Structural Elucidation of Bisulfite Adducts to Pseudouridine That Result in Deletion Signatures during Reverse...

The recent report of RBS-Seq to map simultaneously the epitranscriptomic modifications N¹-methyladenosine, 5-methylcytosine, and pseudouridine (Ψ) via bisulfite treatment of RNA provides a key advance to locate these important modifications. The locations of Ψ were found by a deletion signature generated during cDNA synthesis after bisulfite treatment for which the chemical details of the reaction are poorly understood. In the present work, the bisulfite reaction with Ψ was explored to identify six isomers of bisulfite adducted to Ψ. We found four of these adducts involved the heterocyclic ring, similar to the reaction with other pyrimidines. The remaining two adducts were bonded to the 1' carbon, which resulted in opening of the ribose ring. The utilization of complementary 1D- and 2D-NMR, Raman, and electronic circular dichroism spectroscopies led to the assignment of the two ribose adducts being the constitutional isomers of an S- and an O-adduct of bisulfite to the ribose, and these are the final products after heating. A mechanistic proposal is provided to rationalize chemically the formation and stereochemistries of all six isomeric bisulfite adducts to Ψ; conversion of intermediate adducts to the two final products is proposed to involve E2, S_N2', and [2,3]-sigmatropic shift reactions. Lastly, a synthetic RNA template with Ψ at a known location was treated with bisulfite, leading to a deletion signature after reverse transcription, supporting the RBS-Seq report. This classical bisulfite reaction used for epigenomic and epitranscriptomic sequencing diverges from the C nucleoside Ψ to form stable bisulfite end products that yield signatures for next-generation sequencing.