The isomerization of uridine to pseudouridine (Ψ), known as pseudouridylation, is the most abundant post-transcriptional modification of stable RNAs. Due to technical limitations in pseudouridine detection methods, studies on pseudouridylation have historically focused on ribosomal RNAs, transfer RNAs, and spliceosomal small nuclear RNAs, where Ψs play a critical role in RNA biogenesis and function. Recently, however, a series of deep sequencing methods-Pseudo-seq, Ψ-seq, PSI-seq, and CeU-seq-has been published to map Ψ positions across the entire transcriptome with single nucleotide resolution. These data have greatly expanded the catalogue of pseudouridylated transcripts, which include messenger RNAs and noncoding RNAs. Furthermore, these methods have revealed conditionally-dependent sites of pseudouridylation that appear in response to cellular stress, suggesting that pseudouridylation may play a role in dynamically modulating RNA function. Collectively, these methods have opened the door to further study of the biological relevance of naturally occurring Ψs. However, an in-depth comparison of these techniques and their results has not yet been undertaken despite all four methods relying on the same basic principle: Ψ detection through selective chemical labeling by the carbodiimide known as CMC. In this article, we will outline the currently available high-throughput Ψ-detection methods and present a comparative analysis of their results. We will then discuss the merits and limitations of these approaches, including those inherent in CMC conjugation, and their potential to further elucidate the function of this ubiquitous and dynamic modification.
Keywords: Modification; Next-generation sequencing; Pseudouridine; RNA.
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