Matrix-assisted laser desorption/ionization mass spectrometry screening for pseudouridine in mixtures of small RNAs by chemical derivatization, RNase digestion and signature products.

Pseudouridine (Ψ), the 5-ribosyl isomer of uridine, is the most common post-transcriptionally modified nucleoside in RNA. Pseudouridine is present in all three phylogenetic domains of life and it is found in both large and small non-coding RNAs (ncRNAs).1–3 Pseudouridine is found to be particularly abundant in smaller non-coding RNAs such as small nuclear RNAs (snRNAs) and transfer RNAs (tRNAs).1,4 In snRNAs, pseudouridines are located within regions that are involved in snRNA-snRNA interactions and may play a role in strengthening these intermolecular associations.4 Additionally, their presence in specific regions of snRNAs also suggests a role in the functional assembly of small nuclear ribonucleoproteins (snRNPs) and the spliceosome.5 Among tRNAs, the conservation of pseudouridine at specific positions across all three domains of life is more obvious. For example, the pseudouridine at position 55 (Ψ-55) is universally conserved in almost all tRNAs of known sequence.6,7 Pseudouridines in conserved positions in tRNA exhibit a stabilizing influence on RNA structure by virtue of their extra hydrogen bonding capability and their ability to enhance base stacking.8–10 Pseudouridines may also play an important role in the efficiency of the translational process.11 One focus of tRNA research is geared towards understanding the significance of pseudouridine and other modified nucleosides in mitochondrial tRNA function and mitochondrial diseases. Recent studies demonstrate the association of loss of tRNA pseudouridylation at specific sites with functional defects of mitochondrial tRNAs and pathogenesis in humans.4,12 Additionally, lack of pseudouridine in tRNAs has been found to be linked to deficiencies in the expression of certain genes.13 An essential aspect for clarifying the functional and molecular basis for pseudouridine in RNA is its structural characterization and sequence location. This requires the fractionation and purification of individual RNAs prior to sequencing. Although a number of RNA fractionation techniques have been reported,14 the isolation and purification of small RNAs, such as tRNAs, from a complex cellular pool remains a challenging process.15 In particular, the isolation of mitochondrial tRNAs in amounts sufficient for structural characterization is difficult and laborious. Moreover, a different set of challenges arises upon sequencing pseudouridine because it is an isomer of uridine. The classical approach for identifying pseudouridine in small RNAs such as tRNAs and snRNAs involves radioactive labeling, ribonuclease digestion and two-dimensional thin-layer chromatography.5,13,16 That approach was replaced by the Bakin and Ofengand approach for pseudouridine mapping in RNA,17 which is based on chemical modification of pseudouridine by N-cyclohexyl-N’-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMCT) and reverse transcription. This popular and effective method is most successful with larger ncRNAs such as ribosomal RNAs. A modified CMCT-reverse transcriptase approach for tRNAs has been reported, although this requires the use of poly-adenylation to provide complete sequence coverage of the 3’-end of the molecule.10 More recently, mass spectrometry has begun to play an important role in the identification and localization of pseudouridine.18–24 Unlike conventional biochemical techniques, mass spectrometry offers a fast, sensitive and direct approach to analyze RNA.25 Previously we described the application of CMCT derivatization with mass spectrometry-based detection.20 CMCT derivatization incorporates a 252 Da mass tag onto pseudouridine, facilitating its detection by mass spectrometry. More recently, we improved this approach and incorporated pseudouridine detection into a general RNase mapping strategy.26 However, those prior reports have focused on the identification of pseudouridine within isolated and purified single RNA species. As the RNase mapping approach is extendable to mixtures of tRNAs,27 we sought to determine if the CMCT derivatization strategy could also be extended to mixtures of tRNAs. In the present study, we have developed a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to screen for pseudouridines in a mixture of unfractionated tRNAs. Knowledge of the constituent tRNAs in the mixture is provided by detection of signature endonuclease digestion products.27 The simultaneous detection of pseudouridine would be accomplished by a CMCT-derivatization/MALDI-MS based approach. Because signature digestion products are unique m/z values associated with a particular tRNA and pseudouridine is an isomer of uridine (i.e., pseudouridine does not have a unique mass value), in principle these two approaches should be complementary. For sequence placement of the detected pseudouridine, an RNase mapping strategy with multiple endonucleases should enable the mapping of pseudouridines to the known sequences of those tRNAs that were identified by their signature digestion products. Proof-of-concept studies were conducted using an unfractionated mixture of Escherichia coli tRNAs known to contain pseudouridine.