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| Status |
Public on Jun 23, 2019 |
| Title |
Antibody crossreactivity with mRNA cap accounts for widespread appearance of m1A throughout the transcriptome |
| Organisms |
Homo sapiens; Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
N1-methyladenosine (m1A) was recently identified as a new mRNA modification based on its mapping to the 5’UTRs of thousands of mRNAs with an m1A-binding antibody. More recent studies have questioned the prevalence of m1A. To address this discrepancy, we mapped m1A using ultra-deep RNA-Seq datasets based on m1A-induced misincorporations during reverse transcription. Using this approach, we find m1A only in the mitochondrial MT-ND5 transcript. In contrast, m1A mapping using an m1A-binding antibody showed binding to transcription start nucleotides in mRNA 5’UTRs. Biochemical measurements indicate that m1A is not present at these sites. Instead, we find that the m1A antibody exhibits m1A-independent binding to mRNA cap structures. Our data demonstrate that high-stoichiometry m1A sites are rare in the transcriptome and that previous mapping of m1A to mRNA 5’UTRs reflect an unintended binding to mRNA caps.
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| Overall design |
The data for this study contains the following samples:
m1A miCLIP of HEK 293T cell poly(A) RNA: 2 biological replicates
RNA-Seq of HEK 293T cell poly(A) RNA: 1 biological replicate
m1A miCLIP in mouse brain poly(A) RNA: 6 biological replicates (3 control, 3 AlkB-treated)
Second 1mA miCLIP of HEK 293T cells poly(A) RNA: 6 samples (2 biological replicates per antibody, 3 different antibodies)
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| Contributor(s) |
Grozhik AV, Jaffrey SR, Olarerin-George AO |
| Citation(s) |
31719534 |
| Submission date |
Apr 17, 2017 |
| Last update date |
Nov 19, 2019 |
| Contact name |
Anya Grozhik |
| E-mail(s) |
ang2040@med.cornell.edu
|
| Organization name |
Weill Cornell Medicine
|
| Street address |
1300 York Ave, Room LC-523
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
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| Platforms (3) |
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
|
| Samples (15)
|
| GSM2580141 |
1mA miCLIP mouse brain CTL replicate 1 |
| GSM2580142 |
1mA miCLIP mouse brain CTL replicate 2 |
| GSM2580143 |
1mA miCLIP mouse brain CTL replicate 3 |
| GSM2580144 |
1mA miCLIP mouse brain AlkB replicate 1 |
| GSM2580145 |
1mA miCLIP mouse brain AlkB replicate 2 |
| GSM2580146 |
1mA miCLIP mouse brain AlkB replicate 3 |
| GSM3756486 |
1mA 2nd miCLIP HEK 293T Abcam replicate 1 |
| GSM3756487 |
1mA 2nd miCLIP HEK 293T Abcam replicate 2 |
| GSM3756488 |
1mA 2nd miCLIP HEK 293T Diagenode replicate 1 |
| GSM3756489 |
1mA 2nd miCLIP HEK 293T Diagenode replicate 2 |
| GSM3756490 |
1mA 2nd miCLIP HEK 293T MBL replicate 1 |
| GSM3756491 |
1mA 2nd miCLIP HEK 293T MBL replicate 2 |
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| Relations |
| BioProject |
PRJNA383349 |
| SRA |
SRP104159 |
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