Pseudo-Seq

Abstract

RNA molecules contain a variety of chemically diverse, posttranscriptionally modified bases. The most abundant modified base found in cellular RNAs, pseudouridine (Ψ), has recently been mapped to hundreds of sites in mRNAs, many of which are dynamically regulated. Though the pseudouridine landscape has been determined in only a few cell types and growth conditions, the enzymes responsible for mRNA pseudouridylation are universally conserved, suggesting many novel pseudouridylated sites remain to be discovered. Here, we present Pseudo-seq, a technique that allows the identification of sites of pseudouridylation genome-wide with single-nucleotide resolution. In this chapter, we provide a detailed description of Pseudo-seq. We include protocols for RNA isolation from Saccharomyces cerevisiae, Pseudo-seq library preparation, and data analysis, including descriptions of processing and mapping of sequencing reads, computational identification of sites of pseudouridylation, and assignment of sites to specific pseudouridine synthases. The approach presented here is readily adaptable to any cell or tissue type from which high-quality mRNA can be isolated. Identification of novel pseudouridylation sites is an important first step in elucidating the regulation and functions of these modifications.

CoverageAnalyzer (CAn): A Tool for Inspection of Modification Signatures in RNA Sequencing Profiles

“…The RT signatures in question may comprise nucleotide misincorporation or transcription arrest, and frequently originate from nucleotide modification at the position of interest. Several library preparation protocols have been published that capture cDNA from abortive RT [16,20,22,23] and can therefore be fully exploited by CAn. However, even preparation methods that do not capture abortive cDNA may provide useful information by providing misincorporation signals that may be analyzed by CAn.…”

Characterizing RNA Pseudouridylation by Convolutional Neural Networks

“…Although numerous studies have revealed that pseudouridines in small cellular RNAs are important for maintaining their structures [5,7,11], the functional roles of pseudouridylation in mRNA translation and its contextual sequence features still remain largely unclear. Several high-throughput experimental techniques of characterizing pseudouridines across transcriptome have been developed recently [19][20][21][22], detailed transcriptome-wide landscape, functions and molecular mechanisms of pseudouridines in post-transcriptional regulation are still to be revealed.…”

“…The pseudouridine modification site data were downloaded from the RMBase database [57] which includes the high-throughput profiling data of pseudouridines collected from three recent experimental strudies [19,20,22]. All the labelled pseudouridine sites were separated into a human dataset and a mouse dataset.…”

“…Traditional pseudouridine detection methods are mainly based on the N 3 -CMC labeling and gel electrophoresis experiments [18], which are often labourious and time-consuming. Recently, several high-throughput profiling techniques, including Pseudo-seq, Ψ-seq, PSI-seq, and CeU-seq, have been proposed to map RNA pseudouridine sites to reference transcriptomes [19][20][21][22]. These high-throughput experiments typically combine CMC derivatives with next generation sequencing or deep sequencing techniques to detect pseudouridine sites on a transcriptome-wide scale.…”

Probing the mechanisms underlying human diseases in making ribosomes

“…Current techniques such as pseudo-seq, piloted by Wendy Gilbert and coworkers, 92 will allow for transcriptome-wide mapping of these sites in human cells. These modifications can affect ribosome function, as it has been shown that one pseudouridylation modification present in a stop codon allows for translational read-through by the ribosome 93 .…”