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| Status |
Public on Sep 28, 2015 |
| Title |
m6A-CLIP identified major presence of m6A in last exons |
| Organisms |
Homo sapiens; Mus musculus |
| Experiment type |
Other
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| Summary |
By adapting UV cross-linking immune precipitation for m6A (m6A-CLIP), we developed several novel genomic approaches with single-nucleotide resolution to accurately locate tens of thousands of m6A residues in human cells and mouse brain mRNAs. We found over 70% of these residues in the last exon with a very sharp rise (six-fold) within 150-400 nucleotides of the last exon, which overlaps the beginning of many 3′ untranslated region (UTRs). The 3′ UTRs contain ~two-thirds of the last exon m6A and 40 to 45% of the total of this base modification in mRNA. Contrary to earlier studies, we found no preference for location of m6A sites in the coding sequences around STOP codons. Many mRNA 3′ UTRs contain multiple polyA sites at least some of which are subject to regulated choices when cells change growth rate or differentiation state. The m6A density is at a peak early in the 3′ UTR and gradually diminishes in the more distal region of the last exon suggesting a possible inhibitory role of the proximal m6A residues to allow polyA choice of more distal polyA sites. This possibility was supported by finding that a main switch from distal to proximal polyA site choice is associated with m6A loss after triple knockdown of the m6A methylase complex. There is a significant overlap of m6A with identified binding sites for Ago complexes that carry microRNAs known to regulate mRNA stability which might possibly represent cooperation in function. With higher accuracy of m6A identification it is now more realistic to engage in highly localized mutagenesis to more definitively identify m6A function.
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| Overall design |
Precise mapping m6A sites in mouse brain and liver tissues, human CD8 T cells and A549 cells by m6A-CLIP/IP; PolyA sites mapping by PolyA-Seq (Derti et al., 2012) in the same tissues and cells
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| Contributor(s) |
Darnell JE, Darnell RB |
| Citation(s) |
26404942 |
| Submission date |
Jul 21, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Shengdong Ke |
| E-mail(s) |
sk2410@caa.columbia.edu
|
| Organization name |
The Jackson Laboratory
|
| Lab |
The Ke Lab of Quantitative RNA Biology
|
| Street address |
600 Main Street
|
| City |
Bar Harbor |
| State/province |
ME |
| ZIP/Postal code |
04609 |
| Country |
USA |
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| Platforms (4)
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| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA290463 |
| SRA |
SRP061368 |
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