Transcriptome-wide Mapping Reveals Widespread Dynamic-Regulated Pseudouridylation of ncRNA and mRNA

Abstract

SUMMARY Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of novel sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUSs) uncovers which PUSs modify each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided PUSs. Upon heat shock in yeast, Pus7-mediated pseudouridylation is induced at >200 sites and Pus7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved, but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcritome-wide scope for pseudouridine, and methods to dissect its underlying mechanisms and function.

Publisher's Note

“…On the order of 250 to 300 sites have now been mapped in yeast mRNA at single base resolution. [22][23][24] In human, the reported number of mRNA C sites varies widely from 96 in one study, 23 to 353 in a second, 22 and up to 2084 in a third study. 25 This variation is likely the result of technical differences in experimental design and data analysis.…”

“…In keeping with an emerging theme among mRNA modifications, however, C is dynamic in heat shock, nutrient deprivation, and other stresses. 22,23,25 While 5-methylcytidine (m 5 C) is best known for its central role as an epigenetic mark in DNA, it was also described in early studies of RNA methylation. 3 Mapping of m 5 C in RNA was initially attempted by adapting the bisulfite sequencing technology used for m 5 C in DNA, leading to the identification of approximately 10,000 m 5 C sites in mRNA.…”

Pseudouridine in a new era of RNA modifications

“…Collectively, the papers by Carlile et al [3] and Schwartz et al [4] provide the first global detections of Ψ, and suggest potential regulatory functions of this modification in mRNA. Future research will focus on functional roles of Ψ, either through changing the RNA structure or recoding during translation.…”

“…In the recent two papers, Carlile et al [3] and Schwartz et al [4] each reported a transcriptome-wide sequencing approach for mapping Ψ at single-base resolution. They both took advantage of the known reaction between Ψ and Ncyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulphonate (CMC) to form N3-CMC-Ψ ( Figure 1B).…”

“…Through genetic perturbation of PUS genes, they further revealed that Pus1, Pus2, Pus4 and Pus7 are involved in mRNA pseudouridylation. Schwartz et al [4] used a similar strategy to develop Ψ-seq, but with a focus on quantitative measurement. Instead of performing size selection, they relied on computational method to minimize background and determine the high-confidence Ψ sites, while the addition of synthetic spike-in probes facilities the quantification of the relative stoichiometry.…”