magi3b (NCBI LOC564220 in older builds; ZFIN ZDB-GENE-060503-301, chromosome 6) is one of two zebrafish co-orthologs of vertebrate MAGI3; PANTHER places the duplication at the teleost genome duplication. Its paralog is wu:fi36a10 (magi3a in NCBI RefSeq). It encodes a cytoplasmic membrane-associated guanylate kinase with inverted domain order (MAGI): an N-terminal PDZ domain, a guanylate kinase-like (GK) domain that lacks the nucleotide-binding P-loop and is therefore a protein-interaction module rather than an enzyme, two WW domains, five further PDZ domains and a long disordered C-terminal tail. MAGI proteins act as scaffolds at the cytoplasmic face of cell-cell junctions, where their PDZ domains bind C-terminal motifs of receptors and signalling proteins. In zebrafish, magi3b is expressed in endothelial cells, and F0 crispants for magi3b, like those for wu:fi36a10, have reduced brain vascularization that phenocopies loss of the C-terminal ETTV PDZ-binding motif of the Wnt7 co-receptor Gpr124. A morpholino screen reported shorter kidney motile cilia after knockdown of a magi3 gene attributed to this locus. Public RNA-Seq calls are most prominent in retina, early embryo and ovary, and are narrower than those of wu:fi36a10.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | REMOVE | Summary: Nucleotide binding from UniRule UR000400325 (MAGUK guanylate kinase-like domain). Reason: UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in magi3b (as in wu:fi36a10, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain. Supporting Evidence: PMID:37163606 Unlike other MAGUK GKs, which are composed of three subdomains including guanosine monophosphate (GMP)–binding, LID, and CORE subdomains, MAGI GKs lack the last two subdomains file:DANRE/wu_fi36a10/wu_fi36a10-bioinformatics/RESULTS.md Neither zebrafish copy, nor gar or human MAGI3, has a Walker A (P-loop) motif at the position UniProt annotates as the ATP-binding site |
| GO:0005524 ATP binding | IEA GO_REF:0000104 | REMOVE | Summary: ATP binding from UniRule UR000400325, based on the predicted P-loop of the GK-like domain. Reason: UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in magi3b (as in wu:fi36a10, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain. Supporting Evidence: PMID:37163606 Unlike other MAGUK GKs, which are composed of three subdomains including guanosine monophosphate (GMP)–binding, LID, and CORE subdomains, MAGI GKs lack the last two subdomains file:DANRE/wu_fi36a10/wu_fi36a10-bioinformatics/RESULTS.md Neither zebrafish copy, nor gar or human MAGI3, has a Walker A (P-loop) motif at the position UniProt annotates as the ATP-binding site |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Cytoplasm (IBA, PANTHER PTN000817101 MAGI node). Reason: MAGI proteins are cytoplasmic scaffolds at the inner face of the plasma membrane. magi3b is inside the MAGI clade and keeps all its domains. The target appearing in WITH/FROM (ZFIN:ZDB-GENE-060503-301) reflects its own IDA from PMID:25139857, which is expected for a PAINT node, not circular. Supporting Evidence: PMID:25139857 whereas the remainder appeared to localize to a combination of the cytoplasm, nucleus and cell membrane |
| GO:0005737 cytoplasm | IDA PMID:25139857 Systematic discovery of novel ciliary genes through function... | ACCEPT | Summary: GFP-tagged zebrafish magi3 localized to cytoplasm (plus nucleus and membrane) in Kupffer's vesicle cells; not ciliary. Reason: Direct localization of the tagged zebrafish protein in the embryo. It is a coarse location, but it is correct and consistent with the IBA. Supporting Evidence: PMID:25139857 whereas the remainder appeared to localize to a combination of the cytoplasm, nucleus and cell membrane |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Plasma membrane (UniProt subcellular location, peripheral membrane protein). Reason: MAGI3 is a peripheral protein on the cytoplasmic face of the plasma membrane at cell-cell junctions. In zebrafish it binds the C terminus of the transmembrane receptor Gpr124. Supporting Evidence: PMID:10748157 localizes to epithelial cell tight junctions PMID:35649360 This observation is compatible with a role for Dlg4 and Magi3 as Gpr124 ETTV adaptor proteins, acting in parallel to Dvl. |
| GO:0005911 cell-cell junction | IBA GO_REF:0000033 | ACCEPT | Summary: Cell-cell junction (IBA, PANTHER PTN002569180). Reason: Junctional localization is the conserved property of MAGI1-3. magi3b keeps the full MAGI domain set, and nothing argues for loss of the function. This is the correct level of specificity for a family-level inference. Supporting Evidence: PMID:10748157 localizes to epithelial cell tight junctions |
| GO:0005923 bicellular tight junction | IEA GO_REF:0000044 | ACCEPT | Summary: Bicellular tight junction (UniProt subcellular location). Reason: Human MAGI3 localizes to epithelial tight junctions. There is no zebrafish test of this, but the domain architecture is conserved. It is a reasonable transfer, more specific than the IBA cell-cell junction. Supporting Evidence: PMID:10748157 localizes to epithelial cell tight junctions |
| GO:0007165 signal transduction | IBA GO_REF:0000033 | ACCEPT | Summary: Signal transduction (IBA, PANTHER PTN000817101). Reason: Very general, but correct. MAGI3 scaffolds signalling complexes: PTEN and AKT in mammals, and in zebrafish the Gpr124 ETTV motif in the Wnt7-dependent brain angiogenesis pathway, where magi3b crispants phenocopy loss of that motif. A more specific process term would need evidence beyond F0 crispants. Supporting Evidence: PMID:10748157 cooperate to modulate the kinase activity of AKT/PKB PMID:35649360 This observation is compatible with a role for Dlg4 and Magi3 as Gpr124 ETTV adaptor proteins, acting in parallel to Dvl. PMID:35649360 Targeting magi3a, magi3b, or dlg4b, but not dlg4a, partially impaired brain vascularization (Figures 4H and 4I), in agreement with the expression data. |
| GO:0016301 kinase activity | IEA GO_REF:0000104 | REMOVE | Summary: Kinase activity from UniRule UR000400325. Reason: UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in magi3b (as in wu:fi36a10, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain. Supporting Evidence: PMID:37163606 Unlike other MAGUK GKs, which are composed of three subdomains including guanosine monophosphate (GMP)–binding, LID, and CORE subdomains, MAGI GKs lack the last two subdomains file:DANRE/wu_fi36a10/wu_fi36a10-bioinformatics/RESULTS.md Neither zebrafish copy, nor gar or human MAGI3, has a Walker A (P-loop) motif at the position UniProt annotates as the ATP-binding site |
| GO:0016740 transferase activity | IEA GO_REF:0000104 | REMOVE | Summary: Transferase activity from UniRule UR000400325 (parent of kinase activity). Reason: UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in magi3b (as in wu:fi36a10, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain. Supporting Evidence: PMID:37163606 Unlike other MAGUK GKs, which are composed of three subdomains including guanosine monophosphate (GMP)–binding, LID, and CORE subdomains, MAGI GKs lack the last two subdomains file:DANRE/wu_fi36a10/wu_fi36a10-bioinformatics/RESULTS.md Neither zebrafish copy, nor gar or human MAGI3, has a Walker A (P-loop) motif at the position UniProt annotates as the ATP-binding site |
| GO:0044782 cilium organization | IMP PMID:25139857 Systematic discovery of novel ciliary genes through function... | KEEP AS NON CORE | Summary: Morpholino knockdown of a magi3 gene shortened kidney motile cilia in a Foxj1-target screen. Reason: The evidence is a single morpholino in a 50-gene screen, with no mutant, rescue or second morpholino in the main text. The locus assignment is ZFIN's. MAGI3 is not a known ciliary protein in other species, and the tagged protein was not ciliary. Kept as a curator-made, uncorroborated non-core annotation. cilium organization is the parent of the co-annotated cilium assembly. Supporting Evidence: PMID:25139857 We found that the loss of six genes – BX470211.1, Dr.81747 (GLB1L2 in human), ect2l, magi3, si:ch211-71m22.1 and si:dkey-26i13.8 (KIF18B in human) – showed a significant (P<5.0×10−7, Student's t-test, two-tailed) reduction in the length of motile cilia in the kidney tubules PMID:25139857 We tested the role of the 50 randomly selected FIGs in cilia formation and function by injecting antisense morpholinos into zebrafish embryos PMID:25139857 whereas the remainder appeared to localize to a combination of the cytoplasm, nucleus and cell membrane |
| GO:0060271 cilium assembly | IMP PMID:25139857 Systematic discovery of novel ciliary genes through function... | KEEP AS NON CORE | Summary: Same morphant: shorter motile cilia in kidney tubules. Reason: Same single-morpholino evidence as the cilium organization row. There is no mutant confirmation, and the mechanism may be indirect (the protein is cytoplasmic, not ciliary). Not a core function. Do not propagate it to wu:fi36a10 or to human MAGI3 without mutant data. Supporting Evidence: PMID:25139857 We found that the loss of six genes – BX470211.1, Dr.81747 (GLB1L2 in human), ect2l, magi3, si:ch211-71m22.1 and si:dkey-26i13.8 (KIF18B in human) – showed a significant (P<5.0×10−7, Student's t-test, two-tailed) reduction in the length of motile cilia in the kidney tubules PMID:25139857 whereas the remainder appeared to localize to a combination of the cytoplasm, nucleus and cell membrane |
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Download this section (compressed HTML)Q: Do stable magi3b mutants (ideally RNA-less, to avoid transcriptional adaptation of wu:fi36a10) reproduce the brain vascularization defect seen in F0 crispants?
Q: Does Magi3b bind the Gpr124 ETTV motif directly, as shown for Magi3a by yeast two-hybrid?
Q: Is the kidney cilia phenotype of the magi3 morphant reproduced by a magi3b mutant, and which paralog did the morpholino actually target?
Experiment: Generate stable single, double and compound-heterozygous mutants and count hindbrain central arteries; test cross-rescue by endothelial (kdrl promoter) expression of each coding sequence.
Hypothesis: magi3b and wu:fi36a10 are both required, non-redundantly and dose-dependently, for Gpr124-dependent brain angiogenesis.
Type: genetic interaction and cross-rescue
Experiment: Single-cell or FACS-sorted RNA-seq and in situ hybridization for both paralogs in endothelium, retina and kidney, compared with gar magi3.
Hypothesis: The restricted expression of magi3b relative to wu:fi36a10 reflects expression partition, not reduced function.
Type: expression profiling
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