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The long noncoding RNA SPRIGHTLY acts as an intranuclear organizing hub for pre-mRNA molecules

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Science Advances  03 May 2017:
Vol. 3, no. 5, e1602505
DOI: 10.1126/sciadv.1602505
  • Fig. 1 SPRIGHTLY RNA secondary structure and its binding partners.

    (A) The secondary structure of SPRIGHTLY was determined by RNA structure with the constraints of SHAPE-seq reactivity data. Nucleotides are colored by their normalized reactivities ρ. The probe sequences are labeled P1 to P12. In the text, the 12 primers are termed in three sets: Set D1 is represented by probes P1 to P4, D2 is represented by probes P5 to P8, and D3 is represented by probes P9 to P12. The core pseudoknotted domain overlaps D1, D2, and D3. (B) Histogram of normalized SHAPE-seq reactivities as a function of nucleotide position of SPRIGHTLY. (C) The distribution of RNA sequences within gene bodies corresponding to dChIRP MACS peaks pulled down by the three sets of SPRIGHTLY probes, D1, D2, and D3. dChIRP MACS peaks found in the exonic region including promoter-TSS, exon, 3′UTR, and TTS were plotted. The aggregate plots of RNA dChIRP sequences peaks show the enriched regions distributed across 5000 base pairs (bp) upstream of gene bodies and 5000 bp downstream of the genes. The shades represent the SEM. Green peaks represent RNA pulled down by probes of D1, orange peaks represent RNA pulled down by probes of D2, and purple peaks represent RNA pulled down by probes of D3. (D) SPRIGHTLY binding partner RNAs determined by common MACS peaks. The MACS peaks were mapped to their corresponding genomic loci, and the number of genes was counted. If MACS peaks from individual dChIRP sequencing overlapped or mapped to same gene, then those genes were regarded as SPRIGHTLY binding partners. Six genes have MACS peaks common to all three regions, suggesting that those six genes are most likely to interact with SPRIGHTLY. (E) SPRIGHTLY dChIRP specifically enriches the intronic regions of six genes. SPRIGHTLY dChIRP samples were analyzed by qPCR using primers for representative MACS peak of each gene or using primers for exon-exon junctions. Each intronic region corresponding to MACS peak was enriched >5- to 800-fold over the abundant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. An average of three technical replicates ± SD is shown. (F) The integrated network of six RNA molecules that bind to SPRIGHTLY was constructed by querying integrated gene interaction network data. Green interaction edges represent high-confidence genetic interaction data from Lin et al. (51), and black dashed edges represent consensus miRNA target sequences (52).

  • Fig. 2 SPRIGHTLY hemizygous knockout suppresses anchorage-independent growth in vitro and the tumor growth in vivo.

    (A to G) Colocalization between SPRIGHTLY and intronic regions of SMYD3 and SND1: (A) the differential interference contrast (DIC) image of parental and knockout cells, (B) raw image showing SPRIGHTLY RNA probed using smFISH probes labeled with Texas Red, (C) raw images showing SND1 RNA (top two panels), and SMYD3 RNA (bottom two panels) probed using probes labeled with tetramethylrhodamine (TMR). (D) Merge of RNA spots for SPRIGHTLY are pseudocolored as green, and SND1 and SMYD3 are pseudocolored as red. (E) The spots were identified using custom-written algorithms in MATLAB. WT, wild type; KD, knockdown. The identified spots are overlaid over 4′,6-diamidino-2-phenylindole (DAPI) staining as red (SND1/SMYD3), green (SPRIGHTLY), and yellow (colocalized) circles. (F) Quantification of about 50 cells to determine the copy number. (G) Extent of colocalization of SND1 and SMYD3 with SPRIGHTLY lncRNA. The error bars represent 95% confidence interval. Scale bars, 5 μm. (H) SPRIGHTLY hemizygous knockout decreased the expression levels of both mRNA and pre-mRNA of RNA binding partners. The expression levels were measured in both A375 and SC2-17 cells by qPCR. The expression levels of both mRNA and pre-mRNA of TBP or HPRT1 were not affected by SPRIGHTLY in SC2-17 cells. Delta threshold cycle (ΔCt) was calculated by subtracting the average Ct value of ACTB, TBP, and HPRT1 from the Ct value of target gene. The data were obtained from three independent experiments and are expressed as means ± SD. ns, not significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, Student’s t test. (I) A375 and SC2-17 cells were seeded and cultured for 7 days. Shown is a representative image of impaired colony-forming ability in SC2-17 cells compared to control cells (A375). The MTT assay was used to determine cell number. Results are expressed as means ± SD from six independent experiments. ****P ≤ 0.0001, Student’s t test. (J to L) Five-week-old female SCID mice were subcutaneously injected with either A375 or SC2-17 cells (n = 4 mice per group). (J) Graph depicts tumor volume (volume = (width)2 × length/2) observed for the A375 and SC2-17 groups at different time points until the tumors were excised. (K) Approximately 1 month after implantation, mice were euthanized, and tumors were excised and weighed. Graph depicts tumor weight (in milligrams) of A375 and SC2-17 tumor xenografts. (L) Images of the excised tumors on a scale bar are shown. Student’s t test was performed to detect between-group differences. The data in (J) and (K) are representative of at least three independent experiments with consistent results. **P ≤ 0.01, Student’s t test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1602505/DC1

    fig. S1. The pulldown efficiency of SPRIGHTLY and validation of copurification of RNA binding partners by qPCR.

    fig. S2. Many MACS peaks are composed of repetitive sequences.

    fig. S3. The fold enrichment of dChIRP MACS peaks.

    fig. S4. Gene interaction networks of SPRIGHTLY-interacting RNA molecules.

    fig. S5. SPRIGHTLY knockout using a CRISPR system.

    fig. S6. SPRIGHTLY overexpression recovered the loss of anchorage-independent growth of SC2-17 cells.

    table S1. Probe and qPCR primer sequences.

    table S2. List of common MACS peaks.

    table S3. A list of six genes and corresponding MACS peaks.

    table S4. g:Profiler of all 115 genes.

    table S5. GO term analysis of genes pulled down by D2 and D3 regions.

    table S6. SC2-17 RNA-seq results for six targets.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. The pulldown efficiency of SPRIGHTLY and validation of copurification of RNA binding partners by qPCR.
    • fig. S2. Many MACS peaks are composed of repetitive sequences.
    • fig. S3. The fold enrichment of dChIRP MACS peaks.
    • fig. S4. Gene interaction networks of SPRIGHTLY-interacting RNA molecules.
    • fig. S5. SPRIGHTLY knockout using a CRISPR system.
    • fig. S6. SPRIGHTLY overexpression recovered the loss of anchorage-independent growth of SC2-17 cells.

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    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). Probe and qPCR primer sequences.
    • table S2 (Microsoft Excel format). List of common MACS peaks.
    • table S3 (Microsoft Excel format). A list of six genes and corresponding MACS peaks.
    • table S4 (Microsoft Excel format). gProfiler of all 115 genes.
    • table S5 (Microsoft Excel format). GO term analysis of genes pulled down by D2 and D3 regions.
    • table S6 (Microsoft Excel format). SC2-17 RNA-seq results for six targets.

    Files in this Data Supplement:

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