Abstract
AbstractTemporal 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
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
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.