Pseudouridine (Ψ) is not only the most abundant post-translational modification (PTM) of RNA in all organisms, but it is also amongst the first discovered, way back in the futuristic 50’s, where it was deemed the 5th nucleotide of RNA. Ψ doesn’t discriminate against cellular functions or evolutionary distinct species. And while studied intently in rRNA and tRNA, surprisingly mRNA is about 60 years late to the party.
Now two papers (Carlile et al. in Nature and Schwartz et al. in Cell) describe the development of Pseudo-Seq (Ψ-seq) a single-nucleotide resolution, genome-wide method for detecting Ψ. The authors demonstrate the applicability of this technique in human and yeast, and gain insight into the omic characteristics of Ψ as a PTM.
What’s the Fuss about Pseudouridylation?
At the molecular level, pseudouridylation can change the chemical properties of RNA by rigidifying the phosphate backbone of RNA, leading to increased base stacking. This unique feature means that Ψ is involved in a number of cellular processes, which were discovered in mutants lacking the ability to synthesize Ψ. These mutants had difficulties with translation, delayed growth rates, and just couldn’t compete with the wild-types.
In contrast to the more famous m6A RNA modification, which can be dynamic, it appears that pseudouridylation is a more permanent affair, indicating a difference in role. In order to better understand this modification and the role it plays we need to be able to study it – enter Pseudo-seq!
Pseudo-seq: A New Method for Analyzing Pseudouridylation
So how does Pseudo-Seq (Ψ-Seq) work? In this method Poly(A)-selected RNA are:
- Treated with a chemical (CMC) that has a preference to covalently bind to U and Ψ residues.
- Incubated at an alkaline pH to hydrolyze the U compounds as they are less stable than the Ψ compounds.
- A reverse transcription (RT), which selects for the truncated cDNAs, is primed using clever adaptors that result in premature termination on pseudouridylated sites.
- A second adapter is then added to the cDNA mix, allowing the libraries to be amplified and sequenced.
New Insight Into Pseudouridylation
As well as developing Pseudo-seq and showing it can identify previously known sites, Carlile et al. and Schwartz et al. used variations of this method to unearth new insights into this modification, including the observations that:
- There are 100’s of novel sites in mRNAs and ncRNAs.
- Pseudouridylation is achieved by one of seven conserved pseudouridine synthases (Pus1–4, 6, 7 and 9).
- The majority of Ψ in mRNA is regulated in response to environmental signals, like nutrition.
- mRNA pseudouridinylation depends not only on it’s own special site-specific pseudouridine synthases but also some snoRNA-guided pseudouridine synthases.
- Ψ is a functional element related to transcript stability at over 200 sites upon heat shock in yeast.
Overall, these two papers offer the first omic insights into Ψ. They provide solid observations on the global patterns, characterize the writers (Pus synthases and/or snoRNAs) and also describe some of the cellular functions. Hopefully this new technique will allow us to delve deeper into the mechanisms and functions of pseudouridylation.