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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 15, 2013 |
| Title |
HITS-CLIP analysis of FMRP mRNA binding sites from P11-P25 mouse brain polysomes |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
FMRP loss-of-function causes Fragile X Syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here we use high throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins, and transcripts implicated in autism spectrum disorders
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| Overall design |
Polyribosomes were prepared from UV254-crosslinked pooled littermate FVB mouse brains as described in detail in published paper PMID 21784246. Polyribosomes were dissociated under denaturing conditions using two different protocols to disrupt ribonucleoprotein complexes. In most experiments Fmr1-null littermates were used in parallel to confirm FMRP specificity. After mild RNAse treatment to reduce the size of RNA to 60-100 nucleotides, endogenous FMRP was immunoprecipitated with one of two antibody combinations, either mixed monoclonal antibodies 7G1-1 and 2F5, or polyclonal antibody ab17722 (Abcam). The ages of the mice were P11, P13, P14, P15, and P25. In addition, to assess a different neuronal polysome-associated protein, as another control, lysates prepared from two samples, the P11 and P13 mice, were split in half and one half IPed with a human anti-Hu antisera as the neuronal Hu proteins are polysome-associated in brain. In sum, 7 FMRP HITS-CLIP experiments were performed including (1) P14 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (2) P14 mouse polysomes prepared by Protocol 1 and IPed with ab17722 (3) P15 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (4) P25 mouse polysomes prepared by Protocol 1 and IPed with 7G1-1/2F5 (5) P25 mouse polysomes prepared by Protocol 1 and IPed with ab17722 (6) P11 mouse polysomes prepared by Protocol 2 and IPed with ab17722 and (7) P13 mouse polysomes prepared by Protocol 2 and IPed with ab17722. FMRP-bound or Hu-bound RNA tags were cloned and submitted for high throughput sequencing on the Life Science 454 platform (samples 1-5) or the Illumina platform (samples 6 and 7 and the 2 Hu samples processed in parallel with samples 6 and 7). Full details are given in PMID 21784246.
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| Contributor(s) |
Darnell JC, Zhang C, Darnell RB |
| Citation(s) |
21784246 |
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| Submission date |
Mar 13, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jennifer Darnell |
| E-mail(s) |
darneje@rockefeller.edu
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| Organization name |
The Rockefeller University
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| Street address |
1230 York Ave.
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platforms (2) |
| GPL9250 |
Illumina Genome Analyzer II (Mus musculus) |
| GPL9522 |
454 GS 20 (Mus musculus) |
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| Samples (9)
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| GSM1098051 |
FMRP HITS-CLIP from age P14 mouse brain polysomes, IPed with mixed monoclonal antibodies |
| GSM1098052 |
FMRP HITS-CLIP from age P14 mouse brain polysomes, IPed with a polyclonal antibody |
| GSM1098053 |
FMRP HITS-CLIP from age P15 mouse brain polysomes, IPed with mixed monoclonal antibodies |
| GSM1098054 |
FMRP HITS-CLIP from age P25 mouse brain polysomes, IPed with mixed monoclonal antibodies |
| GSM1098055 |
FMRP HITS-CLIP from age P25 mouse brain polysomes, IPed with a polyclonal antibody |
| GSM1098056 |
FMRP HITS-CLIP from age P11 mouse brain polysomes, IPed with a polyclonal antibody |
| GSM1098057 |
FMRP HITS-CLIP from age P13 mouse brain polysomes, IPed with a polyclonal antibody |
| GSM1098058 |
Hu HITS-CLIP from age P11 mouse brain polysomes, IPed with anti-Hu antisera |
| GSM1098059 |
Hu HITS-CLIP from age P13 mouse brain polysomes, IPed with anti-Hu antisera |
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| Relations |
| BioProject |
PRJNA193166 |
| SRA |
SRP019264 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE45148_RAW.tar |
14.0 Mb |
(http)(custom) |
TAR (of BED, TXT) |
| GSE45148_jendarnell_supplementalinformation_PMID_21784246tableS2C.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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