Adenosine deaminase acting on RNA 2 (sqADAR2a), the longer splice variant of sqADAR2 in the California market squid Doryteuthis opalescens. This enzyme catalyzes the hydrolytic deamination of adenosine to inosine in double-stranded RNA, which is the molecular basis of A-to-I RNA editing. This protein (C1JAR3) is the sqADAR2a variant (786 aa) that uniquely possesses three dsRNA binding domains (dsRBDs) rather than the two found in all other known ADAR2 family members. The extra dsRBD confers unusually high editing activity and resistance to the high intracellular chloride concentrations characteristic of squid neurons. Coleoid cephalopods exhibit the most extensive mRNA recoding by A-to-I editing of any known animal lineage, with over 57,000 recoding sites in the nervous system of the closely related Doryteuthis pealeii, affecting the majority of neural transcripts. sqADAR2 is one of only two catalytically active ADARs in squid (along with sqADAR1) and is the predominant ADAR in non-neural tissues such as the gill. sqADAR2 mRNAs are themselves extensively self-edited, generating additional functional diversity. The sqADAR2 protein is found in both the nucleus and cytoplasm, including the giant axon axoplasm, where it performs spatially regulated RNA editing. Temperature- dependent RNA editing by ADARs in cephalopods is thought to be an adaptive mechanism for neural acclimation to environmental temperature changes.
Curated functional classes representing distinct biological activities. These may be splice variants, cleavage products, or other forms with different functions.
SQADAR2ASQADAR2B| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003723 RNA binding | IEA GO_REF:0000120 | ACCEPT | Summary: ADAR2 binds RNA through its double-stranded RNA binding domains. Reason: RNA binding is required for engagement of the duplex RNA substrate by the ADAR2 dsRNA-binding domains; it describes direct molecular work in editing. The broad RNA-binding term is entailed by directly supported dsRNA binding. It is part of the core substrate-recognition mechanism, not peripheral simply because a more specific binding annotation also exists. Supporting Evidence: PMID:19390115 sqADAR2a differs from sqADAR2b by containing an optional exon that encodes an "extra" dsRBD...Recombinant sqADAR2a and sqADAR2b, produced in Pichia pastoris, are both active on duplex RNA. |
| GO:0003725 double-stranded RNA binding | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter-predicted dsRNA binding is strongly supported by direct experimental evidence. sqADAR2a contains three dsRNA binding domains (dsRBDs), while sqADAR2b has two. The extra dsRBD in sqADAR2a increases RNA binding affinity by 30-fold under vertebrate-like conditions and 100-fold under squid-like high-salt conditions [PMID:22457361]. Both variants bind and edit duplex RNA substrates [PMID:19390115]. dsRNA binding is the essential prerequisite for the catalytic adenosine deaminase activity. Reason: dsRNA binding is a core molecular function of sqADAR2, demonstrated directly using recombinant protein with quantitative binding assays. The three dsRBD architecture of sqADAR2a is a defining and unique feature of this enzyme. This is at the right level of specificity for the binding function. Supporting Evidence: PMID:22457361 the extra dsRBD in sqADAR2a conferred resistance to the high Cl(-) levels found in squid neurons. It does so by increasing the affinity of sqADAR2 for dsRNA by 30- or 100-fold in vertebrate-like or squid-like conditions, respectively. PMID:19390115 Recombinant sqADAR2a and sqADAR2b, produced in Pichia pastoris, are both active on duplex RNA. |
| GO:0003726 double-stranded RNA adenosine deaminase activity | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter-predicted dsRNA adenosine deaminase activity is the core catalytic function of sqADAR2. This has been directly demonstrated using recombinant protein on both perfect duplex RNA and specific mRNA substrates (squid K+ channel mRNAs) [PMID:19390115]. sqADAR2 is one of only two catalytically active ADARs in squid [PMID:37342458]. The enzyme catalyzes the hydrolytic deamination of adenosine to inosine within dsRNA structures, which underlies the unprecedented scale of mRNA recoding in cephalopods (>57,000 sites in the nervous system) [PMID:25569156]. Reason: This is the defining catalytic activity of sqADAR2 and the most important molecular function annotation. It has been demonstrated directly using recombinant enzyme on multiple substrates, with quantitative activity data. The term is at exactly the right level of specificity. Supporting Evidence: PMID:19390115 We next tested the ability of sqADAR2a and sqADAR2b to edit two K+ channel mRNAs in vitro. Both substrates are known to be edited in squid. For each mRNA, sqADAR2a edited many more sites than sqADAR2b. PMID:37342458 Studies using recombinant sqADARs suggest that only sqADAR1 and sqADAR2 are active adenosine deaminases...both on perfect duplex dsRNA and on a squid potassium channel mRNA substrate known to be edited. |
| GO:0004000 adenosine deaminase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the adenosine deaminase domain (IPR002466). While sqADAR2 does catalyze adenosine deamination, this term (GO:0004000) refers to the deamination of free adenosine nucleoside, not adenosine within RNA. The correct and more specific term GO:0003726 (double-stranded RNA adenosine deaminase activity) is already annotated. ADAR enzymes act on adenosines embedded in double-stranded RNA structures, not on free adenosine nucleosides. Reason: GO:0004000 (adenosine deaminase activity) describes the deamination of free adenosine, which is the activity of ADA enzymes, not ADAR enzymes. ADAR2 specifically deaminates adenosine residues within double-stranded RNA. The correct term GO:0003726 is already annotated, so this annotation should be replaced to avoid confusion between ADA and ADAR activities. Proposed replacements: double-stranded RNA adenosine deaminase activity Supporting Evidence: PMID:19390115 Recombinant sqADAR2a and sqADAR2b, produced in Pichia pastoris, are both active on duplex RNA. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Nuclear localization is supported by the general understanding that ADAR enzymes edit pre-mRNAs co-transcriptionally in the nucleus, which is the canonical site of A-to-I editing. However, Vallecillo-Viejo et al. (2020) demonstrated that sqADAR2 is also expressed outside the nucleus in squid neurons, including in the axoplasm [PMID:32201888]. The nuclear localization is correct but does not capture the full picture of sqADAR2 localization, which includes both nuclear and cytoplasmic compartments. Reason: Nuclear localization is well-supported for ADAR2 enzymes generally, as co-transcriptional editing of pre-mRNAs occurs in the nucleus. While sqADAR2 is also found in the cytoplasm, nuclear localization remains valid. Supporting Evidence: PMID:32201888 ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. |
| GO:0005730 nucleolus | IEA GO_REF:0000118 | UNDECIDED | Summary: TreeGrafter-predicted nucleolar localization. In vertebrates, ADAR2 has been shown to accumulate in the nucleolus, and this prediction is based on phylogenetic transfer. However, there is no direct evidence for nucleolar localization of sqADAR2 specifically in squid. The key finding in squid is that sqADAR2 is found in both the nucleus and the cytoplasm/axoplasm [PMID:32201888]. Nucleolar localization may or may not apply to the squid enzyme. Reason: Retain UNDECIDED because the available squid localization evidence resolves nucleus versus cytoplasm but not nucleolus. Vertebrate nucleolar localization can support a phylogenetic hypothesis; missing target-specific microscopy alone is not a refutation. An explicit comparison of nucleolar-targeting determinants would help assess this prediction. Supporting Evidence: PMID:32201888 ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. |
| GO:0005737 cytoplasm | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter-predicted cytoplasmic localization is directly supported by experimental evidence in squid. Vallecillo-Viejo et al. (2020) demonstrated that sqADAR2 is expressed outside the nucleus in squid neurons, and purified axoplasm from the squid giant axon contains active ADAR2 protein that can catalyze A-to-I editing [PMID:32201888]. This cytoplasmic/axonal localization is a key discovery, as RNA editing was previously thought to be restricted to the nucleus. Reason: Cytoplasmic localization is directly demonstrated in squid neurons, where sqADAR2 is found in the axoplasm and is catalytically active. This represents a significant finding that RNA editing occurs outside the nucleus in squid. The term is appropriate. Supporting Evidence: PMID:32201888 ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. |
| GO:0006382 adenosine to inosine editing | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter-predicted A-to-I editing is the core biological process of sqADAR2. This is extensively documented: sqADAR2 catalyzes adenosine to inosine conversion in mRNAs, contributing to the unprecedented >57,000 recoding sites in the squid nervous system [PMID:25569156]. sqADAR2 has been shown to edit specific sites in squid K+ channel mRNAs in vitro [PMID:19390115] and the enzyme performs spatially regulated editing in both the nucleus and axoplasm [PMID:32201888]. Reason: A-to-I editing is the defining biological process for sqADAR2. This is supported by extensive direct evidence from multiple studies using recombinant protein assays and transcriptome-wide editing profiling. The term is at exactly the right level of specificity. Supporting Evidence: PMID:25569156 We identify 57,108 recoding sites in the nervous system, affecting the majority of the proteins studied. PMID:19390115 We next tested the ability of sqADAR2a and sqADAR2b to edit two K+ channel mRNAs in vitro. Both substrates are known to be edited in squid. For each mRNA, sqADAR2a edited many more sites than sqADAR2b. |
| GO:0006396 RNA processing | IEA GO_REF:0000120 | ACCEPT | Summary: Adenosine-to-inosine RNA editing is RNA processing. Reason: RNA processing here is the covalent conversion of substrate adenosine to inosine, not an indirect effect on an RNA pathway. The broad process describes the same direct catalytic work as the accepted A-to-I editing annotation. Its generality is not evidence of a non-core role. Supporting Evidence: PMID:25569156 We identify 57,108 recoding sites in the nervous system, affecting the majority of the proteins studied. |
| GO:0008251 tRNA-specific adenosine deaminase activity | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter-predicted tRNA-specific adenosine deaminase activity. This is very likely an incorrect annotation for sqADAR2. ADAR enzymes (Adenosine Deaminases Acting on RNA) act on double-stranded RNA structures in mRNAs and other transcripts. tRNA-specific adenosine deaminase activity (GO:0008251) is the function of the ADAT family of enzymes (Adenosine Deaminases Acting on tRNAs), which are structurally and functionally distinct from ADARs. All experimental evidence for sqADAR2 demonstrates activity on dsRNA and mRNA substrates, not tRNAs [PMID:19390115, PMID:37342458]. This appears to be a TreeGrafter misannotation arising from the shared adenosine deaminase domain between ADARs and ADATs. Reason: tRNA-specific adenosine deaminase activity is the function of ADAT enzymes, not ADAR enzymes. sqADAR2 has been tested and shown to be active on dsRNA and mRNA substrates. There is no evidence that sqADAR2 edits tRNAs, and the domain architecture (dsRBDs + ADAR-type deaminase domain) is inconsistent with tRNA editing activity. This is a phylogenetic transfer error likely due to the shared deaminase domain superfamily between ADARs and ADATs. The existing OpenScientist report supports the three-dsRBD architecture, while Falcon incorrectly labels C1JAR3 as the two-dsRBD sqADAR2b variant. The cached 786-aa sequence has three dsRNA-binding domains and is consistent with sqADAR2a. Functional divergence is supported, but no exact historical graft error is established from domain architecture alone. Supporting Evidence: PMID:37342458 Studies using recombinant sqADARs suggest that only sqADAR1 and sqADAR2 are active adenosine deaminases...both on perfect duplex dsRNA and on a squid potassium channel mRNA substrate known to be edited. PMID:19390115 We next tested the ability of sqADAR2a and sqADAR2b to edit two K+ channel mRNAs in vitro. file:DOROP/ADAR2/ADAR2-hypotheses/function-hypothesis-go-0008251/openscientist.md Verdict: Refuted — within-superfamily mis-placement (Failure Mode 3) file:DOROP/ADAR2/ADAR2-hypotheses/function-hypothesis-go-0008251/openscientist.md squid ADAR2 contains three double-stranded RNA binding domains (dsRBDs) upstream of its deaminase domain file:DOROP/ADAR2/ADAR2-hypotheses/function-hypothesis-go-0008251/falcon.md recombinant sqADAR2b is catalytically active on duplex RNA substrates |
| GO:0016556 mRNA modification | IDA PMID:19390115 An extra double-stranded RNA binding domain confers high act... | NEW | Summary: Recombinant squid ADAR2 splice variants directly edit potassium-channel mRNAs in vitro, establishing mRNA modification by this enzyme. Reason: PMID:19390115 tests recombinant sqADAR2a and sqADAR2b on two K+ channel mRNAs and demonstrates editing by both variants. This is gene-specific participation evidence, unlike a transcriptome-wide count that can include ADAR1 substrates. GO:0016556 specifies the mRNA substrate class while GO:0006382 specifies A-to-I chemistry; the saved live ancestry responses confirm neither term is an ancestor of the other. Supporting Evidence: PMID:19390115 next tested the ability of sqADAR2a and sqADAR2b to edit two K+ channel mRNAs in vitro. Both substrates are known to be edited in squid. For each mRNA, sqADAR2a edited many more sites than sqADAR2b. |
| GO:0008270 zinc ion binding | NAS file:DOROP/ADAR2/ADAR2-uniprot.txt | NEW | Summary: The deaminase domain of ADAR enzymes coordinates a zinc ion at the catalytic center, which is essential for the hydrolytic deamination of adenosine. The UniProt entry for C1JAR3 lists zinc and metal-binding as keywords. This is a conserved feature of all active ADAR deaminase domains. Reason: Zinc ion binding is an inherent property of the ADAR catalytic deaminase domain. The UniProt entry lists zinc binding as a keyword. While metal ion binding (GO:0046872) is annotated in the UniProt GO cross-references, the more specific zinc ion binding term better captures the biochemistry of the ADAR active site. The supporting source is an electronic UniProt keyword inference, not a direct zinc-binding measurement in PMID:37342458. Supporting Evidence: file:DOROP/ADAR2/ADAR2-uniprot.txt Zinc {ECO:0000256|ARBA:ARBA00022833}. |
| GO:1904115 axon cytoplasm | IDA PMID:32201888 Spatially regulated editing of genetic information within a ... | NEW | Summary: Vallecillo-Viejo et al. (2020) directly demonstrated that sqADAR2 protein is present in the squid giant axon axoplasm and that purified axoplasm has adenosine-to-inosine editing activity [PMID:32201888]. Over 70% of editing sites are edited more extensively in the giant axon than in the cell bodies. This was a landmark finding showing that RNA editing is not restricted to the nucleus in squid neurons. Reason: Axon cytoplasm localization is directly demonstrated for sqADAR2 in the squid giant axon system. This is a key finding that distinguishes sqADAR2 function from the canonical nuclear-only editing paradigm. The term GO:1904115 (axon cytoplasm) is more specific than GO:0005737 (cytoplasm) and accurately captures the demonstrated localization. Supporting Evidence: PMID:32201888 ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. |
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Download this section (compressed HTML)Q: What is the relative contribution of sqADAR2 vs. sqADAR1 to the total editing observed in the squid nervous system? qPCR data suggest sqADAR1 is more abundant in the nervous system and sqADAR2 predominates in the gill, but the relative contribution to specific site editing is unclear.
Q: Is the extra dsRBD of sqADAR2a an adaptation specifically to the high chloride intracellular environment of squid, or does it serve additional functions in substrate selection or editing specificity?
Q: Does temperature-dependent RNA editing by sqADAR2 occur in Doryteuthis opalescens as it does in octopus? The temperature-dependent editing has been demonstrated in Octopus bimaculoides [PMID:37295402] but not yet specifically in D. opalescens.
Experiment: Compare the editing profiles of recombinant sqADAR2a and sqADAR2b on a panel of squid mRNA substrates beyond K+ channel mRNAs. Use high-throughput sequencing of edited products to map site-specific editing levels for each variant. This would reveal whether the extra dsRBD affects not just overall activity but site selectivity.
Hypothesis: sqADAR2a and sqADAR2b have different substrate specificities due to the extra dsRBD.
Type: in vitro editing assay with RNA-seq
Experiment: Acclimate D. opalescens to different temperatures (e.g., 10C, 15C, 20C) and perform transcriptome-wide RNA editing profiling of nervous system tissue. Compare editing levels at known recoding sites to determine if temperature-dependent editing occurs as in octopus [PMID:37295402].
Hypothesis: sqADAR2 performs temperature-dependent editing in D. opalescens nervous system.
Type: in vivo temperature acclimation with RNA-seq
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