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

Abstract

The recent report of RBS-Seq to map simultaneously the epitranscriptomic modifications N 1 -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 Oadduct 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 N 2′, 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.

Nanopore Dwell Time Analysis Permits Sequencing and Conformational Assignment of Pseudouridine in SARS-CoV-2

“… 18 As a result of analyzing sequencing stops, these approaches cannot achieve read-through in order to sequence multiple sites simultaneously in single strands; additionally, RNA structure-induced stops can yield false positives, and quantification of the modification is challenging to conduct accurately using these approaches. 19 , 20 One chemical solution is to treat the suspect RNA with bisulfite to yield a stable sugar adduct on the Ψ nucleotide, 21 , 22 which induces a deletion signature during cDNA synthesis, allowing more than one modification per strand to be sequenced. However, this approach does not yield quantitative deletions, and therefore, the extent of modification at suspected sites is challenging to obtain.…”

“…Pseudouridine (Ψ), the most abundant global RNA modification, is an isomerization product of uridine (U) found at high relative levels (>1%) in eukaryotic tRNA and rRNA, as well as in viral RNA, and lower levels (<1%) in eukaryotic mRNA (Figure A). ,, This modification is tied to critical RNA functions in cells such as reinforcing RNA secondary structure and regulation of translation, and Ψ levels change in response to oxidative, micronutrient, or heat-shock stress. ,− Initially, Ψ was located in RNA via digestion and chromatographic methods followed by mass spectrometric quantification approaches, − and recently, high-throughput NGS using Ψ-specific chemistry has revealed sites in the mammalian transcriptome for Ψ. Pseudouridine is specifically alkylated by the carbodiimide CMC ( N -cyclohexyl- N ′-(2-morpholinoethyl)­carbodiimide metho-p-toluenesulfonate) to yield a stable and bulky adduct to stall reverse transcription, which is found by distinct sequencing read stops in comparison to a nonalkylated matched control. ,− Alternatively, Ψ can be sequenced by the absence of reactivity with hydrazine, while the parent U readily reacts to yield strand breaks detected during NGS . As a result of analyzing sequencing stops, these approaches cannot achieve read-through in order to sequence multiple sites simultaneously in single strands; additionally, RNA structure-induced stops can yield false positives, and quantification of the modification is challenging to conduct accurately using these approaches. , One chemical solution is to treat the suspect RNA with bisulfite to yield a stable sugar adduct on the Ψ nucleotide, , which induces a deletion signature during cDNA synthesis, allowing more than one modification per strand to be sequenced. However, this approach does not yield quantitative deletions, and therefore, the extent of modification at suspected sites is challenging to obtain.…”

Nanopore dwell time analysis permits sequencing and conformational assignment of pseudouridine in SARS-CoV-2

“…17 As a result of analyzing sequencing stops, these approaches cannot achieve read-through in order to sequence multiple sites in single strands; additionally, RNA structure-induced stops can yield false positives, and quantification of the modification is challenging to conduct accurately using these approaches. 18,19 One chemical solution is to treat the suspect RNA with bisulfite to yield a stable sugar adduct on the Ψ nucleotide, 20,21 which induces a deletion signature during cDNA synthesis, allowing more than one modification per strand to be sequenced. However, this approach does not yield quantitative deletions, and therefore, the extent of modification at suspected sites is challenging to obtain.…”

Thioguanosine Conversion Enables mRNA‐Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC‐seq DUAL)

“…These new developments have demonstrated the power of chemical nucleoside conversion to study cellular RNA dynamics upon RNA‐seq . Currently, 4sU is the dominantly used nucleoside for metabolic labeling.…”