The NORAD lncRNA assembles a topoisomerase complex critical for genome stability
The human genome contains thousands of long non-coding RNAs1, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen2–7. A specific long non-coding RNA—non-coding RNA activated by DNA damage (NORAD)—has recently been shown to be required for maintain...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Springer Science and Business Media LLC
2020
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| Online Access: | https://hdl.handle.net/1721.1/126584 |
| _version_ | 1826193441561772032 |
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| author | Munschauer, Mathias Nguyen, Celina T. Sirokman, Klara Hartigan, Christina R. Hogstrom, Larson Engreitz, Jesse Michael Ulirsch, Jacob C. Fulco, Charles P. Subramanian, Vidya Chen, Jenny Schenone, Monica Guttman, Mitchell Carr, Steven A Lander, Eric Steven |
| author2 | Harvard University--MIT Division of Health Sciences and Technology |
| author_facet | Harvard University--MIT Division of Health Sciences and Technology Munschauer, Mathias Nguyen, Celina T. Sirokman, Klara Hartigan, Christina R. Hogstrom, Larson Engreitz, Jesse Michael Ulirsch, Jacob C. Fulco, Charles P. Subramanian, Vidya Chen, Jenny Schenone, Monica Guttman, Mitchell Carr, Steven A Lander, Eric Steven |
| author_sort | Munschauer, Mathias |
| collection | MIT |
| description | The human genome contains thousands of long non-coding RNAs1, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen2–7. A specific long non-coding RNA—non-coding RNA activated by DNA damage (NORAD)—has recently been shown to be required for maintaining genomic stability8, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex—which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)—that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19–CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression—which represent phenotypes that are mechanistically linked to TOP1 and PRPF19–CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex. |
| first_indexed | 2024-09-23T09:39:17Z |
| format | Article |
| id | mit-1721.1/126584 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T09:39:17Z |
| publishDate | 2020 |
| publisher | Springer Science and Business Media LLC |
| record_format | dspace |
| spelling | mit-1721.1/1265842022-09-26T12:52:02Z The NORAD lncRNA assembles a topoisomerase complex critical for genome stability Munschauer, Mathias Nguyen, Celina T. Sirokman, Klara Hartigan, Christina R. Hogstrom, Larson Engreitz, Jesse Michael Ulirsch, Jacob C. Fulco, Charles P. Subramanian, Vidya Chen, Jenny Schenone, Monica Guttman, Mitchell Carr, Steven A Lander, Eric Steven Harvard University--MIT Division of Health Sciences and Technology Massachusetts Institute of Technology. Department of Biology Broad Institute of MIT and Harvard Koch Institute for Integrative Cancer Research at MIT The human genome contains thousands of long non-coding RNAs1, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen2–7. A specific long non-coding RNA—non-coding RNA activated by DNA damage (NORAD)—has recently been shown to be required for maintaining genomic stability8, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex—which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)—that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19–CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression—which represent phenotypes that are mechanistically linked to TOP1 and PRPF19–CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex. 2020-08-14T16:31:29Z 2020-08-14T16:31:29Z 2018-08 2018-01 2020-08-11T15:28:04Z Article http://purl.org/eprint/type/JournalArticle 0028-0836 1476-4687 https://hdl.handle.net/1721.1/126584 Munschauer, Mathias et al. "The NORAD lncRNA assembles a topoisomerase complex critical for genome stability." Nature 561, 7721 (August 2018): 132–136 © 2018 Springer Nature Limited en http://dx.doi.org/10.1038/s41586-018-0453-z Nature Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf application/vnd.openxmlformats-officedocument.spreadsheetml.sheet Springer Science and Business Media LLC Prof. Lander via Courtney Crummett |
| spellingShingle | Munschauer, Mathias Nguyen, Celina T. Sirokman, Klara Hartigan, Christina R. Hogstrom, Larson Engreitz, Jesse Michael Ulirsch, Jacob C. Fulco, Charles P. Subramanian, Vidya Chen, Jenny Schenone, Monica Guttman, Mitchell Carr, Steven A Lander, Eric Steven The NORAD lncRNA assembles a topoisomerase complex critical for genome stability |
| title | The NORAD lncRNA assembles a topoisomerase complex critical for genome stability |
| title_full | The NORAD lncRNA assembles a topoisomerase complex critical for genome stability |
| title_fullStr | The NORAD lncRNA assembles a topoisomerase complex critical for genome stability |
| title_full_unstemmed | The NORAD lncRNA assembles a topoisomerase complex critical for genome stability |
| title_short | The NORAD lncRNA assembles a topoisomerase complex critical for genome stability |
| title_sort | norad lncrna assembles a topoisomerase complex critical for genome stability |
| url | https://hdl.handle.net/1721.1/126584 |
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