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. 2016 Jan 14;164(1-2):69-80.
doi: 10.1016/j.cell.2015.12.017. Epub 2015 Dec 24.

Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins

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Free PMC article

Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins

Sungyul Lee et al. Cell. .
Free PMC article

Abstract

Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here, we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is induced after DNA damage, which we termed "noncoding RNA activated by DNA damage", or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500-1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of mRNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.

Figures

Figure 1
Figure 1. Characterization of NORAD, a highly abundant, conserved mammalian noncoding RNA induced by DNA damage
(A) Schematic representation of NORAD (annotated in RefSeq as LINC00657) with associated UCSC Genome Browser tracks depicting mammalian conservation (PhastCons) as well as ENCODE RNA-seq and H3K4me3 ChIP-seq coverage in human cell lines (Rosenbloom et al., 2013). (B) qRT-PCR analysis of NORAD expression relative to 18S rRNA in p53+/+ and p53−/− HCT116 cells (Bunz et al., 1998) with or without treatment with 1μM doxorubicin for 24 hours. For this and all subsequent qPCR figures, error bars represent standard deviations from 3 independent measurements. (C) Northern blot analysis of NORAD expression in total RNA in HCT116 cells. (D) Absolute quantification of NORAD transcript copy number per cell, determined by qRT-PCR, in various human cell lines with or without treatment with 1μM doxorubicin for 24 hours. (E) Maximum CSF scores of NORAD as well as other known coding and noncoding RNAs determined by analysis with PhyloCSF (Lin et al., 2011). See also Figure S1.
Figure 2
Figure 2. Genetic inactivation of NORAD results in chromosomal instability in human cells
(A) NORAD was inactivated in human cell lines using custom TALEN pairs (represented as scissors) that cleave within the first 300 nucleotides of the gene, thereby stimulating the insertion of a puromycin resistance cassette (PuroR) followed by tandem polyadenylation signals (STOP). Green triangles represent loxP sites. (B) Northern blot analysis of NORAD in HCT116 clones of the indicated genotypes. (C) Flow cytometry histograms showing DNA content, as measured by propidium iodide staining, in representative diploid and tetraploid NORAD−/− HCT116 clones. (D) Metaphase spreads of wild-type HCT116 cells and representative tetraploid and diploid NORAD−/− clones. The number in the lower right corner of each image shows the number of chromosomes present. Abnormal chromosome numbers indicated in red. (E) Representative images of chromosome 7 and 20 FISH in NORAD+/+ and NORAD−/− HCT116 cells. White arrowheads highlight cells with chromosome loss or gain. (F) NORAD−/− cells exhibit significantly elevated levels of aneuploidy. At least 100 interphase nuclei in each of 3 independent knockout clones were assayed for chromosome 7 and 20 using DNA FISH and the frequency of cells exhibiting a non-modal chromosome number was scored. **p<0.005, chi-square test. (G) Representative time-lapse images of mitoses in NORAD+/+ and NORAD−/− HCT116 cells. Time stamp indicates minutes elapsed. (H, I) Quantification of the percentage of mitoses exhibiting the indicated mitotic errors in time-lapse imaging experiments. Values represent the average of 3 independent experiments with 39–100 mitoses imaged per genotype per experiment. Error bars represent standard deviations. *p<0.05; **p<0.01, Student’s t-test. See also Figures S1, S2, and S3.
Figure 3
Figure 3. Chromosomal instability is specifically due to NORAD inactivation
(A) Insertion of a puromycin resistance cassette at the AAVS1/PPP1R12C locus was performed using a published TALEN pair (Hockemeyer et al., 2009; Sanjana et al., 2012) and the frequency of aneuploidy in homozygous targeted HCT116 clones was assessed using DNA FISH as in Figure 2E–F. n.s., not significant (chi-square test). (B) qRT-PCR analysis of NORAD expression, relative to 18S rRNA, in HCT116 cells 48 hours after transfection with control (siNT) or NORAD-targeting siRNAs. (C) Aneuploidy in siRNA-transfected HCT116 cells 12 days after siRNA transfection, assayed as in Figure 2E–F. At least 200 nuclei were scored per condition. P value calculated by chi-square test. (D) Flow cytometry histograms showing DNA content, as measured by propidium iodide staining, in representative HCT116 subclones generated after transfection with the indicated siRNAs. (E) qRT-PCR analysis of NORAD expression in NORAD+/+ and NORAD−/− HCT116 cells with or without adenovirus-Cre infection. (F) Subclones generated from untreated or adenovirus-Cre infected NORAD−/− HCT116 cells were scored for aneuploidy as in Figure 2E–F. P value calculated by Student’s t-test.
Figure 4
Figure 4. NORAD interacts with PUMILIO proteins
(A) Western blot analysis of PUM1 and PUM2 in sense (S) and antisense (AS) NORAD fragment pull-downs. GAPDH served as a negative control. (B, C) Histogram of the total number of CLIP reads per PUM2 target transcript in PAR-CLIP data generated with FLAG-PUM2 (Hafner et al., 2010) (B) or endogenous PUM2 (C). Number of NORAD CLIP reads shown in red text in parentheses. (D) Location, sequence, and conservation of PREs in NORAD. ND, NORAD domain. (E) Location and read depth of endogenous PUM2 (upper) or FLAG-PUM2 (lower) PAR-CLIP clusters mapped to NORAD. Black bars, clusters overlapping PREs; gray bars, non-PRE clusters. See also Figures S4 and S5 and Tables S1 and S2.
Figure 5
Figure 5. Chromosomal instability in NORAD−/− cells is mediated through PUMILIO hyperactivity
(A–C) Cumulative distribution plots depicting behavior of PUM2 CLIP targets, as defined in Hafner et al. and this study, versus non-PUM2-targets in the indicated RNA-seq experiments. P value calculated by Kolmogorov–Smirnov test demonstrates significant repression of PUM2 targets in all tested datasets. (D) PUM1 and PUM2 overexpressing clones were assayed for aneuploidy using chromosome 7/20 FISH as in Figure 2E–F. At least 200 nuclei were scored per clone. n.s., not significant; *p<0.05; **p<0.005; ***p<0.0005, chi-square test. (E, F) Cells of the indicated genotypes were assayed for aneuploidy as in (D). *p<0.05, Student’s t-test, comparing NORAD−/−; PUM1+/+; PUM2+/+ to NORAD−/−; PUM1−/−; PUM2+/+ or NORAD−/−; PUM1+/+; PUM2−/−. (G, H) Quantification of the percentage of mitoses exhibiting the indicated mitotic errors in time-lapse imaging experiments after transfection with control siRNA (siNT) or two distinct sets of siRNAs targeting PUM1 and PUM2. Values represent the average of 3 independent experiments with 85–200 mitoses imaged per condition per experiment. Error bars represent standard deviations. *p<0.05; **p<0.01, Student’s t-test. See also Figures S6 and S7 and Table S3.
Figure 6
Figure 6. Genes required for the maintenance of chromosomal stability are repressed in NORAD−/− and PUM1/2-overexpressing cells
(A) Venn diagram showing overlap of genes that are significantly downregulated in NORAD−/− HCT116 cells (adjusted p value ≤ 0.05; see Table S3) and PUM2 PAR-CLIP targets identified in Hafner et al. and this study. (B) Gene ontology analysis of the 193 PUM2 PAR-CLIP targets that are downregulated in NORAD−/− cells, demonstrating enrichment of genes involved in mitosis, the cell cycle, DNA replication, and DNA repair. (C) qRT-PCR validation of PUM2 PAR-CLIP targets that have a known role in the maintenance of genomic stability (see Table S4) and were downregulated in NORAD−/− cells according to RNA-seq. Gene expression was normalized to 18S rRNA. All genes shown were significantly downregulated in NORAD−/− cells (p≤0.05, Student’s t-test). (D) qRT-PCR demonstrating expression of genes from panel C that are significantly downregulated in both PUM1- and PUM2-overexpressing HCT116 cells (p≤0.05, Student’s t-test) (see also Figure S7G). See also Figure S7 and Table S4.
Figure 7
Figure 7. A NORAD-PUMILIO axis that regulates genomic stability
Due to its abundance and multitude of PUMILIO binding sites, NORAD acts as a potent negative regulator of PUMLIO activity. In the absence of this lncRNA, PUMILIO is released to hyperactively repress a program of genes necessary to maintain chromosomal stability and a euploid state, including key factors required for mitosis, DNA replication, and DNA repair.

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