A-to-I RNA Editing Contributes to Proteomic Diversity in Cancer

Abstract

Adenosine (A) to inosine (I) RNA editing introduces many nucleotide changes in cancer transcriptomes. However, due to the complexity of post-transcriptional regulation, the contribution of RNA editing to proteomic diversity in human cancers remains unclear. Here, we performed an integrated analysis of TCGA genomic data and CPTAC proteomic data. Despite limited site diversity, we demonstrate that A-to-I RNA editing contributes to proteomic diversity in breast cancer through changes in amino acid sequences. We validate the presence of editing events at both RNA and protein levels. The edited COPA protein increases proliferation, migration, and invasion of cancer cells in vitro. Our study suggests an important contribution of A-to-I RNA editing to protein diversity in cancer and highlights its translational potential.

Keywords: ADAR enzyme; CPTAC; RNA editing; RNA-seq; TCGA; amino acid changes; biomarker; cancer drivers; mass spectrometry data; somatic mutations.

Figure 1. Relative contributions of RNA editing and somatic mutations to proteomic diversity in cancer

(A) The flow chart of variant peptide identification using MS data. For each MS set, all missense RNA editing sites and somatic mutations from the corresponding samples were pooled to construct a sample-set-specific database. After the quality control steps, only variants with a uniquely mapped variant peptide were considered as candidates. We manually reviewed each candidate and generated the final lists. (B) Four representative peptide spectrum matches of variant peptides due to RNA editing (Upper: COG3_I635V, COPA_I164V; Lower: IFI30_T233A, IGFBP7_R78G). (C) Information flow during the analysis of CPTAC breast cancer samples. We dissected the genetic information flow from DNA to RNA to protein into seven steps: the number of variants at the DNA level, somatic mutations (variants); number of variants with evidence of expression, both RNA editing and mutations (expressed variants); number of MS detectable variants in theory; number of variants covered by resolved peptides (wild-type or variant); number of variants covered by variant peptides; number of variants covered by uniquely mapped variant peptides; and number of variants passing the manual check. (D) Information flow of simulation analysis with the same editing sites but different nucleotide changes (A-to-C and A-to-T). See also Tables S1–3, Figure S1.

Figure 2. Experimental validation of RNA editing events with peptide support at the RNA level

(A) The relative expression (top) and the editing level changes (bottom) after transfection of wild-type ADAR enzymes (ADAR1/2 WT) and catalytically-inactive ADAR enzymes (ADAR1/2 MUT), GFP serves as negative control. Error bars denote ± SD. (B) ADAR-perturbed RNA-seq experiment. The editing levels of the six RNA editing sites with sufficient coverage (×10) in different perturbed conditions, and GFP, mock and RISC_free served as negative controls. See also Figure S2, Figure S3.

Figure 3. Validation of variant peptides caused by RNA editing sites through LC-MS/MS with heavy isotope labeling synthetic peptides

(A–D) Annotated MS (A, B) and retention times (C, D) for EVSLDLKK (COG3_I635V) (A, C) and VWDVSGLR (COPA_I164V) (B, D). The results of unlabeled endogenous variant peptide are shown at the top whereas the results of the spiked, labeled synthetic peptide are shown at the bottom of each panel. The heavy isotope labeled amino acids are shown in red.

Figure 4. Clinical relevance of A-to-I RNA editing events with peptide support

(A) Normal-tumor comparison of RNA editing levels. Paired t test was used to assess statistical significance. (B) The upper quartile values of RNA editing levels of four RNA editing sites. For each editing site, only the top five cancer types with the highest editing levels are shown. (C) Clinically relevant patterns of RNA editing sites with peptide evidence in different cancer types. For each cancer type, grey boxes indicate not significant, red boxes indicate significantly differential editing levels among tumor subtypes (Kruskal-Wallis or Wilcoxon rank sum test, FDR < 0.05, editing level difference > 3%), orange boxes indicate significantly differential editing levels among stage (Kruskal-Wallis or Wilcoxon rank sum test, FDR < 0.05, editing level difference > 3%), and blue boxes indicate significant associations of editing level with progression-free survival times (log-rank or Cox model test, FDR < 0.05, editing level difference >3%). (D) Differential editing level of COPA_I164V (left) and IGFBP7_R78G (right) in stomach adenocarcinoma (STAD) subtypes (left) and lung Adenocarcinoma (LUAD) subtypes (right). Kruskal-Wallis test was used to assess statistical significance. (E) Correlations of editing level in COPA_I164V (left) and IGFBP7_R78G (right) with patient progression-free survival time in kidney renal clear cell carcinoma (KIRC). Log-rank test was used to assess statistical significance. (F) The association of editing level at COG3_I635V with the drug sensitivity of fluorouracil and austocystin D. (G) The association of editing level at COPA_I164V with the sensitivity to austocystin D and lapatinib. (F) and (G) Wilcoxon rank sum test was used to assess statistical significance. In (A), (D), (F) and (G), the horizontal line in the box is the median, the bottom and top of the box are the first and third quartiles, and the whiskers extend to 1.5 IQR of the lower quartile and the upper quartile, respectively. In (D)–(G), numbers in parentheses indicate the sample numbers included in each comparison group. See also Table S4, Table S5, Figure S4.

Figure 5. Functional effects of RNA editing at COPA_I164V

(A) Impact of I164V on folding of COPA protein. (B) The annotated LC-MS/MS of the edited COPA peptide in the MDA-MB-231 cell line that overexpressed the edited COPA protein. (C–F) Effects of COPA editing on cell viability (C), wound healing (D), migration (E), and invasion (F) in CRISPR/cas9 COPA knockout MDA-MB-231 cells. (G–I) Effects of the edited COPA on cell viability (G) migration (H), and invasion (I) in three wild-type cell lines MCF10A, MDA-MB-231 and SLR25. All scale bars = 100 µm. All error bars denote ± SD. t test, *, p < 0.05; **, p < 0.01; ***, p < 0.001. All functional assays were performed simultaneously. See also Figure S5, Figure S6, Table S6.