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| Status |
Public on May 07, 2018 |
| Title |
Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.
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| Overall design |
HEK293T rRNA was treated with CMC or mock-treated, then reverse transcribed under 3 different conditions (HIV RT; SIII, Mn; SIII, Mg) for a total of 6 samples.
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| Contributor(s) |
Zhou KI, Clark WC, Pan DW, Eckwahl MJ, Dai Q, Pan T |
| Citation(s) |
29683381 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| RM1 HG008935 |
Center for dynamic RNA epitranscriptomes |
UNIVERSITY OF CHICAGO |
CHUAN HE |
| F30 GM120917 |
Role of m6A in HNRNPG protein localization and HNRNPG-regulated alternative splicing |
UNIVERSITY OF CHICAGO |
Katherine Ismei Zhou |
| K01 HG006699 |
Investigation of 5-hmC, 5fC and 5-caC Modifications in Genome DNA |
UNIVERSITY OF CHICAGO |
Qing Dai |
| T32 GM007281 |
Medical Scientist National Research Service Award |
UNIVERSITY OF CHICAGO |
Marcus Ramsay Clark |
| P30 CA014599 |
Cancer Center Support Grant |
UNIVERSITY OF CHICAGO |
MICHELLE M LE BEAU |
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| |
| Submission date |
Feb 06, 2018 |
| Last update date |
Mar 21, 2019 |
| Contact name |
Tao Pan |
| E-mail(s) |
[email protected]
|
| Phone |
(773) 702-4179
|
| Organization name |
University of Chicago
|
| Department |
Biochemistry and Molecular Biology
|
| Street address |
929 E. 57th Street
|
| City |
Chicago |
| State/province |
Illinois |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA433264 |
| SRA |
SRP132322 |
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