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

Abstract

Molecular mechanisms by which long noncoding RNA (lncRNA) molecules may influence cancerous condition are poorly understood. The aberrant expression of SPRIGHTLY lncRNA, encoded within the drosophila gene homolog Sprouty-4 intron, is correlated with a variety of cancers, including human melanomas. We demonstrate by SHAPE-seq and dChIRP that SPRIGHTLY RNA secondary structure has a core pseudoknotted domain. This lncRNA interacts with the intronic regions of six pre-mRNAs: SOX5, SMYD3, SND1, MEOX2, DCTN6, and RASAL2, all of which have cancer-related functions. Hemizygous knockout of SPRIGHTLY by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 in melanoma cells significantly decreases SPRIGHTLY lncRNA levels, simultaneously decreases the levels of its interacting pre-mRNA molecules, and decreases anchorage-independent growth rate of cells and the rate of in vivo tumor growth in mouse xenografts. These results provide the first demonstration of an lncRNA's three-dimensional coordinating role in facilitating cancer-related gene expression in human melanomas.

Keywords: gene regulation; long noncoding RNA; melanoma; pre-mRNA.

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 (ΔC_t) was calculated by subtracting the average C_t value of ACTB, TBP, and HPRT1 from the C_t 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.