Effects of ADARs on functional activities of mature miRNAs processed from pri-mir-376a2. (A) Inhibition of 3′ miRNA activity. A value more than 1 means that the miRNA is less active when the ADAR is present than when the pri-mir-376a2 is expressed alone. (B) Retargeting of 3′ miRNA activity. A value less than 1 means that the miRNA is more active in the presence of the ADAR than when the pri-mir-376a2 is expressed alone. Pre-edited mir-376a2 (a177g) indicates a construct expressing pri-mir-376a2 with the +44 site (position 177) mutated from an adenosine to a guanosine. (C) Inhibition of 5′ miRNA activity. (D) Retargeting of 5′ miRNA activity. The activity for pre-edited mir-376a2 (a137g) is from a construct expressing pri-mir-376a2 with the +4 site (position 137) mutated from an adenosine to a guanosine.

Deaminase-inactive ADAR2 fails to retarget miRNAs but still inhibits their processing. (A) Top panel: effects of normal versus deaminase mutant ADAR2 on production of mature unedited and edited miRNA activities. Inactive ADAR2 E319A retained the inhibitory effect of ADAR2 on the unedited 5′ miRNA activity but was unable to retarget the activity by editing. Bottom panel: effects of cytoplasmic ADAR2 ΔN and ADAR2 ΔN E319A on the activity of mir-376a2. ADAR2 ΔN E319A did not affect the 5′ reporter, indicating a loss in retargeting. Neither hADAR2 ΔN nor hADAR2 ΔN deaminase mutant affected the 3′ mature miRNA activity. (B) Effect of ADAR2 E319A on processing of mir-376a2 in cotransfected HEK 293T cells. Small RNA northerns of RNA from cotransfected cells probed with antisense probes to the 3′ miRNA. Blots were also probed with a 5S rRNA-specific probe as a loading control (data not shown). Deaminase mutant ADAR2 E319A expression reduced both pre-miRNA and mature miRNA levels. Percentage of pre-miRNA signal relative to pri-mir-376a2 expressed alone is noted below the blot. (C) Effects of main isoforms of ADAR1 and ADAR2 on functional activities of 5′ and 3′ miRNAs expressed from a construct bearing mir-376a1 alone. As in the case of mir-376a2, ADAR2 was able to inhibit the activity of mir-376a1 independently of deaminase activity. (D) The ADAR2 E319A deaminase domain mutant reduced the level of pre-mir-376a1 in cotransfected HEK 293T cells. Small RNA northern blot of RNA from transfected cells probed with antisense probe to the 3′ miRNA. The percentage of pre-mir-376a1 signal relative to pri-mir-376a1 expressed alone is listed below the blot.

Editing levels in the human mir-376 cluster. (A) Diagram of the mir-376 cluster; nomenclature is from miRBASE http://microrna.sanger.ac.uk/sequences. Numbers at bottom indicate intervening lengths of sequences. Bold text regions are the mature microRNAs. Boxes indicate major sites of editing. There is no evidence of editing in mir-654. (B) Editing in mir-376a1 expressed from a complete mir-376 cluster. (C) Editing in mir-376a2. Ratio G/(A+G) indicates the ratio of the guanosine signal to the total (adenosine+guanosine) chromatogram signal. +4 and +44 are positions within mir-376a2 indicated in A. (D) ADAR1 and ADAR2 protein domains showing the positions and features used in the construction of the hADAR expression vectors. hADAR1 p150 contains two Z-DNA-binding domains, three dsRBDs and one deaminase domain. hADAR1 p110 begins at amino-acid position 296 of hADAR1 p150. hADAR2 has two dsRBDs and one deaminase domain. hADAR2 ΔN is a deletion mutant of hADAR2 with amino acids from positions 4–72 removed. A key glutamate residue in the deaminase domain is amino acid 912 for ADAR1 and 319 for ADAR2. Mutation of this residue to alanine renders the ADAR catalytically inactive.

Effects of ADARs on processing of pri-mir-376a2. (A, B) Effects of ADARs on processing in HEK 293T cells. Small RNA northern on RNA from cotransfected HEK 293T cells probed with antisense probes to (A) the 3′ miRNA and (B) the 5′ miRNA. Nothern blots were also probed with a 5S rRNA-specific probe as a loading control (data not shown). ADAR2 expression reduced both the pre-miRNA and the mature 3′ and 5′ miRNA signals. The numbers below the blot indicate the amount of both pre-mir-376a2 or mature 376a2 signal in each lane relative to that in pri-mir-376a2 expressed alone. (C) Recombinant ADAR2 inhibits Drosha cleavage in HeLa extracts in vitro. Drosha cleavage assays performed in the presence of recombinant ADAR2 and either pri-let-7a-1 or pri-mir-376a2. A double plus in the ADAR2 table indicates that a higher amount of ADAR2 was added. Cleavage of pri-mir-376a2 but not pri-let-7a-1 was inhibited by ADAR2, indicating that ADAR2 directly and specifically effects Drosha processing of mir-376a2. The percentage of pre-miRNA and mature 376a2 signal relative to cleavage in the absence of ADAR2 is indicated below the blot. (D) Lack of effect of RNA editing base changes in pri-mir-376a2 on Drosha cleavage in HeLa extracts in vitro. Drosha cleavage was not impaired by point mutations mimicking RNA editing events in mir-376a2. a137g mimics editing of the +4 site. a177g mimics editing of the +44 site. c188t closes the A–C mismatch at the +4 site in pri-mir-376a2. The amount of pre-miRNA signal relative to wild-type pri-mir-376a2 cleavage is indicated below the blot.

Human ADAR1 antagonizes white hairpin-mediated RNA interference in Drosophila. Effects of human ADAR proteins on silencing of white⁺ gene expression in Drosophila eyes by a co-expressed cytoplasmic white RNA hairpin. These flies are female progeny of homozygous w+; GMR-GAL4, UAS-whiteIR (GMR>wIR) crossed to w1118; UAS construct lines. (A) w+; GMR>wIR alone, (B) ADAR p110 (line 19 on Chr III), (C) ADAR p150 (line 25 on Chr III), (D) ADAR2 (line 22 on Chr II), (E) Dicer-2 (line D2 on Chr II), (F) Dicer-2 (line D2) and ADAR1 p150 (line 25), (G) ADAR1 p150 E319A (line 34 on Chr III), (H) ADAR1 p150 E319A (line 35 on Chr II).