10.1002/anie.201707465
Angewandte Chemie International Edition
COMMUNICATION
temperature or at 50°C, respectively. In one instance (see Figure 3B), the
RNA was denatured at 92°C for 2 min before treatment with OsO4.
The same analysis was applied to the endogenous cell cycle-
related transcripts showing strongest transcription for CcnB1
and CcnE1 with ~6.2% new transcripts, while CcnD1,
p21/Cdkn1A and CcnA2 appeared to be more weakly
transcribed with 4.9%, 3.6% and 1.6% new transcripts,
respectively (Figure 4C, Supporting Table 3). This conclusion is
based on the assumption that the contribution of RNA decay
within the labeling period is negligible. Cyclin mRNA half-lifes
have been estimated to range from 2 to 4.5 h for mouse
fibroblast cells.25 Therefore, mRNA decay likely plays a minor
role in our 1 h labeling period.
After OsO4/NH4Cl treatment, the reaction mixture was transferred into
Vivaspin 500 (MWCO 3000, PES) centrifugal concentrators (Sartorius,
Göttingen, Germany), washed 4 times with H2O (500 µL) and the
concentrate was lyophilized or directly used in the next step (see
Supporting Information for all other methods).
Acknowledgements
Taken together, our experiments demonstrate that OsO4/NH4Cl
mediated conversion of thiolated uridines allows for direct
sequencing-based analysis of metabolically labeled or naturally
modified RNA. The high selectivity and specificity of the osmium
reaction combined with nearly quantitative yields, make TUC-
seq a promising new method (with a very low number of
processing steps) to study the cellular dynamics of various types
of RNA. It is important to note that the OsO4/NH4Cl mediated
4sU-to-C conversion is a clean and mild reaction that otherwise
leaves the RNA intact; it does not result in unwanted
modification of canonical nucleobases nor in unwanted (e.g.
hydrolyzed or alkylated) 4sU byproducts. Therefore, interference
with any downstream processing steps, such as reverse
transcription, that might cause biased results can be excluded.
For the proof of principle examples shown here, we have used
PCR-based sequencing methods. Clearly, our approach is
amenable for direct RNA-seq methods, and we expect that
RNA-seq of OsO4-treated samples (TUC-seq) will find a broad
field of applications. TUC-seq enables the simultaneous and
accurate quantification of labeled and unlabeled RNA. Together
with properly designed pulse and pulse-chase labeling
conditions, it will greatly facilitate transcriptome-wide analyses of
RNA synthesis and decay rates or of RNA processing events.
Moreover, the method will be highly useful for the identification
of novel sites of endogenous 4sU in RNA of any organism.
Funding from the Austrian Science Fund FWF (P27947 to R.M.;
P27024-BBL to A.L.) are acknowledged.
Keywords: metabolic labeling • thiouridine • RNA modification •
osmiumtetroxide • oligonucleotides • transcription • cyclin
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