Mass spectrometry of the fifth nucleoside: A review of the identification of pseudouridine in nucleic acids

Abstract

Pseudouridine, the so-called fifth nucleoside due to its ubiquitous presence in ribonucleic acids (RNAs), remains among the most challenging modified nucleosides to characterize. As an isomer of the major nucleoside uridine, pseudouridine cannot be detected by standard reverse-transcriptase-based DNA sequencing or RNase mapping approaches. Thus, over the past 15 years, investigators have focused on the unique structural properties of pseudouridine to develop selective derivatization or fragmentation strategies for its determination. While the N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide p-tosylate (CMCT)-reverse transcriptase assay remains both a popular and powerful approach to screen for pseudouridine in larger RNAs, mass-spectrometry-based approaches are poised to play an increasingly important role in either confirming the findings of the CMCT-reverse transcriptase assay or in characterizing pseudouridine sequence placement and abundance in smaller RNAs. This review includes a brief discussion of pseudouridine including a summary of its biosynthesis and known importance within various RNAs. The review then focuses on chemical derivatization approaches that can be used to selectively modify pseudouridine to improve its detection, and the development of mass-spectrometry-based assays for the identification and sequencing of pseudouridine in various RNAs.

Detection of RNA modifications

“…71,72 CMCT (Table 1) acylates G, U, inosine and Ψ residues under physiological conditions, but the acyl-moieties can be removed from G, I and U residues by alkaline treatment, leaving only N3-acylated pseudouridines. 36 The latter arrest reverse transcription and can thus be detected by primer elongation techniques similar to other modifications (compare Fig. 5).…”

“…5). In addition cyanoacrylate and methylvinylsulfone, which have been mentioned in context MS-based detection of Ψ, 36,40 4-methylbromo-7-methoxy-coumarine has also been reported to specifically react with Ψ residues. 73 Recent method development makes use of the chemical tagging for detection by mass spectrometry in RNA fragments obtained after digestion e.g., by RNase T1.…”

“…34 Pseudouridine, the most abundant modification known thus far, shows unusual fragmentation behavior, because its glycosidic bond is an untypical und very stable carbon-carbon bond. 35,36 Interestingly, in addition to standard detection Upon phosphate removal from nucleotides, e.g., by alkaline phosphatase, the resulting nucleosides can be separated by ion exchange. Initial work combined LC with UV detection for the rapid determination of the base composition of unknown RNA.…”

“…38,39 Yet another recently evolved method is nucleoside-specific derivatization (e.g., of pseudouridine with acrylonitrile, with N-cyclohexyl-N'-β-(4-methylmorpholinium) ethylcarbodiimide p-tosylate (CMCT), or with methylvinylsulfone, see also Table 1) followed by MS analysis. 36 A particularly sophisticated instrumental setup was implemented by Emmerechts et al 40 using LC-Q-TOF-MS/MS. One of the most interesting latest developments was published by Hossain and Limbach 2009.…”

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

“…To overcome this limitation, 2 general methods for mapping pseudouridines onto primary RNA sequences by mass spectrometry, derivitization or fragmentationbased identification have been developed. [64][65][66][67][68][69] The fragmentation-based method is very convenient because it does not require special sample treatment, but does require more sample to be analyzed for the detection of specific product ions from fragmentation that will indicate a pseudouridine is present on the end of the oligonucleotide or in the middle of the oligonucleotide. 64,67 The derivitization method typically involves treating the sample to chemically label pseudouridines and not uridines, with methylvinylsulfone, acrylonitrile or 1-cyclohexyl-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT), which was originally used for biochemical pseudouridine detection by strong stops during primer extension.…”