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| Status |
Public on Apr 24, 2024 |
| Title |
Chemical manipulation of m1A mediates its detection in human tRNA |
| Organism |
Homo sapiens |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
|
| Summary |
N1-methyl adenosine (m1A) is a wide-spread RNA modification present in tRNA, rRNA and mRNA. m1A modification sites in tRNAs are evolutionary conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick base pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red‑m1A-seq to sequence human small RNA and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-ATT and 5.8S rRNA.
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| |
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| Overall design |
Detect m1A sites on tRNA and rRNA in HeLa cells using new chemical manipulation method
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| |
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| Contributor(s) |
Lyu R, Pajdzik K, Dai Q, He C |
| Citation(s) |
38531647 |
| Submission date |
Jan 19, 2024 |
| Last update date |
Apr 25, 2024 |
| Contact name |
Ruitu Lyu |
| E-mail(s) |
lvruitu@uchicago.edu
|
| Organization name |
The University of Chicago
|
| Department |
Chemistry
|
| Lab |
Chuan He
|
| Street address |
929 E 57th Street
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
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| Platforms (1) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA1066722 |
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