Altered editing in RNA editing adenosine deaminase ADAR2 gene transcripts of systemic lupus erythematosus T lymphocytes

Abstract

Adenosine Deaminases that act on RNA (ADARs) edit gene transcripts through site-specific conversion of adenosine to inosine by hydrolytic deamination at C6 of the adenosine. ADAR2 gene transcripts are substrates for the ADAR1 and ADAR2 enzymes and their expression is regulated by editing at the - 1 and - 2 sites. Our previous experiments demonstrated up-regulation of type I interferon (IFN) inducible 150 kDa ADAR1 in systemic lupus erythematosus (SLE) T cells. In this study we investigate the role of ADAR1 and ADAR2 in editing of ADAR2 gene transcripts of healthy controls and SLE patients. The ADAR2 gene transcripts were cloned into pCR2.1-TOPO vectors. A total of 150 clones from SLE and 150 clones from controls were sequenced. Sequence analysis demonstrated A to I editing at - 1, + 10, + 23 and + 24 in normal T cells. In SLE clones site-selective editing of the - 2 site was observed as a result of type I IFN-inducible 150 kDa ADAR1 expression. These results are confirmed by analysing ADAR2 transcripts of normal T cells activated with type I IFN-alpha. Editing of the + 23 and + 24 sites was decreased in SLE T cells compared to normal controls. In addition to A to G changes, U to C discrepancies were observed in normal and SLE T cells. In SLE cells, positions - 6 and + 30 were frequently edited from U to C compared to normal controls. Taken together, these results demonstrate altered and site-selective editing in ADAR2 transcripts of SLE patients. Based on these results, it is proposed that altered transcript editing contributes to the modulation of gene expression and immune functions in SLE patients.

Figure 1

ADAR2 transcript editing observed in the T cells of the SLE patients. ADAR2 gene transcripts of the normal and SLE patients were amplified and cloned into TA cloning vectors (pCR2.1) and sequenced using an automated DNA sequencer. Figure represents normal sequence and the normal counterparts of A to G mutated bases at the − 16, − 2, − 1, + 10, + 13, + 23 and + 24 positions in different clones are boxed (yellow) and indicated by dots. Edited thymidines are indicated in purple boxes.

Figure 2

A to G editing observed in ADAR2 gene transcript from the T cells of the SLE patients. Panels (a) (c) (f) and (i) represent normal sequence. Normal counterparts of A to G mutated bases at the − 16, − 2, − 1, + 10, + 13, + 23 and + 24 positions in different clones are indicated by dots. Panels (b) (d) (e) (g) (h) and (j) demonstrates edited bases at the − 16, − 2, − 1, + 10, + 13, + 23 and + 24 positions, respectively, in different clones. All edited bases are indicated by arrows (↑). All sequences represent sense strands.

Figure 3

The graph represents A to I editing frequencies observed at site − 2, − 1, + 10, + 23, + 24 and other base positions of ADAR2 gene transcripts from normal and SLE T lymphocytes. The P-values are indicated at appropriate places.

Figure 7

Predicted secondary structure of human ADAR2 pre-mRNA with editing events. The nucleotide sequence is shown with its secondary structure as predicted by mfold. The positions of observed A to G transitions (filled arrow heads) as well as observed U to C transitions (open arrow head) are indicated. The site co-ordinates are relative to the 3′-splice junction that is known to be created through A to I editing.,

Figure 4

ADAR2 transcript editing observed in the T cells of the SLE patients. Figure represents normal sequence and the normal counterparts of T to C mutated bases at the − 14, − 6, + 19 and + 30 positions in different clones are boxed (yellow) and indicated with dots. Edited adenosines are indicated in purple boxes.

Figure 5

T to C editing observed in ADAR2 gene transcript from the T cells of the SLE patients. Panels (a) (c) (e) and (g) represent normal sequence. Normal counterparts of T to C mutated bases at the − 14, − 6, + 19 and + 30 positions in different clones are indicated by dots. Panels (b) (d) (f) and (h) demonstrate edited bases at the − 14, − 6, + 19 and + 30 positions, respectively, in different clones. All edited bases are indicated by arrows (↑). All sequences represent sense strands.

Figure 6

The graph represents U to C editing frequencies observed at site − 6, + 19, + 30, + 32 and other base positions of ADAR2 gene transcripts from normal and SLE T lymphocytes. The P-values are indicated at appropriate places.