Drosha versus ADAR: wrangling over pri-miRNA.
Processing of a mammalian hematopoietic microRNA precursor is suppressed when it is edited by ADARs. The edited pri-miRNA can subsequently be degraded by Tudor-SN.
Adenosine Deaminase↗
Biomedical subjects
Liam P Keegan
Publications and source records attributed to Liam P Keegan.
SUMO-1 modification alters ADAR1 editing activity.
We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.
Adenosine Deaminase↗
Tuning of RNA editing by ADAR is required in Drosophila.
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.
Adenosine Deaminase↗
Adenosine deaminases acting on RNA (ADARs): RNA-editing enzymes.
Adenosine deaminases acting on RNA (ADARs) were discovered as a result of their ability extensively to deaminate adenosines in any long double-stranded RNA, converting them to inosines. Subsequently, ADARs were found to deaminate adenosines site-specifically within the coding sequences of transcripts encoding ion-channel subunits, increasing the diversity of these proteins in the central nervous system. ADARI is now known to be involved in defending the genome against viruses, and it may affect RNA interference. ADARs are found in animals but are not known in other organisms. It appears that ADARs evolved from a member of another family, adenosine deaminases acting on tRNAs (ADATs), by steps including fusion of two or more double-stranded-RNA binding domains to a common type of zinc-containing adenosine-deaminase domain.
Adenosine Deaminase↗
Purification and assay of recombinant ADAR proteins expressed in the yeast Pichia pastoris or in Escherichia coli.
ADARs are found in Metazoans but are not present in yeasts. We have found that the methanol-utilizing yeast Pichia pastoris can be used to efficiently express enzymatically active epitope-tagged ADARs. We describe plasmid construction and protein expression procedures for producing Drosophila ADAR in this system.ADAR expression in Pichia pastoris uses the methanol-inducible alcohol oxidase AOX1 promoter for induction. A Zeocin resistance gene on the plasmid is used to select high copy number tandem integrations of the plasmid constructs. Preparation of extracts by grinding cultures in liquid nitrogen and purification protocols using 6 x HIS and FLAG epitope tags are described. Procedures for preparing radiolabeled dsRNA and for assaying the non-specific RNA editing activity of ADARs are described.ADARs produced in Escherichia coli are not enzymatically active. We describe expression of the ADAR dsRNA binding domains in E. coli using current versions of the T7 promoter based Studier vectors as well as the purification of the domains.
Adenosine Deaminase↗
An ADAR that edits transcripts encoding ion channel subunits functions as a dimer.
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.
Adenosine Deaminase↗
Dynamic association of RNA-editing enzymes with the nucleolus.
ADAR1 and ADAR2 are editing enzymes that deaminate adenosine to inosine in long double stranded RNA duplexes and specific pre-mRNA transcripts. Here, we show that full-length and N-terminally truncated forms of ADAR1 are simultaneously expressed in HeLa and COS7 cells owing to the usage of alternative starting methionines. Because the N-terminus of ADAR1 contains a nuclear export signal, the full-length protein localizes predominantly in the cytoplasm, whereas the N-terminally truncated forms are exclusively nuclear and accumulate in the nucleolus. ADAR2, which lacks a region homologous to the N-terminal domain of ADAR1, localizes exclusively to the nucleus and similarly accumulates in the nucleolus. Within the nucleolus, ADAR1 and ADAR2 co-localize in a novel compartment. Photobleaching experiments demonstrate that, in live cells, ADAR1 and ADAR2 are in constant flux in and out of the nucleolus. When cells express the editing-competent glutamate receptor GluR-B RNA, endogenous ADAR1 and ADAR2 de-localize from the nucleolus and accumulate at sites where the substrate transcripts accumulate. This suggests that ADAR1 and ADAR2 are constantly moving through the nucleolus and might be recruited onto specific editing substrates present elsewhere in the cell.
Adenosine Deaminase↗
Micro-processing events in mRNAs identified by DHPLC analysis.
Post-transcriptional processes such as alternative splicing and RNA editing have a huge impact on the diversity of the proteome. Detecting alternatively spliced transcripts is difficult when they are rare. In addition, edited transcripts often differ from the genomic sequence by only a few nucleotides. Denaturing high performance liquid chromatography (DHPLC) is routinely used for single nucleotide polymorphism detection and we used this method to detect alternatively spliced or edited transcripts. As the sites of RNA editing appear to be conserved within gene families, we investigated whether editing sites are conserved in the murine homologue of the Drosophila cacophony transcript encoding the alpha1 subunit of a voltage-gated calcium channel that is edited at 10 independent positions. Although DHPLC analysis detects RNA editing at as low as 3% in control transcripts, no evidence of RNA editing was found in the analysed murine transcript. However an alternative exon was identified at the 3' end of the mouse Cacna1alpha transcript and an alternative micro-exon encoding only two amino acids (NP) was found in the extracellular loop before the IVS4 helix in the same transcript. In the homologous Drosophila transcript a micro-exon also encoding two amino acids was found at the same position before the IVS4 helix.
Alternative Splicing↗