Adenosine-to-inosine (A-to-I) editing modifies RNA transcripts from their genomic blueprint. A prerequisite for this process is a double-stranded RNA (dsRNA) structure. Such dsRNAs are formed as part of the microRNA (miRNA) maturation process, and it is therefore expected that miRNAs are affected by A-to-I editing. Editing of miRNAs has the potential to add another layer of complexity to gene regulation pathways, especially if editing occurs within the miRNA–mRNA recognition site. Thus, it is of interest to study the extent of this phenomenon. Current reports in the literature disagree on its extent; while some reports claim that it may be widespread, others deem the reported events as rare. Utilizing a next-generation sequencing (NGS) approach supplemented by an extensive bioinformatic analysis, we were able to systematically identify A-to-I editing events in mature miRNAs derived from human brain tissues. Our algorithm successfully identified many of the known editing sites in mature miRNAs and revealed 17 novel human sites, 12 of which are in the recognition sites of the miRNAs. We confirmed most of the editing events using in vitro ADAR overexpression assays. The editing efficiency of most sites identified is very low. Similar results are obtained for publicly available data sets of mouse brain-regions tissues. Thus, we find that A-to-I editing does alter several miRNAs, but it is not widespread.

The availability of complete genome sequences has made it clear that gene number is not the sole determinant of the complexity of the proteome. Additional complexity that is not readily detected by genome analysis is present in the number and types of RNA transcript that can be derived from each locus. Although alternative splicing is a well-recognized method of generating diversity, the more subtle mechanism of RNA editing is less familiar.

Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1.

Grade IV astrocytoma or glioblastoma multiforme (GBM) is one of the most aggressive and lethal tumors affecting humans. ADAR2-mediated A-to-I RNA editing, an essential post-transcriptional modification event in brain, is impaired in GBMs and astrocytoma cell lines. However, the role of ADAR2 editing in astrocytomas remains to be defined. Here, we show that ADAR2 editing rescue in astrocytomas prevents tumor growth in vivo and modulates an important cell cycle pathway involving the Skp2/p21/p27 proteins, often altered in glioblastoma. We demonstrate that ADAR2 deaminase activity is essential to inhibit tumor growth. Indeed, we identify the phosphatase CDC14B, which acts upstream of the Skp2/p21/p27 pathway, as a novel and critical ADAR2 target gene involved in glioblastoma growth. Specifically, ADAR2-mediated editing on CDC14B pre-mRNA increases its expression with a consequent reduction of the Skp2 target protein, as shown both in vitro and in vivo. We found that, compared to normal brain, both CDC14B editing and expression are progressively impaired in astrocytomas from grade I to IV, being very low in GBMs. These findings (1) demonstrate that post-transcriptional A-to-I RNA editing might be crucial for glioblastoma pathogenesis, (2) identify ADAR2-editing enzyme as a novel candidate tumor suppressor gene and (3) provide proof of principle that ADAR2 or its substrates may represent a suitable target(s) for possible novel, more effective and less toxic approaches to the treatment of GBMs.

Since alterations in post-transcriptional events can contribute to the appearance and/or progression of cancer, we investigated whether RNA editing, catalyzed by the ADAR (adenosine deaminases that act on RNA) enzymes, is altered in pediatric astrocytomas. We find a decrease in ADAR2 editing activity that seems to correlate with the grade of malignancy in children. Despite the loss of ADAR2 editing activity in tumor tissues, the high grade astrocytomas do not exhibit alterations in ADAR2 expression when compared with their specific control tissues. However, high expression levels of ADAR1 and ADAR3 were found in tumors when compared with normal tissues dissected in the same area of the brain. We reintroduced either ADAR2 or the inactive version of ADAR2 in three astrocytoma cell lines (U118, A172, U87). The "reverted" editing status is necessary and sufficient for a significant decrease in cell malignant behavior as measured by proliferation, cell cycle, and migration assays. We show that elevated levels of ADAR1, as found in astrocytomas, do indeed interfere with ADAR2 specific editing activity. Furthermore, we show that the endogenous ADAR1 can form heterodimers with ADAR2 in astrocytes.The ADARs 3 (adenosine deaminases that act on RNA) are enzymes responsible for adenosine (A) to inosine (I) conversion in pre-mRNA. This deamination is the most widespread type of RNA editing in higher eukaryotes. Since the translation machinery reads inosine as guanosine (1), the ADARs can modify protein codons and thus modulate protein sequence and function of several gene products. The editing of a specific adenosine is not usually 100% efficient and as a consequence, the ADARs can generate different protein isoforms in the same cell.

RNA editing increases during development in more than 20 transcripts encoding proteins involved in rapid synaptic neurotransmission in Drosophila central nervous system and muscle. Adar (adenosine deaminase acting on RNA) mutant flies expressing only genome-encoded, unedited isoforms of ion-channel subunits are viable but show severe locomotion defects. The Adar transcript itself is edited in adult wild-type flies to generate an isoform with a serine to glycine substitution close to the ADAR active site. We show that editing restricts ADAR function since the edited isoform of ADAR is less active in vitro and in vivo than the genome-encoded, unedited isoform. Ubiquitous expression in embryos and larvae of an Adar transcript that is resistant to editing is lethal. Expression of this transcript in embryonic muscle is also lethal, with above-normal, adult-like levels of editing at sites in a transcript encoding a muscle voltage-gated calcium channel.

In this report, we establish that Drosophila ADAR (adenosine deaminase acting on RNA) forms a dimer on double‐stranded (ds) RNA, a process essential for editing activity. The minimum region required for dimerization is the N‐terminus and dsRNA‐binding domain 1 (dsRBD1). Single point mutations within dsRBD1 abolish RNA‐binding activity and dimer formation. These mutations and glycerol gradient analysis indicate that binding to dsRNA is important for dimerization. However, dimerization can be uncoupled from dsRNA‐binding activity, as a deletion of the N‐terminus (amino acids 1—46) yields a monomeric ADAR that retains the ability to bind dsRNA but is inactive in an editing assay, demonstrating that ADAR is only active as a dimer. Different isoforms of ADAR with different editing activities can form heterodimers and this can have a significant effect on editing in vitro as well as in vivo. We propose a model for ADAR dimerization whereby ADAR monomers first contact dsRNA; however, it is only when the second monomer binds and a dimer is formed that deamination occurs.

Highlights d CD27 dull and CD27 bright MBCs share their VH repertoire but have different functions d CD27 dull MBCs are the long-lived substrate of selected and specific CD27 bright MBCs d The interplay between CD27 dull and CD27 bright MBCs preserves B cell memory d In pregnancy, MBCs decline, but persisting CD27 dull MBCs re-expand after delivery