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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.

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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.
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f1: 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.

Mentions: ADAR1 and ADAR2 have similar domain structures (Figure 1D) but mainly display distinct editing site specificities—although their editing activities do overlap at some sites. There are two major isoforms of ADAR1 because of alternative promoter usage, the longer shuttling p150 isoform and the shorter nuclear p110 isoform. ADAR1 p150 is upregulated by interferon and can compete with PKR for double-stranded (ds)RNA. At the amino terminus ADAR1 p150 has two Z-DNA-binding domains and a nuclear export signal, which allows it to shuttle in and out of the nucleus, however it is predominantly found in the cytoplasm. Both isoforms have three dsRNA-binding domains, the third one overlaps with a nuclear localization signal (NLS) and the catalytic deaminase domain is at the carboxy terminus (Strehblow et al, 2002). ADAR1 is widely expressed throughout the body with the exception of skeletal muscle. ADAR2, on the other hand, is most highly expressed in the central nervous system, has two dsRNA-binding domains and a carboxy-terminal deaminase domain. It is localized to the nucleus by an NLS present in the N-terminal region (Desterro et al, 2003; Wong et al, 2003). To what extent each of the different domains of ADAR1 and ADAR2 contributes to their biological activities is an open question. Two other members of the ADAR family in vertebrates are catalytically inactive, ADAR3 that is brain specific and TENR that is expressed in the testis (Melcher et al, 1996; Connolly et al, 2005). Drosophila has a single Adar gene encoding a deaminase with the same domain structure as ADAR2. Adar mutant flies are viable with severe locomotion defects and loss of RNA editing in transcripts expressed in the CNS (Palladino et al, 2000a, 2000b).


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)

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

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

f1: 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.
Mentions: ADAR1 and ADAR2 have similar domain structures (Figure 1D) but mainly display distinct editing site specificities—although their editing activities do overlap at some sites. There are two major isoforms of ADAR1 because of alternative promoter usage, the longer shuttling p150 isoform and the shorter nuclear p110 isoform. ADAR1 p150 is upregulated by interferon and can compete with PKR for double-stranded (ds)RNA. At the amino terminus ADAR1 p150 has two Z-DNA-binding domains and a nuclear export signal, which allows it to shuttle in and out of the nucleus, however it is predominantly found in the cytoplasm. Both isoforms have three dsRNA-binding domains, the third one overlaps with a nuclear localization signal (NLS) and the catalytic deaminase domain is at the carboxy terminus (Strehblow et al, 2002). ADAR1 is widely expressed throughout the body with the exception of skeletal muscle. ADAR2, on the other hand, is most highly expressed in the central nervous system, has two dsRNA-binding domains and a carboxy-terminal deaminase domain. It is localized to the nucleus by an NLS present in the N-terminal region (Desterro et al, 2003; Wong et al, 2003). To what extent each of the different domains of ADAR1 and ADAR2 contributes to their biological activities is an open question. Two other members of the ADAR family in vertebrates are catalytically inactive, ADAR3 that is brain specific and TENR that is expressed in the testis (Melcher et al, 1996; Connolly et al, 2005). Drosophila has a single Adar gene encoding a deaminase with the same domain structure as ADAR2. Adar mutant flies are viable with severe locomotion defects and loss of RNA editing in transcripts expressed in the CNS (Palladino et al, 2000a, 2000b).

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