Frontiers I - HP-F3 - Part 3: Epigenetics and Epitranscriptomics

Paper Presentation

A-to-I RNA Editing Contributes to

Proteomic Diversity in Cancer
Xinxin Peng, Xiaoyan Xu, Yumeng Wang, David H. Hawke, Shuangxing Yu, Leng Han, Zhicheng Zhou, and
Gordon B. Mills, Peng et al., 2018, Cancer Cell 33, 1-12 May 14, 2018

Supervisors
Prof. Dr. Nina Papavasiliou
Dr. Riccardo Pecori

Toros Taşgın
Master Programme “Molecular Biosciences“, Major “Cancer Biology“ University of
Heidelberg and DKFZ
 What is RNA editing?

• DNA sequence independent alteration of RNA sequence.

• ADAR enzyme: Double stranded RNA-specific adenosine deaminase
enzyme (ADAR1, ADAR2, and ADAR3)1
- dsRBMs: double stranded RNA binding domain
- C terminal catalytic domain
• Binding specificity: structure and the context (not so clear) 2
• Various RNAs (tRNAs, rRNAs, mRNAs, miRNAs) are subjected to RNA
editing.3
• The most prevalent RNA editing is adenosine(A) to inosine (I)4
conversion.
• Almost all metazoans express ADAR enzymes.5

Adenosine Inosine
What may be the consequences of RNA editing?

• Direct change of amino acid sequence

• Change in miRNA binding site (seed sequence)
• Alteration of alternative splicing pattern of pre-mRNA
• May affect RNA stability

Overall: Higher complexity than mere DNA sequence

differences

Biological-role of RNA editing:

• Innate immunity
• Viral defense
• Metabolic regulation
RNA editing and Cancer

• AZIN1 editing in liver cancer

• CDC14B editing in glioblastoma
• RHOQ editing in colorectal cancer
• PODXL editing in gastric cancer
• GABRA3 editing in breast cancer
Objective
Problem: Due to the complexity of post-transcriptional regulation, contribution of
RNA editing to the proteomic diversity in cancer is unclear.

They aimed to address to what extent genetic information altered by missense A-to-I
editing is translated to protein sequences, thus contributing to proteomic diversity in
cancer.

RNA editing
Results 1: Relative Contributions of RNA editing and Somatic Mutations to Cancer Proteomic Diversity
Results 1: RNA editing events with confident variant peptide support

BRCA: 34 RNA editing events with variant peptide support

OV: 1 RNA editing events with variant peptide support
CRC:2 RNA editing events with variant peptide support
Results 1: Detailed analysis of BRCA dataset
Results 1: Simulation analysis for estimating false-positive rate
 1th Validation by RNA
Results 2: Independent Validation of Peptide Supported Editing Events editing finger print assay:
• Four of the RNA editing sites with variant peptide support
(COG3_I635V, COPA_I164V, FLNB_Q232R, IGFBP7_R78G) have
been previously reported in humans and mice (Pinto et al.,
2014).
• COPA_I164V is the only editing event can be seen in all cancer
datasets.
Hs578T cell line

Figure 2A: 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.
Results 2: Independent Validation of Peptide Supported Editing Events
2nd Validation by siRNA perturbation:

Figure 2B: ADAR-perturbed RNA-seq experiment. The editing

levels of the six RNA editing sites with sufficient coverage (310) in
different perturbed conditions, and GFP,
mock, and RISC_free served as negative controls.
Results 2: Detection of variant proteins in independent samples
immunoprecipitation
+
OVCAR-8 cancer cells
COPA and COG3 proteins Synthetic COPA and COG3 proteins

MS/MS

Figure 3: Annotated MS (A and B) and retention times (C

and D) for EVSLDLKK (COG3_I635V) (A and C) and
VWDVSGLR (COPA_I164V) (B and 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.
Results 2: Detection of variant proteins in independent samples
Results 3: Clinically Relevant Patterns of Editing
Main Question: whether they are “driver mutations” or “passenger” events.

Figure 4A: Normal-tumor comparison of RNA editing levels. Paired t test was used to assess statistical significance.
Results 3: Clinically Relevant Patterns of Editing
Broad Scale Investigation of Editing patterns in multiple cancer types: Pan-cancer analysis

Figure 4B: 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.
Results 3: Clinically Relevant Patterns of Editing

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. 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.
Results 3: Correlation between Drug Resistance and Editing levels
Cancer Cell Line Encyclopedia (CCLE): Pharmacological characterization

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 assessstatistical significance.
Results 4: Functional Effects of COPA Editing on Tumor Cells
Why COPA?
• Variant peptide prevalence across four MS datasets.
• Relatively high editing level
• Extensive clinical correlations across cancer types.
• Large predicted free-energy change on protein conformation.

Figure 5A: Impact of I164V on folding of

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.
Results 4: Functional Effects of COPA Editing on Tumor Cells

(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.
Results 4: Functional Effects of COPA Editing on Tumor Cells
COPA (WT/edited) transfected MDA-MB-231: parental RNA
depletion by small hairpin RNA (shRNA)

(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.
Results 4: Functional Effects of COPA Editing on Tumor 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.
Results 4: Functional Effects of COPA Editing on Tumor 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.
Conclusions

• The transcripts with higher editing levels have higher ion intensities: RNA editing can
introduce amino acid changes into proteins at least in breast cancer.

• Level of edited COPA and COG3 proteins are unignorably high in multiple cancer types.
Although it is not so easy to determine the required level of variant protein for gain of
function activity in cancer, this research showed that the RNA editing has a power to
cause significant effects on protein function and this effect cannot be ignored by low
level of editing.

• Editing in COPA protein at a specific site is sufficient to drive the growth and migration
of cancer cells, in a manner similar to driver somatic mutations.

• Overall, this study suggested that A-to-I RNA editing contributes to the protein
heterogeneity at least in some cancer types.
References:

1,2,3,4,5-Rosenthal, Joshua J C. “The emerging role of RNA editing in plasticity.” The Journal of experimental
biology vol. 218,Pt 12 (2015): 1812-21. doi:10.1242/jeb.119065

Kung, Che-Pei, et al. “The Role of RNA Editing in Cancer Development and Metabolic Disorders.” Frontiers,
Frontiers, 3 Dec. 2018, www.frontiersin.org/articles/10.3389/fendo.2018.00762/full.

Riedmann, Eva M, et al. “Specificity of ADAR-Mediated RNA Editing in Newly Identified Targets.” RNA (New York,
N.Y.), Cold Spring Harbor Laboratory Press, June 2008, www.ncbi.nlm.nih.gov/pmc/articles/PMC2390793/.

Bass, B., Nishikura, K., YA. Savva, L., P. Danecek, C., J. Rodriguez, J., S. Garrett, J., . . . K. Wang, M. (1970, January
01). Mammalian conserved ADAR targets comprise only a small fragment of the human editosome. Retrieved
December 05, 2020, from https://genomebiology.biomedcentral.com/articles/10.1186/gb-2014-15-1-r5