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. 2016 Jan;48(1):53-8.
doi: 10.1038/ng.3452. Epub 2015 Nov 23.

The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer

Zurab Siprashvili  1 , Dan E Webster  1 , Danielle Johnston  1 , Rajani M Shenoy  1 , Alexander J Ungewickell  1 , Aparna Bhaduri  1 , Ross Flockhart  1 , Brian J Zarnegar  1 , Yonglu Che  1 , Francesca Meschi  2 , Joseph D Puglisi  2 , Paul A Khavari  1   3
Affiliations

The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer

Zurab Siprashvili et al. Nat Genet. 2016 Jan.

Abstract

Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification. To explore their role in cancer, we compared 5,473 tumor-normal genome pairs to identify snoRNAs with frequent copy number loss. The SNORD50A-SNORD50B snoRNA locus was deleted in 10-40% of 12 common cancers, where its loss was associated with reduced survival. A human protein microarray screen identified direct SNORD50A and SNORD50B RNA binding to K-Ras. Loss of SNORD50A and SNORD50B increased the amount of GTP-bound, active K-Ras and hyperactivated Ras-ERK1/ERK2 signaling. Loss of these snoRNAs also increased binding by farnesyltransferase to K-Ras and increased K-Ras prenylation, suggesting that KRAS mutation might synergize with SNORD50A and SNORD50B loss in cancer. In agreement with this hypothesis, CRISPR-mediated deletion of SNORD50A and SNORD50B in KRAS-mutant tumor cells enhanced tumorigenesis, and SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types. SNORD50A and SNORD50B snoRNAs thus directly bind and inhibit K-Ras and are recurrently deleted in human cancer.

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Figures

Figure 1
Figure 1
Frequent deletion of SNORD50A/B in human cancers, SNORD50A/B expression and patient survival. (a) Schematic of the approach to identify altered snoRNA-encoding genomic loci in cancer using TCGA data. All somatic deletion segments, including those spanning contiguous oncogenes or tumor-suppressor genes, are shown in gray, whereas filtered deletions that do not also span known cancer-associated genes are shown in blue. (b) Somatic SNORD50A/B deletion in individual cancer types. (c) SNORD50A/B locus deletion frequency in 21 TCGA tumor types unfiltered with CNAs involving cancer-associating genes (gray) or filtered (blue). (d) Overall patient survival in the TCGA invasive breast cancer cohort as a function of filtered SNORD50A/B deletion. Analysis of statistical significance was performed using the log-rank (Mantel-Cox) test. (e–h) Expression of the SNORD50A/B host gene, SNHG5, in TCGA breast cancer data (62 tumor-matched normal breast tissue pairs) (e), published lymphoma data (10 tumor and 28 normal T cell samples) (f), published melanoma data (2 tumor and 2 normal melanocyte samples) (g) and published colon cancer data (45 tumor and 39 normal colon tissue samples) (h). Statistical significance was determined using an unpaired two-sided t test, except in the case of breast adenocarcinoma, where the samples are patient matched and a paired t test was used. In all cases, data represent means ± s.d. (i) Overall survival as a function of SNHG5 expression from a breast cancer cohort annotated in GEO series GSE6532.
Figure 2
Figure 2
SNORD50A and SNORD50B directly bind K-Ras. (a) Schematic of hybridization to microarrays containing 9,125 recombinant human proteins. (b) Protein microarray signal at the K-Ras4A protein isoform for SNORD50A and SNORD50B. Enlarged duplicate spotted K-Ras signals plus adjacent protein controls are shown. (c) Top gene ontology (GO) terms for SNORD50Aand SNORD50B-binding proteins. The significance of gene term enrichment calculated with a modified Fisher’s exact test using control spot subtracted 9,125 proteins on ProtoArray as a background universe. (d) CLIP assay demonstrates K-Ras association with SNORD50A and SNORD50B in vivo. The dotted line represents a value of 1.0, of no enrichment over input (n = 3, mean of three biological replicates ± s.d.). (e) Electromobility gel shift assay of SNORD50A and SNORD50B versus scrambled RNA control with increasing amounts of purified recombinant human K-Ras protein. (f) Quantification of the electromobility gel shift assay. Data are represented as the means ± s.d. of four independent experiments. (g) Intracellular RP-PLA in normal human epidermal keratinocytes identifying extranuclear association of SNORD50A and SNORD50B with endogenous K-Ras protein (orange); K-Ras binding to the SNORD33 snoRNA, scrambled RNA and no RNA represent controls. Scale bars, 100 µm. (h) Quantification of PLA (means ± s.d., n = 3).
Figure 3
Figure 3
Impact of SNORD50A and SNORD50B loss on K-Ras. (a) RNA blot demonstrating depletion of cellular SNORD50A and SNORD50B RNA by delivery of separate ASO specific to SNORD50A and SNORD50B (SNORD50A/B). (b) Quantification of RNA blot analysis. (c) Quantitative PCR demonstrating depletion of cellular SNORD50A and SNORD50B (means ± s.d., n = 4). (d) SNORD50A and SNORD50B depletion in normal human epidermal keratinocytes increases levels of active phosphorylated ERK1/2 MAPKs. (e) SNORD50A and SNORD50B depletion in multiple cancer cell lines with intact Ras-ERK1/2 MAPK signaling components increases levels of active ERK1/2. (f) Quantification of active, phosphorylated ERK1/2 as a function of SNORD50A and SNORD50B depletion in primary epidermal keratinocytes. (g) Quantification of active phosphorylated ERK1/2 as a function of SNORD50A and SNORD50B depletion in cancer cell lines. Data in f and g are represented as means ± s.d. of three independent experiments. (h) PCR analysis of genomic DNA from CRISPR-edited CHL-1 melanoma cells (CRISPR-50A/B) demonstrating deletion of the adjacent SNORD50A and SNORD50B genes in duplicate independent CHL-1 clones. (i) CRISPR-mediated SNORD50A/B deletion increases levels of active ERK1/2. (j) Quantification of active, phosphorylated ERK1/2 as a function of SNORD50A and SNORD50B depletion. (k) Tumor growth kinetics in vivo assessed by bioluminescent imaging of wild-type CHL-1 parental melanoma cells and two independent SNORD50A/B-deleted clones (n = 3 mice/group, means ± s.d.). Explanted tumor xenografts at day 28 are shown at right.
Figure 4
Figure 4
Impact of SNORD50A and SNORD50B on K-Ras function. (a) Depletion of SNORD50A and SNORD50B increases cellular levels of active, GTP-bound K-Ras protein. (b) Quantification of GTP-bound K-Ras protein. (c) Enforced expression of SNORD50A and SNORD50B suppresses the increased levels of phosphorylated ERK1/2 induced by mutant oncogenic K-Ras Gly12Val. (d) Quantification of phosphorylated ERK1/2 as a function of combined SNORD50A and SNORD50B (SNORD50A/B) overexpression. (e) RNA blot demonstrating enforced expression of exogenous SNORD50A and SNORD50B RNA. (f) Quantification of RNA blot analysis. (g) The effects of SNORD50A and SNORD50B depletion on K-Ras protein farnesylation and association with FTase. Orange shows PLA signal for farnesylated K-Ras (left column), FTase-K-Ras proximity (middle column) and ubiquitinated K-Ras (right column). Scale bars, 100 µm. (h) PLA quantification. Data are representative of two (b,d) and at least three (h) independent experiments and are shown as means ± s.d.
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