Cellular Translocation Of ADAR1 And Identification Of It's New Isoforms

BACKGROUND AND AIMS: ADAR1 is a RNA-dependent adenosine deaminase thatspecifically modifies pre-mRNA by conversion of adenosine to inosine, termed A-to-l RNA editase. ADAR1 is known to edit pre-mRNAs of ion channel proteins as well potassium channel proteins in the central nervous system. Studies have demonstrated that editing by ADAR1 regulates the functions of glutamate and serotonin receptors and plays crucial role in excitatory neuron transmission, neuron degeneration and behavior. The function of ADAR1 in other systems such as immune system is still under investigation and the mechanism for ADAR1 expression, regulation and cellular translocation are also obscure. Recently, studies from our laboratory have demonstrated that various ADAR1 isoforms are induced and regulated in immune organs such as spleen and thymus during acute inflammation. These isoforms are classified into the long and short variants due to inflammation-induced alternative splicing. Interestingly, the regulated ADAR1 isoforms contain different nuclear importing/exporting signals that may lead them to function in distinct cellular compartments and edit different pre-mRNAs. Therefore, we proposed the following studies to identify the signals for nuclear importation/expeortation and elucidate the mechanisms for ADAR1 translocation in cytoplasm, nucleus and nucleolus. METHODS Following strategies were applied in the proposed studies. 1) Identification of the cellular localization. To this end, we constructed a series of chimeric ADAR1-EGFP genes by subcloning the ADAR1 fragments and EGFP cDNA in frame. The chimeras were transfected into the target cells with the help of liposome. By examining the green fluorescence, the localization of the transiently expressed protein was identified. 2) Confirmation of the nucleolar sub-structures. This was performed by double staining of the transiently expression proteins and the nucleolar specific proteins. 3) Directly detection of the cellular expression of ADAR1 by Western blotting. Over and above, Western blotting is a very powerful means to detect thedifference of ADAR1 expression. 4) Location of ADAR1 isoforms in different cell types and mouse organs. We further investigated the expression of ADAR1 in organs and tissues from normal or endotoxin-stimulated mice. 5) Finally, the expression of various ADAR1 isoforms in human and mouse tumor cell lines were analyzed by immunoblotting. Regulation of ADAR1 isoforms in naive splenocytes and thymocytes after stimulation with mitogens (such as PHA, ConA, LPS) and cytokines (such as TNF-a IL-2) were also studied by Western blotting. In addition, RNA editing activity was examined by an assay that is based on thin layer chromatography (TLC). RESULTS AND CONCLUSIONS: 1: It was confirmed that ADARIi (long form of ADAR1) is not a protein that is repeatively shuttling between cytoplasm and nucleus. Instead, it is translated in cytoplasm, translocated into nucleus and exported back to cytoplasm. It supports that either ADARI's nuclear exporting signal (NESs) functions dominantly over nuclear importing signal, and/or ADAR1 is modified in the nucleus which blocks the modified ADAR1 to be imported back to the nucleus. The latter indicates that ADARIi and ADARIc could play different roles in different subcelluar structures. 2: We identified the minimum motif of nucleolar binding domain in ADAR1, which sequence is APNKIRRIGEL 3. We systematically analyzed the expression of different ADAR1 variants at the protein level in mouse organs. It was demonstrated that ADAR1 is highly expressed not only in brain but also in testis, thymus and spleen, could be produced and they were detected by five different anti-ADAR1 antibodies. 5. Our data indicated that ADAR1 might exist in phosphorylated forms. The un-phosphrylated ADARIc might either associate to malignant proliferation of tumor cells, or development of normal immature cells. 6. Our data demonstrated that ADAR1 is up regulated during the development and/or differentiation of splenic and thymic lymphocytes. In su...