smad3b (SMAD family member 3b) is one of two zebrafish co-orthologs of vertebrate SMAD3, retained from the teleost-specific genome duplication; its paralog is smad3a. It encodes a receptor-regulated SMAD with an MH1 DNA-binding domain, a linker and an MH2 domain ending in the receptor-phosphorylated SSVS motif. It mediates TGF-beta/activin/nodal signals by forming complexes with SMAD4 that regulate transcription. smad3b is the faster-evolving copy: it differs from human SMAD3 mainly in the linker and carries asparagine at the position of the CK1 phosphosite S418, while keeping the Zn-binding, DNA-binding and receptor-phosphorylation residues. It is maternally supplied and zygotically expressed at the gastrula margin, tail bud, lateral mesoderm, brain regions, retina, spinal cord, somites and heart; unlike smad3a it is not expressed with a circadian rhythm. Overexpressed smad3b induces mesoderm and organizer genes such as goosecoid, and constitutively active Smad3b increases cardiomyocyte proliferation and activates Smad3 target genes. Knockdown of smad3b, like smad3a, alters neural differentiation. Combined loss of smad3a and smad3b enlarges the ventral aorta but is compatible with adult survival.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000976 transcription cis-regulatory region binding | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA electronic transfer of cis-regulatory region binding, a parent of the SMAD-binding element binding captured by the IBA to GO:0000978. Reason: The MH1 domain of smad3b keeps the Zn-coordinating residues and K40/K41 of human SMAD3 (94% MH1 identity), and a Smad3 reporter built on SMAD-binding elements responds to Smad3b. Correct though general. Supporting Evidence: PMID:25286120 Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. |
| GO:0000978 RNA polymerase II cis-regulatory region sequence-specific DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference from the SMAD family node that R-SMADs bind RNA polymerase II cis-regulatory DNA. Reason: smad3b is inside the node and keeps every annotated MH1 functional residue of human SMAD3 (smad3a-bioinformatics). No smad3b-specific loss is known. Supporting Evidence: file:DANRE/smad3a/smad3a-bioinformatics/RESULTS.md Conserved in both copies and in gar |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference that SMAD proteins are sequence-specific RNA polymerase II transcription factors. Reason: Core function. Smad3b activates a Smad3 reporter and target genes (constitutively active Smad3b raises Smad3 target-gene expression in heart). It was weaker than Smad3a on the myf5 promoter, a quantitative difference that does not remove the activity. Supporting Evidence: PMID:25286120 Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. PMID:29196619 an upregulation in Smad3 target gene expression in caSmad3a and caSmad3b hearts |
| GO:0005515 protein binding | IPI PMID:16890162 Tob1 controls dorsal development of zebrafish embryos by ant... | REMOVE | Summary: Interaction with Tob1a, which the paper reports inhibits Smad3 transcriptional activity. Reason: Generic protein binding carries no functional information. The Tob1a interaction is not disputed, but the cached abstract gives no evidence for a more informative Smad3b activity term. Treated identically on smad3a. Supporting Evidence: PMID:16890162 Although Tob1a can also inhibit the transcriptional activity of the Nodal effector Smad3, its role in limiting dorsal development is executed primarily by antagonizing the beta-catenin signal. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: Nuclear location mapped from the UniProt subcellular-location rule for SMADs. Reason: Activated R-SMADs act in the nucleus; Smad3b drives nuclear reporter and target-gene transcription. |
| GO:0005667 transcription regulator complex | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the MH1 domain. Reason: R-SMADs act in transcription regulator complexes with SMAD4; the IBA to heteromeric SMAD protein complex is more specific. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Cytoplasmic location mapped from the UniProt subcellular-location rule. Reason: Unstimulated R-SMADs are cytoplasmic and shuttle to the nucleus after receptor phosphorylation. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the SMAD/MH1/MH2 domains. Reason: Correct but general; the IBA to positive regulation of transcription by RNA polymerase II is more specific. |
| GO:0007179 transforming growth factor beta receptor signaling pathway | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference at the SMAD2/3 node; smad3a (ZFIN:ZDB-GENE-000509-3) is among the descendant evidence. Reason: Core process. The receptor-phosphorylated C-terminal SSVS motif is conserved in smad3b, and the Smad3 reporter that responds to Smad3b is blocked by ALK4/ALK5 inhibitors. Supporting Evidence: PMID:25286120 Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. |
| GO:0009653 anatomical structure morphogenesis | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Very broad family-level PAINT process. Reason: Not wrong for a SMAD, but uninformative and describes downstream outcomes rather than what smad3b does. |
| GO:0009880 embryonic pattern specification | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA transfer of embryonic patterning. Reason: Smad3b overexpression induces mesoderm and organizer genes, and dominant-negative Smad3b blocks mesendoderm induction and neural induction; these reagents act on Smad2/3 signalling generally. Plausible but non-core. Supporting Evidence: PMID:12112463 We show that zebrafish Smad3b, in contrast to the related zebrafish Smad2, can induce mesoderm independently of TGF-beta signaling. PMID:19580801 Collectively, these data indicate that Smad2/3 activities are required for neural induction and neuroectodermal posteriorization in zebrafish. |
| GO:0009953 dorsal/ventral pattern formation | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA transfer of dorsal/ventral patterning, a SMAD family-wide rule dominated by BMP-pathway SMADs. Reason: Dorsal/ventral patterning in zebrafish is driven mainly by BMP-SMAD1/5. Smad3b activates goosecoid when overexpressed, but no smad3b loss-of-function evidence shows a dorsal/ventral patterning defect. Treated identically on smad3a. Supporting Evidence: PMID:12112463 Although mammalian Smad3 was shown to inhibit expression of the organizer-specific genes goosecoid, zebrafish smad3b activates organizer genes such as goosecoid. |
| GO:0030154 cell differentiation | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Broad family-level PAINT process. Reason: Correct in general (smad3b knockdown alters neural differentiation) but uninformative. Supporting Evidence: PMID:25286120 Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. |
| GO:0030509 BMP signaling pathway | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA transfer of BMP signalling, a SMAD family-wide rule. Reason: SMAD3 is an R-SMAD of the TGF-beta/activin/nodal branch; in zebrafish the Smad3 reporter that responds to Smad3b is blocked by ALK4/ALK5 inhibitors. No evidence places smad3b downstream of BMP receptors. Treated identically on smad3a. Supporting Evidence: PMID:25286120 Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. |
| GO:0032924 activin receptor signaling pathway | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference at the SMAD2/3 node for activin signalling. Reason: SMAD2/3 are the R-SMADs of the activin/nodal branch; dominant-negative Smad3b blocks nodal-induced mesendoderm, and smad3b keeps the receptor-phosphorylation motif. Supporting Evidence: PMID:18025082 Overexpression of these mutants abolished mesendoderm induction by ectopic Nodal signaling in zebrafish embryos. |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference at the SMAD2/3 node. Reason: Smad3b activates transcription of organizer and Smad3 target genes; it is inside the node with no loss of functional residues. Supporting Evidence: PMID:29196619 an upregulation in Smad3 target gene expression in caSmad3a and caSmad3b hearts |
| GO:0060395 SMAD protein signal transduction | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference at the SMAD family node. Reason: Core process for an R-SMAD. |
| GO:0070411 I-SMAD binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference that SMADs bind inhibitory SMADs (SMAD6/7). Reason: MH2 is 97.9% identical to human SMAD3; zebrafish smad7 is regulated by Smad3. No smad3b-specific loss is known. Supporting Evidence: PMID:12112463 Furthermore, we show that Smad3 and BMP signals activate smad7. |
| GO:0071144 heteromeric SMAD protein complex | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference that R-SMADs form heteromeric complexes with SMAD4. Reason: Conserved mechanism; smad3b keeps the MH2 domain and C-terminal SSVS motif. No smad3b-specific data. |
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Download this section (compressed HTML)Q: Do smad3b single mutants have any phenotype, and is smad3a upregulated in smad3b PTC mutants (transcriptional adaptation)?
Q: Does the S418N substitution or the diverged linker change Smad3b activity or its regulation by CK1/MAPK/CDK phosphorylation?
Q: Is the brain and spinal cord expression of smad3b (Pogoda and Meyer 2002) shared by smad3a, which has not been surveyed at the same resolution?
Experiment: Equal-dose SBE-reporter and target-gene assays of Smad3a, Smad3b, Smad3b N418S and linker-swapped chimeras in smad3a;smad3b double mutant embryos.
Hypothesis: Smad3b is transcriptionally weaker than Smad3a because of linker divergence.
Type: cross-rescue
Experiment: Double fluorescent in situ or single-cell RNA-seq comparison of smad3a and smad3b across embryonic and adult tissues.
Hypothesis: The two copies overlap in expression at cellular resolution.
Type: expression analysis
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