HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA

Abstract

Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed ‘HydraPsiSeq’, a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10–50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20–25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.

Current Practice in Bicistronic IRES Reporter Use: A Systematic Review

“…The protein composition or ribosomal RNA (rRNA) post-transcriptional modifications (PTM) affect its function [ 26 , 27 ]. Novel techniques to quantify small changes in rRNA PTM open up new possibilities for epitranscriptomics [ 28 , 29 ], which are expected to become increasingly complex when more types of RNA PTMs can be accurately quantified [ 30 , 31 ]. New insights in ribosome heterogeneity in stem cell differentiation [ 32 ], human diseases related to single gene defects (ribosomopathies [ 33 ]), or complex multifactorial diseases such as cancer [ 34 ] underline the importance of robust reporter assays to interrogate ribosome function.…”

Non-Redundant tRNA Reference Sequences for Deep Sequencing Analysis of tRNA Abundance and Epitranscriptomic RNA Modifications

“…This is also a feature of the RiboMethSeq protocol, which is now extensively used for analysis of tRNA 2′-O-methylations [ 20 , 21 ]. Other protocols, such as AlkAnilineSeq for detection of m 7 G/m 3 C/D/ho 5 C [ 38 ] and HydraPsiSeq for pseudouridine mapping and quantification [ 39 ], also result in relatively short tRNA reads.…”

Nopp140-chaperoned 2’-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity

“…Apparently, the modifications of these two guanosines exhibit a more basic function in splicing than the remaining ones of U2 and of all other snRNAs. Similarly, pseudouridines cannot be lost from snRNAs, although to determine if this is true for all pseudouridines will require a more detailed genome-wide approach, perhaps using the recently developed HydraPsiSeq (Marchand et al, 2020).…”