The NORAD lncRNA assembles a topoisomerase complex critical for genome stability
The NORAD lncRNA assembles a topoisomerase complex critical for genome stability
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DOI：
10.1038/s41586-018-0453-z
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发表时间：
2018-09-06
期刊：
影响因子：
64.8
通讯作者：
Lander, Eric S.
中科院分区：
文献类型：
--
作者：
Munschauer, Mathias;Nguyen, Celina T.;Lander, Eric S.
人类基因组包含数千个长的非编码RNA 1，但仅发现了大约十几个特定的生物学功能和生化机制。一种特定的长非编码RNA-DNA损伤激活的非编码RNA（NORAD）-最近被证明是维持基因组稳定性所必需的（8），但其分子机制尚不清楚。在这里，我们结合联合收割机RNA反义纯化和定量质谱，以确定蛋白质，直接与NORAD在活细胞中。我们表明，NORAD与蛋白质参与DNA复制和修复在稳态细胞和本地化后，与复制应激或DNA损伤的刺激细胞核。特别是，NORAD与DNA损伤反应的一个组成部分RBMX相互作用，并在转录组中包含最强的RBMX结合位点。我们证明，NORAD控制RBMX组装核糖核蛋白复合物的能力，我们称之为NORAD激活的核糖核蛋白复合物1（NARC 1），其中包含已知的抑制基因组不稳定性拓扑异构酶I（TOP1），MATRIX REF和PRPF 19-CDC 5L复合物。细胞耗尽NORAD或RBMX显示染色体分离缺陷的频率增加，复制叉速度降低和细胞周期进程改变-这代表了与TOP 1和PRPF 19-CDC 5L功能机械相关的表型。NORAD的反式表达可以挽救由NORAD耗尽引起的缺陷，但当NORAD中的RBMX结合位点被删除时，挽救会显著受损。我们的研究结果表明，NORAD和RBMX之间的相互作用是重要的NORAD功能，NORAD是需要组装以前未知的拓扑异构酶复合物NARC 1，这有助于保持基因组的稳定性。此外，我们发现了一个以前未知的功能，长的非编码RNA在调节RNA结合蛋白组装一个高阶核糖核蛋白复合物的能力。
The human genome contains thousands of long non-coding RNAs1, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen(2-7). A specific long non-coding RNA-non-coding RNA activated by DNA damage (NORAD)-has recently been shown to be required for maintaining genomic stability(8), 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.