RNA editing by adenosine deamination has been shown to generate multiple isoforms of several neural receptors, often with profound effects on receptor function. However, little is known about the regulation of editing activity during development. We have developed a large-scale RNA sequencing protocol to determine adenosine-to-inosine (A-to-I) editing frequencies in the coding region of genes in the mammalian brain. Using the 454 Life Sciences (Roche) Amplicon Sequencing technology, we were able to determine even low levels of editing with high accuracy. The efficiency of editing for 28 different sites was analyzed during the development of the mouse brain from embryogenesis to adulthood. We show that, with few exceptions, the editing efficiency is low during embryogenesis, increasing gradually at different rates up to the adult mouse. The variation in editing gave receptors like HTR2C and GABA A (gamma-aminobutyric acid type A) a different set of protein isoforms during development from those in the adult animal. Furthermore, we show that this regulation of editing activity cannot be explained by an altered expression of the ADAR proteins but, rather, by the presence of a regulatory network that controls the editing activity during development.

Pancreatic islet transplantation has emerged as a therapy for type 1 diabetes and is today performed using both freshly isolated and cultured islets. Islet blood vessels are disrupted during islet isolation; therefore, proper revascularization of the transplanted islets is of great importance for islet graft function and survival. We have studied intraislet endothelial cells after islet isolation, during islet culture, and following islet transplantation. By isolating islets from the transgenic Tie2-GFP (green fluorescent protein) mouse, characterized by an endothelial cell-specific expression of GFP, living endothelial cells could be studied in intact islets utilizing two-photon laser-scanning microscopy (TPLSM). Intraislet endothelial cells were found to survive islet transplantation but to rapidly disappear during islet culture. By transplanting freshly isolated Tie2-GFP islets and applying a novel ex vivo model for simultaneous perfusion and TPLSM imaging of the graft-bearing kidneys, GFP fluorescent endothelial cells were found to extensively contribute to vessels within the islet graft vasculature. Real-time imaging of the flow through the islet graft vasculature confirmed that the donor-derived vessels were functionally integrated. Hence, intraislet endothelial cells have the capability of participating in revascularization of pancreatic islets subsequent to transplantation. Therefore, preservation of intraislet endothelial cell mass may improve long-term graft function.

Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the ␣3 subunit of the GABA A receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABA A receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in ␣3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of ␣3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for ␣3 in combination with either the ␤2 or the ␤3 subunit. Further, an amino acid substitution at the corresponding I/M site in the ␣1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the ␣3 protein abundance. Our results suggest that editing controls trafficking of ␣3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of ␣ subunits in the adult brain. Adenosine to inosine (A-to-I)2 RNA editing is a mechanism used in the mammalian nervous system to provide alterations in the protein sequence by co-transcriptional modification of single nucleotides. This modification is catalyzed by adenosine deaminases that act on RNA (ADAR1 and ADAR2) that can selectively modify adenosine to inosine residues within double stranded pre-mRNAs. Within mRNA transcripts, inosine is read as guanosine by the translation machinery. Therefore, this mechanism has the potential to change the amino acid sequence and thereby the function of the protein. Several gene products encoding proteins involved in neurotransmission have been shown to be A-to-I edited, including ligand-and voltage-gated ion channels as well as a G-proteincoupled receptor and thereby creating diverse isoforms of proteins essential for balanced neuronal kinetics (reviewed in Ref. 1).One of the most well studied substrates for editing in the brain is the transcript coding for the AMPA glutamate receptor (GluA). AMPA receptors consist of four subunits (GluA1-GluA4) in different combinations. Changing a codon for glutamine to arginine in GluA2 is essential to the organism and required for a normal brain development (2, 3).We have previously found that the mouse Gabra-3 transcript, coding for the ␣3 subunit of the GABA A receptor undergoes site-selective A-to-I editing causing an isoleucine to methionine (I/M) change in the third transmembrane region (TM3) (4). The chloride-permeable (GABA A ) receptors are the main mediators of fast inhibitory neurotransmission in the mammalian central nervous system (reviewed in Ref. 5). These heteropentameric ligand-gated chloride ion channels can be f...

Adenosine to inosine (A-to-I) RNA editing is important for a functional brain, and most known sites that are subject to selective RNA editing have been found to result in diversified protein isoforms that are involved in neurotransmission. In the absence of the active editing enzymes ADAR1 or ADAR2 (also known as ADAR and ADARB1, respectively), mice fail to survive until adulthood. Nuclear A-to-I editing of neuronal transcripts is regulated during brain development, with low levels of editing in the embryo and a dramatic increase after birth. Yet, little is known about the mechanisms that regulate editing during development. Here, we demonstrate lower levels of ADAR2 in the nucleus of immature neurons than in mature neurons. We show that importin-α4 (encoded by Kpna3), which increases during neuronal maturation, interacts with ADAR2 and contributes to the editing efficiency by bringing it into the nucleus. Moreover, we detect an increased number of interactions between ADAR2 and the nuclear isomerase Pin1 as neurons mature, which contribute to ADAR2 protein stability. Together, these findings explain how the nuclear editing of substrates that are important for neuronal function can increase as the brain develops.