Identification and mapping of post-transcriptional modifications on the HIV-1 antisense transcript Ast in human cells

LC-MS based detection of Ast RNA nucleoside modifications. Extracted ion chromatograms (XICs) corresponding to specific mass-to-charge (m/z) values for each class of the modified nucleosides are shown. (A) The cytidine modifications, 5-methylcytide and 2′-O-methylcytidine (m⁵C and Cm, respectively) share identical m/z values but differ in their retention times. (B,C) Uridine (U) modifications, Ψ (pseudouridine, shorter retention time than uridine, panel B), and 3-methyluridine and 2′-O-methyluridine (m³U and Um, respectively, panel C), which are indistinguishable by retention time and m/z values. (D) Guanosine modifications, 7-methylguanosine, 2′-O-methylguanosine, and 2-methylguanosine (m⁷G, Gm, and m²G, respectively) differentiated by their retention time and mass spectral behavior. (E,F) Adenosine modifications, 1-methyladenosine, 2′-O-methyladenosine, and N⁶-methyladenosine (m¹A, Am, and m⁶A, respectively, panel E) and inosine (I, panel F) are distinguished by their retention time and MS spectra.