Identification of a selective nuclear import signal in adenosine deaminases acting on RNA

Abstract

The adenosine deaminases acting on RNA (ADARs) comprise a family of RNA editing enzymes that selectively modify single codons within RNA primary transcripts with often profound impact on protein function. Little is known about the mechanisms that regulate nuclear RNA editing activity. Editing levels show cell-type specific and developmental modulation that does not strictly coincide with observed expression levels of ADARs. Here, we provide evidence for a molecular mechanism that might control nuclear import of specific ADARs and, in turn, nuclear RNA editing. We identify an in vivo ADAR3 interaction partner, importin alpha 1 (KPNA2) that specifically recognizes an arginine-rich ADAR3 sequence motif and show that it acts as a functional nuclear localization sequence. Furthermore, whereas KPNA2, but not KPNA1 or KNPA3, recognizes the ADAR3 NLS, we observe the converse binding specificity with ADAR2. Interestingly, alternative splicing of ADAR2 pre-mRNA introduces an ADAR3-like NLS that alters the interaction profile with the importins. Thus, in vivo RNA editing might be regulated, in part, through controlled subcellular localization of ADARs, which in turn is governed by the coordinated local expression of importin alpha proteins and ADAR protein variants.

Figure 1.

(A) The ADAR3 construct used as bait in the yeast two-hybrid interaction hunt (amino acids 1–133) is depicted in relation to full-length ADAR3 (amino acids 1–746) and the domain structures of ADAR1 and ADAR2. ADAR1 refers to the human main splice form ADAR1a and ADAR2 corresponds to the human main splice form ADAR2a (57). dsRBD, double-stranded RNA-binding domain; deaminase, catalytic adenosine deaminase domain; R-domain, arginine-rich sequence motif. (B) The library clone Y-7, isolated from the two-hybrid screen as ADAR3-binding protein is outlined in relation to the full-length human importin α1 (KPNA2) sequence (Genbank Accession NM_002266). (C) The point mutant ADAR3Nmut, where two arginines within the R-domain have been replaced by serines, is shown in comparison to the ADAR3N construct. The binding activity to KPNA2 importin alpha according to the yeast interaction assay is indicated.

Figure 2.

In vitro interaction of ADAR3N with KPNA2 but not KPNA1. (A) Expression of in vitro transcribed importin alpha proteins KPNA1 and KPNA2, as well as the ADAR3 protein A3N. Importin proteins carry the HA epitope tag, the ADAR protein a myc epitope tag. (B) Co-immunoprecipitation of importin/ADAR mixtures using a monoclonal anti-HA-tag antibody. In addition to KPNA2 protein, ADAR3N is co-immunoprecipitated (lane 1), but not when omitting the HA-tag specific antibody (lane 3). KPNA1 is not able to co-precipitate ADAR3N (lane 2).

Figure 3.

The R-domain of ADAR3 confers nuclear localization on GFP protein. HEK293 cells cultured on glass coverslips and transfected with expression plasmids EGFP (A), EGFP-NLS (B) or EGFP-NLSmut (C) were fixed after 24 h and examined by fluorescent microscopy. The native EGFP protein was preferentially observed in the cytoplasm (A), whereas the EGFP-NLS fusion protein with the ADAR3 R-domain sequence accumulated in the nucleus with strongest staining in the nucleoli (B). The mutated NLS sequence prevents nuclear accumulation of EGFP with some staining still seen in the nucleoli (C).

Figure 4.

Interaction profiles of ADARs with importin alpha proteins. Depicted are ADAR3 (A) as well as ADAR 1, and 2 (B) expression constructs used in yeast two-hybrid interaction assays. Asterisks indicate the location of putative nuclear-localization signals; the dsRBD of ADAR1 that mediates transportin 1 shuttling (22) is indicated by an arrow. The binding activities of the listed constructs to KPNA1 to 3 importin alpha proteins according to a β-galactosidase assay on yeast colonies expressing pairs of proteins are indicated. In most cases, the results were either strongly positive (developing a blue color in less than 20 min) or clearly negative (no color after 24 h at 30°C). Occasionally, a weak signal developed after 12–24 h which is designated as ± in the table. dsRBDs: double-stranded RNA-binding domains; deaminase, catalytic adenosine deaminase domain; R-domain, arginine-rich sequence motif.