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| Status |
Public on Nov 15, 2015 |
| Title |
miRNA-target chimeras identified with CLEAR-CLIP reveal miRNA 3' end pairing as a major determinant of Argonaute binding in vivo [mouse cortex CLEAR-CLIP] |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate post-transcriptional silencing of target mRNAs. Despite their importance in many biological processes, rules governing AGO-miRNA targeting are only partially understood. We use a modified AGO HITS-CLIP strategy, termed CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs) CLIP that enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ~130,000 endogenous miRNA-target interactions in mouse brain and ~40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3’ ends. This work provides a means for explicit biochemical identification of miRNA sites in vivo, leading to the discovery that miRNA 3’ end pairing is a general determinant of AGO binding specificity.
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| Overall design |
In vivo microRNA-target interactions were identified with CLEAR-CLIP, a modified HITS-CLIP approach that allows direct ligation of microRNA and target within purified, endogenous AGO-RNA complexes. Native AGO-RNA complexes were purified under stringent conditions selectively preserve cross-linked AGO-target interactions, then microRNA and target mRNA were ligated within the AGO complex to form miRNA-target chimeras in addition to standard AGO CLIP reads. AGO-bound RNAs were cloned by addition of a pre-adenylated 3' linker, a 5' RNA linker containing a 4 nt degenerate barcode followed by a G (NNNNG), and RT-PCR amplification. A second round of PCR amplification added a 4 nt index (for multiplexing) followed by a 16 nt common priming sequence. Raw data files have been de-multiplexed, but are otherwise not modified. Samples BR4 and BR5 are no-ligase controls in which chimera ligation was omitted. Several 'mixing' controls experiments were also done to measure ligation of microRNAs to non-cross-linked targets due to post-lysis re-association by adding E. coli RNA (BR17-20) or Drosophila S2 cell lysates (BR21, BR22) into mouse brain lysates.
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| Contributor(s) |
Moore M, Scheel T, Rice C, Darnell R |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Sep 15, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael John Moore |
| Organization name |
The Rockefeller University
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| Department |
Molecular Neuro-Oncology
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| Lab |
Robert Darnell
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| Street address |
1230 York Ave Box 226
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platforms (2) |
| GPL16417 |
Illumina MiSeq (Mus musculus) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
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| Samples (26)
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| GSM1881519 |
cortex CLEAR-CLIP, biological rep 4 |
| GSM1881520 |
cortex CLEAR-CLIP, biological rep 5 |
| GSM1881521 |
cortex CLEAR-CLIP, biological rep 6, Miseq run |
| GSM1881522 |
cortex CLEAR-CLIP, biological rep 6, Hiseq run |
| GSM1881523 |
cortex CLEAR-CLIP, biological rep 7 |
| GSM1881524 |
cortex CLEAR-CLIP, biological rep 8 |
| GSM1881525 |
cortex CLEAR-CLIP, biological rep 9 |
| GSM1881526 |
cortex CLEAR-CLIP, biological rep 10a |
| GSM1881527 |
cortex CLEAR-CLIP, biological rep 10b |
| GSM1881528 |
cortex CLEAR-CLIP, biological rep 11 |
| GSM1881529 |
cortex CLEAR-CLIP, biological rep 12a |
| GSM1881530 |
cortex CLEAR-CLIP, biological rep 12b |
| GSM1881531 |
cortex CLEAR-CLIP, biological rep 13 |
| GSM1881532 |
cortex CLEAR-CLIP, biological rep 14 |
| GSM1881533 |
cortex CLEAR-CLIP, biological rep 15 |
| GSM1881534 |
cortex CLEAR-CLIP, biological rep 16 |
| GSM1881535 |
cortex CLEAR-CLIP, biological rep 17 |
| GSM1881536 |
cortex CLEAR-CLIP, biological rep 18 |
| GSM1881537 |
cortex CLEAR-CLIP, biological rep 19 |
| GSM1881538 |
cortex CLEAR-CLIP, biological rep 20 |
| GSM1881539 |
cortex CLEAR-CLIP, biological rep 21 |
| GSM1881540 |
cortex CLEAR-CLIP, biological rep 22 |
| GSM1881541 |
cortex CLEAR-CLIP, biological rep 23 |
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| This SubSeries is part of SuperSeries: |
| GSE73059 |
miRNA-target chimeras identified with CLEAR-CLIP reveal miRNA 3' end pairing as a major determinant of Argonaute binding in vivo |
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| Relations |
| BioProject |
PRJNA296129 |
| SRA |
SRP063858 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE73058_Mouse_cortex_AGO_peak_information.xlsx |
11.5 Mb |
(ftp)(http) |
XLSX |
| GSE73058_Mouse_cortex_miRNA_chimera_interactions.xlsx |
21.5 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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