1. Chromosomes and Gene Expression
  2. Genetics and Genomics
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PUMILIO, but not RBMX, binding is required for regulation of genomic stability by noncoding RNA NORAD

  1. Mahmoud M Elguindy
  2. Florian Kopp
  3. Mohammad Goodarzi
  4. Frederick Rehfeld
  5. Anu Thomas
  6. Tsung-Cheng Chang
  7. Joshua T Mendell  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
Research Advance
Cite this article as: eLife 2019;8:e48625 doi: 10.7554/eLife.48625
7 figures, 1 table, 1 data set and 2 additional files

Figures

NORAD localizes predominantly to the cytoplasm.

(A) RNA FISH in HCT116 cells using a panel of 11 probes tiling the entire NORAD transcript reveals a predominantly cytoplasmic signal that is absent in NORAD–/– cells with all probes except probe 7, which produces a nonspecific signal likely due to the presence of an Alu repeat element. NORAD FISH signal in red, DAPI counterstain in blue. Locations of PREs indicated by arrowheads. ND1-ND5 represent repetitive NORAD domains, as previously described (Lee et al., 2016). (B) RNA FISH image using probe 3 showing a wider field of cells. (C) Subcellular fractionation followed by qRT-PCR in HCT116 cells using primers located at the 3′ or 5′ end of NORAD, in GAPDH (cytoplasmic control), or in NEAT1 (nuclear control). n = 3 biological replicates each with three technical replicates.

https://doi.org/10.7554/eLife.48625.002
Figure 2 with 1 supplement
NORAD remains predominantly in the cytoplasm after treatment with DNA damaging agents.

(A) RNA FISH in HCT116 cells using the indicated NORAD probes following a 12 hr treatment with doxorubicin or camptothecin. (B) NORAD RNA FISH (probe 5) after the indicated drug treatments. Images captured with identical microscope settings. (C–D) Subcellular fractionation followed by qRT-PCR in HCT116 cells after treatment with camptothecin (C) or camptothecin plus actinomycin D (D) for the indicated number of hours. n = 3 biological replicates each with three technical replicates. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; one-tailed t-test comparing each sample to the 0 hr time-point.

https://doi.org/10.7554/eLife.48625.003
Figure 2—figure supplement 1
NORAD remains predominantly cytoplasmic following doxorubicin-induced DNA damage.

(A–B) Immunofluorescence (A) or western blot (B) analysis of the DNA damage marker γ-H2AX in HCT116 cells treated with or without doxorubicin (1 μM) or camptothecin (200 nM) for 12 hr. (C–D) Subcellular fractionation followed by qRT-PCR in HCT116 cells after treatment with doxorubicin (C) or doxorubicin plus actinomycin D (D) for the indicated number of hours. n = 3 biological replicates each with three technical replicates. ns, not significant; *p<0.05; **p<0.01; one-tailed t-test comparing each sample to the 0 hr time-point.

https://doi.org/10.7554/eLife.48625.004
Figure 3 with 1 supplement
Generation and stable expression of NORAD constructs.

(A) Schematic depicting wild-type or mutant NORAD constructs. NORAD sequence conservation in mammals (UCSC Genome Browser Hg38 PhastCons track) highlights the strong conservation of the region of NORAD harboring PREs (arrowheads). PREmut contains 18 UGU to ACA mutations in PREs (gray arrowheads); 5′ deletion (5′ del) lacks the RBMX binding site (nt 1–898) (Munschauer et al., 2018); 5′ fragment (5′ frag) spans the RBMX binding site (nt 33–898); ND4 construct represents the most conserved segment of NORAD (nt 2494–3156). (B) Schematic depicting insertion of constructs into the AAVS1/PPP1R12C locus using TALENs. (C) qRT-PCR analysis of expression of each NORAD construct in HCT116 CRISPRi cells after infection with control or endogenous NORAD-targeting sgRNAs. Expression was normalized to endogenous NORAD level, represented by expression in AAVS1-GFP cells infected with sgControl (replicate 1 samples normalized to sgControl AAVS1-GFP replicate 1; replicate 2 samples normalized to sgControl AAVS1-GFP replicate 2). The data in the left graph were generated with a primer pair in ND4 that does not amplify the 5′ fragment, while the right graph used primers at the NORAD 5′ end. Replicates represent two independently-derived AAVS1 knock-in and sgRNA-infected cell lines. Values normalized to GAPDH expression. n = 3 technical replicates per sample.

https://doi.org/10.7554/eLife.48625.005
Figure 3—figure supplement 1
Reanalysis of NORAD RAP-MS data.

Analysis of previously published NORAD RAP-MS data (Munschauer et al., 2018) using a combined three search engine algorithm (MS Amanda, Sequest HT, Mascot) identifies isoforms of PUM1 (green), PUM2 (red), and RBMX (blue) as significantly enriched NORAD interactors. Volcano plot showing the average fold-change compared to control RMRP pull-down and significance from two biological replicates.

https://doi.org/10.7554/eLife.48625.006
Figure 4 with 1 supplement
RNA immunoprecipitation and localization of NORAD constructs.

(A) UV crosslinking and RNA immunoprecipitation (RIP) was used to assess PUM1, PUM2, and RBMX interactions with GFP mRNA or the indicated NORAD constructs. After knock-in of the indicated constructs to the AAVS1 locus in HCT116 CRISPRi cells, endogenous NORAD was silenced with a lentivirally-expressed sgRNA. qRT-PCR was used to assess NORAD or GAPDH recovery in each RIP sample, expressed as fold-enrichment over pull-down with IgG. The data in the left graphs were generated with a primer pair in ND4 that does not amplify the 5′ fragment, while the right graphs used primers at the NORAD 5′ end. n = 2 biological replicates, each measured with three technical replicates. (B) Representative RNA FISH images of wild-type or mutant NORAD transcripts expressed from the AAVS1 locus in HCT116 CRISPRi cells after knockdown of endogenous NORAD. Probe 10 was used for full-length NORAD, PREmut, and 5′ del constructs; probe 1 was used for 5′ frag; and probe 6 was used for ND4.

https://doi.org/10.7554/eLife.48625.007
Figure 4—figure supplement 1
Representative western blots of PUM1, PUM2, and RBMX in RIP experiments.

PUM1 migrates as a doublet in HCT116, possibly indicating post-translational modification or alternative splicing. We have confirmed that both bands represent PUM1 protein using siRNA knockdown experiments (data not shown).

https://doi.org/10.7554/eLife.48625.008
PUMILIO, but not RBMX, binding to NORAD is necessary for genome stability.

(A) Representative DNA FISH images for chromosome 7 (green) and 20 (red) showing examples of cells with modal (2 n) and non-modal (2n ± 1) chromosome numbers. Arrowheads indicate chromosome gain or loss. (B) HCT116 CRISPRi cells stably expressing the indicated AAVS1 knock-in construct were infected with lentivirus expressing control or endogenous NORAD-targeting sgRNA. Aneuploidy was assayed 18–21 days later using DNA FISH for chromosome 7 and 20, and the frequency of interphase cells exhibiting a non-modal (2 n) chromosome number was scored. Replicates represent two independently-derived AAVS1 knock-in and sgRNA-infected cell lines. 200 nuclei were scored per sample. The dotted line denotes the highest level of background aneuploidy observed in sgControl-infected cells. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, chi-square test comparing sgNORAD to sgControl for each replicate. (C) Representative images of anaphase cells with normal or abnormal (arrowheads) chromosome segregation in DAPI-stained HCT116 CRISPRi cells. (D) The frequency of mitotic cells exhibiting chromosome segregation defects was determined in both biological replicates of each cell population (100 anaphase cells assayed per sample). The dotted line denotes the highest percentage of chromosome segregation defects observed in sgControl-infected cells. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, chi-square test comparing all sgNORAD replicate 1 samples to sgControl GFP replicate 1 and all sgNORAD replicate 2 samples to sgControl GFP replicate 2.

https://doi.org/10.7554/eLife.48625.009
RBMX is not required for NORAD expression or localization.

(A) qRT-PCR analysis of RBMX and NORAD transcript levels in HCT116 CRISPRi cells after introduction of the indicated lentivirally-expressed sgRNA with or without doxorubicin treatment (1 μM for 24 hr). Quantification relative to GAPDH. n = 3 technical replicates. (B) Subcellular fractionation and qRT-PCR of NORAD, GAPDH (cytoplasmic control), or NEAT1 (nuclear control) following introduction of control or RBMX-targeting sgRNAs. n = 3 biological replicates each with three technical replicates.

https://doi.org/10.7554/eLife.48625.010
Author response image 1
Co-immunoprecipitation of RBMX and TOP1 in NORADwild-type and knockout cells.

FLAG-tagged RBMX was stably expressed in HCT116 cells and immunoprecipitated using identical conditions as that described in Munschauer et al., 2018.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (Homo sapiens)NORAD (LINC00657)NAEnsembl:ENSG00000260032
Cell line (Homo sapiens)HCT116ATCCCCL-247, RRID:CVCL_0291
Cell line (Homo sapiens)NORAD-/- HCT116Lee et al., 2016
Cell line (Homo sapiens)HCT116 CRISPRithis paper; see Materials and methods section Cell culture and generation of HCT116 CRISPRi cell line
AntibodyAnti-PUM1 (polyclonal rabbit)Santa Cruzsc-135049, RRID:AB_10610604RIP
AntibodyAnti-PUM2 (polyclonal goat)Santa Cruzsc-31535, RRID:AB_654939RIP
AntibodyAnti-RBMX (monoclonal rabbit)Cell Signaling#14794, RRID:AB_2798614RIP,
WB (1:1000)
AntibodyAnti-PUM2 (monoclonal rabbit)Abcamab92390, RRID:AB_10563318WB (1:1000)
AntibodyAnti-PUM1 (monoclonal rabbit)Abcamab92545, RRID:AB_10563695WB (1:1000)
AntibodyAnti-GAPDH (monoclonal rabbit)Cell Signaling#2118, RRID:AB_561053WB (1:5000)
AntibodyAnti-phospho-histone H2A.X (Ser139)Cell Signaling#2577, RRID:AB_2118010IF (1:500),
WB (1:1000)
AntibodyIRDye 800CW anti-rabbit (donkey)Licor925–32213, RRID:AB_2715510WB (1:10000)
AntibodyAnti-Digoxigenin (monoclonal mouse)Roche11333062910, RRID:AB_514495RNA FISH
AntibodyAnti-Mouse IgG, Cy3 (polyclonal goat)EMD MilliporeAP124CRNA FISH
Recombinant DNA reagentAAVS1/PPP1R12C targeting vectorAddgene#22072, RRID:Addgene_22072
Recombinant DNA reagenthAAVS1 1L TALEN; hAAVS1 1R TALENAddgene#35431, RRID:Addgene_35431; #35432, RRID:Addgene_35432
Recombinant DNA reagentpU6-sgRNA EF1a-PuroR-T2A-BFPAddgene#60955, RRID:Addgene_60955CRISPRi-mediated knockdown
Sequence-based reagentChromosome enumeration Probe Chr. 7 (green)Empire GenomicsCHR07-10-GRDNA FISH
Sequence-based reagentChromosome enumeration Probe Chr. 20 (red)Empire GenomicsCHR20-10-REDNA FISH
Software, algorithmPrism 7GraphPad Software
Software, algorithmProteome DiscovererThermo Fisher
Software, algorithmLimma package for RSmyth, 2004

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

The following previously published data sets were used
  1. 1
    MassIVE
    1. M Munschauer
    2. CT Nguyen
    3. K Sirokman
    4. CR Hartigan
    5. L Hogstrom
    6. JM Engreitz
    7. JC Ulirsch
    8. CP Fulco
    9. V Subramanian
    10. J Chen
    11. M Schenone
    12. M Guttman
    13. SA Carr
    14. ES Lander
    (2018)
    The NORAD lncRNA assembles a topoisomerase complex critical for DNA replication and genome stability.

Additional files

Supplementary file 1

Sequences of oligonucleotides used in this study.

https://doi.org/10.7554/eLife.48625.011
Transparent reporting form
https://doi.org/10.7554/eLife.48625.012

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