Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL).

Catherina Gasser,Lukas Trixl,Dietmar Rieder,Eva Neuner,Isabel Delazer,Katharina Pascher,Sarah Klotz,Karl Brillet,Alexandra Lusser,Ronald Micura,Eric Ennifar,Maximilian Himmelstoß

doi:10.1002/anie.201916272

Abstract

Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4Cl/OsO4‐based conversion of 6‐thioguanosine (6sG) into A′, where A′ constitutes a 6‐hydrazino purine derivative. A′ retains the Watson–Crick base‐pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4‐thiouridine (4sU) into C, the combination of both modified nucleosides in dual‐labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC‐seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA‐lifetime evaluation with unprecedented precision.

Highlights

RNA maintains many important functions in the cell with most of the RNA-involving processes being tightly regulated
Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay
We assumed that the conditions that we previously elaborated for quantitative 4sU-to-C conversions (TUC-seq),[17,18] namely the application of osmium tetroxide in ammonium chloride buffer, would result in the formation of 2-aminoadenosine, which should be decoded as adenine in RNA-sequencing experiments

Summary

Introduction

RNA maintains many important functions in the cell with most of the RNA-involving processes being tightly regulated. The isolated total RNA was thiol- biotinylated, followed by separation into newly transcribed and pre-existing, unlabeled RNA.[16] With TUC-seq, we have recently published the first protocol that eliminates bothersome RNA enrichment steps by quantitatively converting 4-thiouridine into native cytosine using oxidative OsO4-based substitution chemistry, allowing for direct detection of newly synthesized transcripts containing U-to-C mutations by sequencing.[17,18] Two related methods, SLAM-seq and TimeLapse-seq,[19,20] have been reported; both approaches modify 4sU to a non-native pyrimidine analogue, an S-alkylated 4sU thioiminoester and an N-trifluoroethyl-modified cytosine, respectively. Such an experimental design should be adaptable to the existing 4sU-based sequencing approaches because handling of complementary U-to-C and Gto-A data sets in bioinformatics is expected to be straightforward

Results and Discussion

Conclusion

Conflict of interest