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| Status |
Public on Feb 21, 2018 |
| Title |
Enzymatic or in vivo installation of propargyl groups in combination with click chemistry enables enrichment and detection of methyltransferase target sites in RNA |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
|
| Summary |
m6A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3-METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise transcriptome-wide assignment of m6A sites is of utmost importance. However, m6A does not interfere with Watson-Crick base pairing making polymerase-based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65 % at m6A sites after bioconjugation and purification, hence enabling detection of METTL3-METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl-L-selenohomocysteine and validated different types of known rRNA methylation sites.
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| Overall design |
enrichment of methylated nucleotides by bioconjugation of propagyl groups in synthetic oligos and rRNA
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| Contributor(s) |
Hartstock K, Nilges BS, Ovcharenko A, Cornelissen N, Püllen N, Lawrence-Dörner A, Leidel SA, Rentmeister A |
| Citation(s) |
29461645 |
| |
| Submission date |
Jan 17, 2018 |
| Last update date |
Dec 28, 2018 |
| Contact name |
Benedikt Sebastian Nilges |
| E-mail(s) |
[email protected]
|
| Organization name |
Max Planck Institute for Molecular Biomedicine
|
| Department |
RNA Biology Group
|
| Street address |
Von-Esmarch-Straße 54
|
| City |
Muenster |
| ZIP/Postal code |
48149 |
| Country |
Germany |
| |
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| Platforms (2) |
| GPL15456 |
Illumina HiScanSQ (Homo sapiens) |
| GPL24519 |
Illumina HiScanSQ (synthetic construct) |
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| Samples (19)
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| Relations |
| BioProject |
PRJNA430372 |
| SRA |
SRP131073 |
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