ADAR2 regulates RNA stability by modifying access of decay-promoting RNA-binding proteins.

Adenosine deaminases acting on RNA (ADARs) catalyze the editing of adenosine residues to inosine (A-to-I) within RNA sequences, mostly in the introns and UTRs (un-translated regions). The significance of editing within non-coding regions of RNA is poorly understood. Here, we demonstrate that association of ADAR2 with RNA stabilizes a subset of transcripts. ADAR2 interacts with and edits the 3΄UTR of nuclear-retained Cat2 transcribed nuclear RNA (Ctn RNA). In absence of ADAR2, the abundance and half-life of Ctn RNA are significantly reduced. Furthermore, ADAR2-mediated stabilization of Ctn RNA occurred in an editing-independent manner. Unedited Ctn RNA shows enhanced interaction with the RNA-binding proteins HuR and PARN [Poly(A) specific ribonuclease deadenylase]. HuR and PARN destabilize Ctn RNA in absence of ADAR2, indicating that ADAR2 stabilizes Ctn RNA by antagonizing its degradation by PARN and HuR. Transcriptomic analysis identified other RNAs that are regulated by a similar mechanism. In summary, we identify a regulatory mechanism whereby ADAR2 enhances target RNA stability by limiting the interaction of RNA-destabilizing proteins with their cognate substrates.

Observational Study of Clindamycin Phosphate and Tretinoin Gel for the Treatment of Acne

INTRODUCTION: Acne vulgaris can cause pain/discomfort and have a negative impact on quality of life (QOL). Clin-RA is an acne treatment consisting of clindamycin phosphate 1.2% and tretinoin 0.025%, which has been proven effective and well tolerated in clinical studies. This prospective, non-interventional study aimed to capture data on previous treatment, acne severity, and QOL in patients with acne treated with Clin-RA and assess the efficacy and tolerability of Clin-RA in routine clinical practice. METHODS: The study was performed at 18 centers in Sweden and enrolled patients aged ≥15 years with acne, who were prescribed Clin-RA for the first time. The observation period was ~12 weeks. The primary objective was to assess the patient's perception of their facial acne severity before and during Clin-RA treatment using a visual analog scale (VAS; 100 mm scale). Secondary objectives included QOL evaluation before and after treatment, using the Dermatology Life Quality Index (DLQI) questionnaire. RESULTS: 84 patients were enrolled and eligible for analyses (79.8% female; mean age 22.6 years). Patient-assessed VAS scores for acne severity decreased continuously during the study, indicating improvement: the median percentage reduction from baseline for VAS score was 17.6% at week 4 and 63.8% at week 12, with changes from baseline being statistically significant (P=0.0004 at week 4; P<0.0001 at weeks 8 and 12). Overall, QOL improved after Clin-RA treatment, reflected by a decrease in the mean (standard deviation) DLQI sum score from 8.8 (5.8) on day 0 to 4.9 (4.2) at week 12. Seventy percent of patients were satisfied/very satisfied with treatment. Clin-RA was well tolerated, with no serious adverse drug reactions reported. CONCLUSIONS: Treatment with Clin-RA resulted in continuous improvement of facial acne over the course of 12 weeks, along with improved QOL and a tolerable safety profile, supporting the use of Clin-RA in clinical practice. J Drugs Dermatol. 2019;18(4):328-334.

Adenosine-to-inosine RNA editing affects trafficking of the gamma-aminobutyric acid type A (GABA(A)) receptor.

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 α3 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 α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the ß2 or the ß3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 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 α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain.

Increased A-to-I RNA editing of the transcript for GABAA receptor subunit α3 during chick retinal development.

Adenosine-to-inosine (A-to-I) RNA editing is a cotranscriptional or posttranscriptional gene regulatory mechanism that increases the diversity of the proteome in the nervous system. Recently, the transcript for GABA type A receptor subunit α3 was found to be subjected to RNA editing. The aim of this study was to determine if editing of the chicken α3 subunit transcript occurs in the retina and if the editing is temporally regulated during development. We also raised the question if editing of the α3 transcript was temporally associated with the suggested developmental shift from excitation to inhibition in the GABA system. The editing frequency was studied by using Sanger and Pyrosequencing, and to monitor the temporal aspects, we studied the messenger RNA expression of the GABAA receptor subunits and chloride pumps, known to be involved in the switch. The results showed that the chick α3 subunit was subjected to RNA editing, and its expression was restricted to cells in the inner nuclear and ganglion cell layer in the retina. The extent of editing increased during development (after embryonic days 8-9) concomitantly with an increase of expression of the chloride pump KCC2. Expression of several GABAA receptor subunits known to mediate synaptic GABA actions was upregulated at this time. We conclude that editing of the chick GABAA subunit α3 transcript in chick retina gives rise to an amino acid change that may be of importance in the switch from excitatory to inhibitory receptors.

A method for finding sites of selective adenosine deamination.

Single sites of selective adenosine (A) to inosine (I) RNA editing with functional consequences on the proteome are rarely found in mammals. Here we describe a method that can be used to detect novel site-selective A-to-I editing in various tissues as well as species. The method utilizes immunoprecipitation of intrinsic RNA-protein complexes to extract substrates subjected to site-selective in vivo editing. We show that known single sites of A-to-I editing are enriched utilizing an antibody against the ADAR2 protein. We propose that this method is suitable for identification of novel substrates subjected to site-selective A-to-I editing.

A method to find tissue-specific novel sites of selective adenosine deamination.

Site-selective adenosine (A) to inosine (I) RNA editing by the ADAR enzymes has been found in a variety of metazoan from fly to human. Here we describe a method to detect novel site-selective A to I editing that can be used on various tissues as well as species. We have shown previously that there is a preference for ADAR2-binding to selectively edited sites over non-specific interactions with random sequences of double-stranded RNA. The method utilizes immunoprecipitation (IP) of intrinsic RNA-protein complexes to extract substrates subjected to site-selective editing in vivo, in combination with microarray analyses of the captured RNAs. We show that known single sites of A to I editing can be detected after IP using an antibody against the ADAR2 protein. The RNA substrates were verified by RT-PCR, RNase protection and microarray. Using this method it is possible to uniquely identify novel single sites of selective A to I editing.

Editing modifies the GABA(A) receptor subunit alpha3.

Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain development.