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| Status |
Public on Aug 04, 2013 |
| Title |
Top3β is an RNA topoisomerase that works with Fragile X syndrome protein to promote synapse formation |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Topoisomerases are crucial to solve DNA topological problems, but they have not been linked to RNA metabolism. Here we show that human topoisomerase 3β (Top3β) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein deficient in Fragile X syndrome and known to regulate translation of mRNAs important for neuronal function and autism. Notably, the FMRP-Top3β interaction is abolished by a disease-associated FMRP mutation, and several human genetic studies link Top3β mutation to schizophrenia and intellectual disability. Top3β binds multiple mRNAs encoded by genes with neuronal functions related to schizophrenia and autism. Expression of one such gene, ptk2/FAK, is reduced in neuromuscular junctions of Top3β mutant flies. Synapse formation is defective in Top3β mutant flies and mice, as observed in FMRP mutant animals. Our findings suggest that Top3β acts as an RNA topoisomerase and works with FMRP to promote expression of mRNAs critical for neurodevelopment and mental health.
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| Overall design |
We have identified Top3β as the first RNA topoisomerase in eukaryotes. To study whether Top3β binds mRNAs in vivo and also to identify its genome-wide RNA targets, we performed HITS-CLIP (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation; Licatalosi et al. Nature 456, 464-469, 2008). In this method, proteins are covalently crosslinked with bound RNAs in vivo, thus allowing specific mapping of the sites of interaction of the protein with the RNA. We cultured a HeLa cells stably-expressing HF-Top3β and irradiated the cells with UV to crosslink RNA and proteins in vivo. Following cell lysis, RNA was partially digested using various concentrations of RNase A. The RNA crosslinked to HF-Top3β was then immunoprecipitated with anti-Flag M2 argarose beads (Sigma). As a control, we also performed a mock immunoprecipitation using HeLa cells that do not express HF-Top3β. The 3’ ends of the purified RNAs were ligated to a p32-labeled RNA adapter, fractionated by SDS-PAGE, and transferred to a nitrocellulose membrane. The p32-labeled RNA-Top3β complexes were detected by autoradiography and recovered from the membrane. The complexes were treated with Proteinase K to remove proteins. The RNA was ligated at their 5’end to another RNA adaptor, and reverse-transcribed to cDNA. The cDNA obtained was fractionated a denaturing 6% TBE-Urea gel (InVitrogen), amplified by PCR, and subjected to high-throughput sequencing by Genome Analyzer II (Illumina). Sequenced tags were mapped to the human genome (hg18, NCBI/NIH) and to human RefSeq genes using ELAND program (Illumina).
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| Contributor(s) |
Xu D, Shen W, Wang W |
| Citation(s) |
23912945 |
| Submission date |
May 30, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Minoru S.H. Ko |
| E-mail(s) |
kom@mail.nih.gov
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| Phone |
410-558-8359
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| Organization name |
NIH
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| Department |
National Institute on Aging
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| Lab |
Lab of Genetics
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| Street address |
251 Bayview Blvd, Suite 100, 10C
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21224 |
| Country |
USA |
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| Platforms (1) |
| GPL9115 |
Illumina Genome Analyzer II (Homo sapiens) |
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| Samples (1) |
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| Relations |
| BioProject |
PRJNA205898 |
| SRA |
SRP023351 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE47502_RAW.tar |
156.3 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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