Quantitative profiling of pseudouridylation landscape in the human transcriptome

Abstract

Pseudouridine (ψ) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, transcriptome-wide measurement of individual ψ sites remains unaddressed. Here, we develop "PRAISE", via selective chemical labeling of ψ by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the ψ landscape in the human transcriptome. Unlike traditional RNA/DNA bisulfite treatment, our approach is based on quaternary base mapping and revealed a ~10% median modification level for 2,714 confident ψ sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7 and TRUB1, with TRUB1 mRNA targets showing the highest modification stoichiometry. In addition, we identified and quantified known and novel ψ sites in mitochondrial mRNA, catalyzed by a mitochondria-localized isoform of PUS1. Collectively, we provide a sensitive and convenient method to measure transcriptome-wide ψ ; we envision this quantitative approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

“…12,52 The occupancy of Ψ across the transcriptome is organism, cell-type, and stress-dependent, suggesting that this U isomer has a significant role in cellular processes. 4,12,22,23,37 Determination of these roles will require accurate and quantitative sequencing of RNA, in which nanopore direct RNA sequencing is a candidate method to achieve this goal. 37,53 Our work identified that nanopore signatures for Ψ are highly sequence context dependent and that using a consensus of nanopore current, helicase dwell time, and base miscalling is an approach to sequence this U isomer.…”

“…37,53 Our work identified that nanopore signatures for Ψ are highly sequence context dependent and that using a consensus of nanopore current, helicase dwell time, and base miscalling is an approach to sequence this U isomer. 3 The pH 7 bisulfite reaction to yield Ψ adducts results in detection of adducts via a deletion signature in traditional sequencing after reverse transcription, 1,22,23 and it is found as an indel in nanopore direct RNA sequencing. 51 In the nanopore data, the indel results from the current levels being poorly recognized by the software.…”

“…Direct RNA sequencing allows sequencing modifications in their native form without the application of chemicals or antibodies selective for the modifications to raise a signal from the background. 1,22,23 The commercial nanopore sequencer is comprised of a lipid-bilayer embedded protein nanopore sensor with a central constriction zone slightly larger than single-stranded RNA for recording current levels as the RNA passes the pore (Figure 3a). 28 Within the central constriction zone resides ∼5 nts of RNA that contribute to the current levels, referred to as a k-mer.…”

“…21 Nevertheless, there is sufficient reactivity of Ψ at pH 7 to develop highly efficient sequencing protocols for Ψ alone, and precisely that was done recently in both the He laboratory with BID-seq 22 and shortly thereafter in the Yi laboratory where the PRAISE protocol was developed (Figure 2b). 23…”

Disease Diagnosis Based on Nucleic Acid Modifications

“…On the basis that bisulfite treatment could be utilized to cause a deletion signature at the Ψ sites during RT, two bisulfite-based detection strategies, PRAISE and BID- seq, have been developed to realize more sensitive profiling of Ψ sites in the mammalian transcriptome. 51,52 Chemical labeling-based strategies are also exploited in the profiling of other abundant RNA modifications, such as Nm. The unmodified 3′-end of RNA is supposed to be oxidized into dialdehydes by NaIO 4 , followed by β-elimination with alkaline cleavage to remove the terminal nucleotide, according to Nmseq.…”