The cell line A-to-I RNA editing catalogue

Abstract

Adenosine-to-inosine (A-to-I) RNA editing is a common post transcriptional modification. It has a critical role in protecting against false activation of innate immunity by endogenous double stranded RNAs and has been associated with various regulatory processes and diseases such as autoimmune and cardiovascular diseases as well as cancer. In addition, the endogenous A-to-I editing machinery has been recently harnessed for RNA engineering. The study of RNA editing in humans relies heavily on the usage of cell lines as an important and commonly-used research tool. In particular, manipulations of the editing enzymes and their targets are often developed using cell line platforms. However, RNA editing in cell lines behaves very differently than in normal and diseased tissues, and most cell lines exhibit low editing levels, requiring over-expression of the enzymes. Here, we explore the A-to-I RNA editing landscape across over 1000 human cell lines types and show that for almost every editing target of interest a suitable cell line that mimics normal tissue condition may be found. We provide CLAIRE, a searchable catalogue of RNA editing levels across cell lines available at http://srv00.recas.ba.infn.it/atlas/claire.html, to facilitate rational choice of appropriate cell lines for future work on A-to-I RNA editing.

Figure 1.

Variability of editing in Cell-lines. (A) We study 933 cell line samples from the CCLE dataset (63) and 675 samples from the GCLB dataset (64). Data for 460 cell lines is found in both datasets. (B) The cell lines analyzed originate from a variety of tissues, mostly from lung, and hematopoietic and lymphoid tissues. (C) Ratios of AEI values calculated for two biological replicated of each of 460 cell lines (same cell line type appearing in both datasets, cultured and sequenced in different labs) follow a log-normal distribution. The distribution is compared with that of 211,140 comparisons of non-duplicate cell lines (ratios of AEI values for different cell-lines from a different dataset, cultured and sequenced in different labs). Since the ratios follow a log-normal distribution, we plot the distribution for the logarithm of the ratio, log₂(editing of cell line from CCLE/editing of cell line from GCLB), which is approximately normal. The half width at half maximum (HWHM) of the distribution of log₂(ratio) for duplicates is 0.26, compared with a HWHM of 0.56 for pairs of non-duplicate cell lines. (D) Ratios of editing levels at specific sites calculated for two biological replicated of each of 460 cell lines (same cell line type appearing in both datasets, cultured and sequenced in different labs), and compared with the distribution of ratios obtained for specific sites in each of the 211,140 non-duplicate cell line pairs. For each sample, only well covered sites were considered (minimum coverage of 20 reads with at least 10 counts of ‘G’ in each of the duplicates; median number of sites analyzed per sample is 6), and for each pair ratios were calculated only for sites that are well covered in both pair mates. Altogether, we considered 3170 ratios between duplicates, and 788,484 for non-duplicate cell line pairs. The HWHM of the log₂(ratio) for duplicates is 0.59, compared to a HWHM of 0.82 for non-duplicate cell lines.

Figure 2.

Expression levels of the ADAR enzymes in cell lines and normal tissues. ADAR expression levels (TPM) were calculated for all cell lines in the two datasets. Normal levels were calculated for matching GTEx normal tissue samples (72) when available. (A) ADAR1 expression levels are overall higher in cell lines than in normal tissues (B) but ADAR2 levels mostly resemble those of normal tissues. Note the logarithmic scale.

Figure 3.

Editing in cell lines is lower compared to normal tissues (A) Global level of editing, measured by the AEI, for cell lines and matched normal tissues. (B) The distribution of the average editing level at the evolutionarily conserved coding sites (Supplementary Table S1) (71) is presented for cell lines and normal tissue. Note the logarithmic scale. (C) Distributions of editing levels at specific evolutionarily conserved coding sites in cell lines and normal tissues. Note that for each site, a number of cell lines exhibit editing levels comparable to those found in normal tissues. The distribution of editing levels was calculated only for samples with at least 10 reads supporting the site.

Figure 4.

Choosing the right cell line for the target of interest. (A) A heatmap of editing levels in cell lines that are edited most strongly at evolutionarily conserved coding sites (Supplementary Table S1) (71), as well as the commonly used HEK293 and HeLa cells, present a non-uniform pattern of editing. Yellow indicates low coverage (<10 reads). Even the cell lines showing strong editing in multiple coding sites are not suitable for all sites. For each of these, there are many conserved coding sites that are poorly expressed or poorly edited. (B) Using our dataset, we have selected three cell lines predicted to yield appreciable editing levels at the AZIN1 recoding site, grown and sequenced to verify the editing levels. Chromatograms of DNA and RNA sanger sequencing for the cell lines CAMA-1, NCI-H1573 and ZR-75-1 show RNA editing levels of 42%, 36% and 67% respectively. DNA sequencing of the same cells confirm no variability at the genomic level.