results indicate that the above three apolipoproteins (i.e HDL-BGBP and both the large and the small subunit of dLp) are translated as a large precursor. Cleavage at the furin type sites releases two subunits forming a heterodimeric dLP particle, while the remaining part forms an HDL-BGBP whose relationship with other lipoproteins as well as specific functions are yet to be elucidated.