The NORAD Lncrna Assembles a Topoisomerase Complex Critical for Genome Stability Mathias Munschauer1*, Celina T
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Corrected: Publisher Correction LETTER https://doi.org/10.1038/s41586-018-0453-z The NORAD lncRNA assembles a topoisomerase complex critical for genome stability Mathias Munschauer1*, Celina T. Nguyen1, Klara Sirokman1, Christina R. Hartigan1, Larson Hogstrom1, Jesse M. Engreitz1, Jacob C. Ulirsch1,2,3, Charles P. Fulco1, Vidya Subramanian1, Jenny Chen1,4, Monica Schenone1, Mitchell Guttman5, Steven A. Carr1 & Eric S. Lander1,6,7* The human genome contains thousands of long non-coding RNAs1, cytoplasmic extracts, which may not accurately represent the protein but specific biological functions and biochemical mechanisms contacts of NORAD in living cells (Supplementary Note 1). have been discovered for only about a dozen2–7. A specific long To reveal the direct interactions of NORAD with proteins in live non-coding RNA—non-coding RNA activated by DNA damage cells, we captured and identified NORAD-interacting proteins by (NORAD)—has recently been shown to be required for maintaining combining RNA antisense purification (RAP) with quantitative liquid genomic stability8, but its molecular mechanism is unknown. Here chromatography–mass spectrometry using isobaric mass tag quantifi- we combine RNA antisense purification and quantitative mass cation (RAP MS) (Fig. 1a). HCT116 colon carcinoma cells were treated spectrometry to identify proteins that directly interact with NORAD with 365-nm light after 4-thiouridine labelling10, which covalently in living cells. We show that NORAD interacts with proteins involved crosslinks proteins to RNA but not to other proteins. lncRNA– in DNA replication and repair in steady-state cells and localizes protein complexes were purified by RNA hybrid selection with to the nucleus upon stimulation with replication stress or DNA antisense oligonucleotides that target NORAD, under denaturing damage. In particular, NORAD interacts with RBMX, a component and reducing conditions at high temperature to minimize the co- of the DNA-damage response, and contains the strongest RBMX- purification of indirectly bound proteins2 (Fig. 1a). To identify specific binding site in the transcriptome. We demonstrate that NORAD interactors with NORAD, we quantitatively compared the resulting pro- controls the ability of RBMX to assemble a ribonucleoprotein teins to those captured in purifications with antisense oligonucleotides complex—which we term NORAD-activated ribonucleoprotein that target the well-characterized ‘RNA component of mitochondrial complex 1 (NARC1)—that contains the known suppressors of RNA processing endoribonuclease’ (RMRP), which is not expected to genomic instability topoisomerase I (TOP1), ALYREF and the interact with the same proteins as NORAD 11. We analysed biological PRPF19–CDC5L complex. Cells depleted for NORAD or RBMX replicate purifications in a single 4-plex iTRAQ cassette, quantifying display an increased frequency of chromosome segregation 1,361 proteins that each had more than two unique peptides (Fig. 1b). defects, reduced replication-fork velocity and altered cell-cycle The control purification captured about 85% of RMRP transcripts progression—which represent phenotypes that are mechanistically (Extended Data Fig. 1) and enriched the target RNA approximately linked to TOP1 and PRPF19–CDC5L function. Expression of 550-fold versus input RNA. We found 12 strongly enriched proteins NORAD in trans can rescue defects caused by NORAD depletion, (mean log2(iTRAQ ratio (NORAD/RMRP)) < −1.6, P < 0.05, moder- but rescue is significantly impaired when the RBMX-binding site ated t-test) (Fig. 1c), including 8 of the 10 known core components of in NORAD is deleted. Our results demonstrate that the interaction the RMRP complex11 and one previously identified candidate rRNA- between NORAD and RBMX is important for NORAD function, and and/or tRNA-processing factor12. that NORAD is required for the assembly of the previously unknown We then analysed NORAD antisense purifications. Experiments topoisomerase complex NARC1, which contributes to maintaining captured 82% of endogenous NORAD (Extended Data Fig. 1) (about genomic stability. In addition, we uncover a previously unknown 80-fold enrichment versus input RNA). We reproducibly identified function for long non-coding RNAs in modulating the ability of an 45 proteins that met our enrichment criteria (mean log2(iTRAQ ratio RNA-binding protein to assemble a higher-order ribonucleoprotein (NORAD/RMRP)) > 1.6, P < 0.05, moderated t-test) (Fig. 1b). This set complex. of proteins is highly specific to NORAD, in that 41 out of the 45 proteins NORAD stands out among long non-coding RNAs (lncRNAs) (Fig. 1c) were not among 219 promiscuous binders (Supplementary because it (1) is highly conserved relative to other lncRNAs, (2) is Note 3). The RNA-binding protein PUMILIO2 (PUM2) was indeed abundantly expressed in many cell types, (3) is upregulated upon DNA present in our NORAD interactome, but it ranked 185th out of damage and (4) induces chromosomal instability and aneuploidy when the 265 proteins we detected (mean log2(iTRAQ ratio (NORAD/ deleted. This phenotype is intriguing as little is known about the roles RMRP)) > 0.5) and did not meet our cut-off for strongly enriched of lncRNAs in maintaining a stable genome. A model for lncRNA proteins. function suggests that lncRNAs can serve as assembly scaffolds for Notably, many of the 41 NORAD-interacting proteins have key roles ribonucleoprotein complexes6,7, yet this model has been explored in in nuclear processes such as DNA unwinding, replication and repair only a few cases. The mechanism that connects the NORAD lncRNA (including PURA, PURB, TAF15, ALYREF, SFPQ, SRSF1, RBM14, to chromosomal instability remains unknown. DDX17, RBMX and its retrogene RBMXL1). Twenty-nine (71%) of Two recent studies have reported PUMILIO, a highly abundant cyto- the forty-one proteins localize to the nucleus, nucleoplasm or chro- plasmic RNA-binding protein with no known role in genomic stability, matin, whereas only two (5%) localize exclusively to the cytoplasm as the sole NORAD-interacting protein8,9. However, these results were (Fig. 1d). The interactome thus points towards an important nuclear obtained from in vitro mixing of exogenous NORAD fragments with function of NORAD. 1Broad Institute of MIT and Harvard, Cambridge, MA, USA. 2Division of Hematology/Oncology, Boston Children’s Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. 3Program in Biological and Biomedical Sciences, Harvard University, Cambridge, MA, USA. 4Division of Health Sciences and Technology, MIT, Cambridge, MA, USA. 5Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA. 6Department of Biology, MIT, Cambridge, MA, USA. 7Department of Systems Biology, Harvard Medical School, Boston, MA, USA. *e-mail: [email protected]; [email protected] 132 | NATURE | VOL 561 | 6 SEPTEMBER 2018 © 2018 Springer Nature Limited. All rights reserved. LETTER RESEARCH 4 a Human 365 nm LC–MS/MS b RBMXL1 cell CSTF2 Ultraviolet crosslink TAF15 HNRNPR 4-thiouridine 2 RBMX PURA eplicate 2) KHDRBS3 ) r Identication Cell lysis, Quantication Biotin y antisense- RMRP / capture –4 –2 24 Intensit POP1 Denaturing RPP40 NORAD RPP25L m/z ( purication C18ORF21 –2 RPP14 POP4 POP5 Protein elution Trypsin NORAD RMRP NORAD RMRP –4 by benzonase digest rep 1 rep 1 rep 2 rep 2 (iTRAQ ratio RMRP mean log2 ratio < –1.6, P < 0.05 2 NORAD mean log2 ratio > 1.6, P < 0.05 Isobaric mass tag labelling log log2(iTRAQ ratio (NORAD/RMRP) replicate 1) c 10 RMRP enriched proteins NORAD enriched proteins NORAD interactome iTRAQ (RMRP/NORAD) 10 iTRAQ (NORAD/RMRP) d 100% Cytoplasm Nucleus & 5 80% 5 cytoplasm Chromatin iTRAQ ratio 60% Nucleoplasm 0 0 1 1 9 3 3 L 2 3 7 2 5 0 8 4 0 A R 40% LY A0 AB F15 TR3 FUS RA YREF DDX5 POP4 POP5 POP1 SFPQ PURA PURB RBM3 TA RBMX SF3A CIRBP Nucleus U2AF2 CSTF2 ALPK1 RPP2 RPP4 RPP3 RPP1 RPP3 SRSF SRSF SRSF DDX17 DDX3X PTPR PSPC MA 20% SUGP RBM47 RBM45 RBM14 KHSRP CHERP AL RPP25L SRSF10 AKAP8 DAZAP1 PPHLN1 LSM14B RBMXL1 IGF2BP IGF2BP HNRNP C3ORF17 HNRNP KHDRBS HNRNP C18ORF21 APOBEC3F 0% Fig. 1 | NORAD directly binds many nuclear proteins in living cells. of RMRP- (blue) and NORAD- (red) enriched proteins. Columns represent a, Schematic overview of RAP MS. b, Quantification of NORAD- and the mean of two biological replicate experiments, individual data points RMRP-interacting proteins. Scatter plot of log2-transformed iTRAQ ratios are shown (Supplementary Tables 1, 2). d, Subcellular localization of from two biological replicates is shown. Adjusted P value, two-tailed NORAD-interacting proteins. moderated t-test (Supplementary Note 2). Rep, replicate. c, iTRAQ ratios Given the overrepresentation of nuclear proteins, we used single- sequenced RNA crosslinked to RBMX. RBMX displays unusually molecule RNA fluorescent in situ hybridization (smRNA FISH) to strong and specific binding to the 5′ end of NORAD (Fig. 2e). The assess the subcellular localization of NORAD in intact cells. In contrast RBMX-binding site in NORAD extends over more than 800 nucleotides to previous reports that characterized NORAD as being located exclu- and covers about 15% of NORAD—making it eight times larger than sively in the cytoplasm8, we found that on average 40–50% of NORAD the majority of RBMX-binding sites (Extended Data Fig. 3d) and the transcripts in HCT116 cells reside in the nucleus (Fig. 2a and Extended strongest RBMX-binding region in the transcriptome (Fig. 2f). This Data