Limits...
Editing independent effects of ADARs on the miRNA/siRNA pathways.

Heale BS, Keegan LP, McGurk L, Michlewski G, Brindle J, Stanton CM, Caceres JF, O'Connell MA - EMBO J. (2009)

Bottom Line: Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity.We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity.These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, UK.

ABSTRACT
Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.

Show MeSH
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2735678&req=5

f2: 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.

Mentions: Having established conditions to maximize silencing activities of mature miRNAs from the transfected pri-mir-376a2 construct and to determine their target site preferences we cotransfected the mir-376a2 reporter with constructs expressing ADARs and ADAR mutations to assess how ADARs affect the silencing activities of resulting miRNAs. Consistent with their editing of pri-mir-376a2 at position +44, both hADAR1 p110 and hADAR1 p150 increased the silencing activity corresponding to the edited form of the 3′ miR (Figure 2B). This change was largely editing dependent, as the deaminase mutant ADARs (ADAR p150 E912A and ADAR p110 E912A) did not have this effect (Figure 2B); the effect was not dependent on the Z-DNA-binding domains of ADAR1 (ADAR1 443-end). Further, the Z-DNA-binding domains of ADAR1 (ADAR1 1-442) were not sufficient on their own to significantly increase the silencing activity of edited 3′ miRNA.


Editing independent effects of ADARs on the miRNA/siRNA pathways.

Heale BS, Keegan LP, McGurk L, Michlewski G, Brindle J, Stanton CM, Caceres JF, O'Connell MA - EMBO J. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2735678&req=5

f2: 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.
Mentions: Having established conditions to maximize silencing activities of mature miRNAs from the transfected pri-mir-376a2 construct and to determine their target site preferences we cotransfected the mir-376a2 reporter with constructs expressing ADARs and ADAR mutations to assess how ADARs affect the silencing activities of resulting miRNAs. Consistent with their editing of pri-mir-376a2 at position +44, both hADAR1 p110 and hADAR1 p150 increased the silencing activity corresponding to the edited form of the 3′ miR (Figure 2B). This change was largely editing dependent, as the deaminase mutant ADARs (ADAR p150 E912A and ADAR p110 E912A) did not have this effect (Figure 2B); the effect was not dependent on the Z-DNA-binding domains of ADAR1 (ADAR1 443-end). Further, the Z-DNA-binding domains of ADAR1 (ADAR1 1-442) were not sufficient on their own to significantly increase the silencing activity of edited 3′ miRNA.

Bottom Line: Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity.We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity.These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, UK.

ABSTRACT
Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.

Show MeSH