smad3a (SMAD family member 3a) is one of two zebrafish co-orthologs of vertebrate SMAD3, retained from the teleost-specific genome duplication; its paralog is smad3b. It encodes a receptor-regulated SMAD (R-SMAD) with an N-terminal MH1 DNA-binding domain, a linker carrying regulatory phosphosites and a C-terminal MH2 domain ending in the SSVS motif that TGF-beta/activin/nodal type I receptors phosphorylate. On activation it forms a heteromeric complex with SMAD4, enters the nucleus and binds SMAD-binding elements to regulate transcription of target genes. smad3a is the more conserved of the two copies (97% identical to human SMAD3). Its mRNA is supplied maternally and is broadly expressed, with higher levels in paraxial mesoderm and newly formed somites. Unlike smad3b, it is expressed with a circadian rhythm and is a direct target of Clock1a. With smad2 and smad3b it mediates nodal/TGF-beta signals in mesendoderm induction. Overexpressed smad3a activates the myf5 promoter together with Smad4, and constitutively active Smad3a promotes cardiomyocyte proliferation. 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 more precisely by the IBA to GO:0000978. Reason: The MH1 domain and its DNA-contacting residues are conserved (smad3a-bioinformatics), and Smad3a occupies SMAD-binding elements of the myf5 promoter in vivo. The term is correct though less specific than GO:0000978. Supporting Evidence: PMID:21159776 However, two PCR products with 218 bp containing SBE1 and SBE2 were found in embryos injected with either pHis-Smad3a or pHis-Smad4 |
| 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 (SMAD-binding elements). Reason: The node is sound and smad3a sits within it with all MH1 DNA-binding and Zn-coordinating residues conserved. In vivo ChIP places Smad3a on SBEs of the myf5 promoter. Supporting Evidence: PMID:21159776 However, two PCR products with 218 bp containing SBE1 and SBE2 were found in embryos injected with either pHis-Smad3a or pHis-Smad4 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: This is the core molecular function of an R-SMAD. smad3a overexpression activates the myf5 promoter in zebrafish embryos, and a Smad3-responsive reporter is activated by both zebrafish Smad3 paralogs. Supporting Evidence: PMID:21159776 Using a luciferase assay to detect myf5 promoter activity when smad2 , smad3a , smad3b , or smad4 mRNAs were overexpressed, we found that excessive smad2 and smad3a showed enhanced myf5 promoter activity but not excessive smad3b or smad4 mRNA. PMID:25286120 Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. |
| GO:0000981 DNA-binding transcription factor activity, RNA polymerase II-specific | TAS PMID:28887217 Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm... | ACCEPT | Summary: ZFIN TAS from the Eaf1/2 paper, which describes Smad-containing TGF-beta transcriptional complexes repressed by Eaf1/2. Reason: The activity is correct for smad3a; the contributes_to qualifier reflects that R-SMADs act in heteromeric complexes with SMAD4 and cofactors. The cached paper is abstract-only, so the curator's reading is deferred to; the function itself is well supported by other data. Supporting Evidence: PMID:28887217 Furthermore, Eaf1/2 might co-localize and interact with TGF-Ξ² transcriptional factors in the transcriptional complex as repressors to target and suppress TGF-Ξ² signaling activity. |
| GO:0001714 endodermal cell fate specification | IMP PMID:28887217 Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm... | KEEP AS NON CORE | Summary: ZFIN IMP from the Eaf1/2 study of mesoderm and endoderm formation. Reason: Nodal-Smad2/3 signalling specifies endoderm in zebrafish, and dominant-negative Smad3a blocks mesendoderm induction (PMID:18025082). The cached paper is abstract-only, and the loss-of-function reagent recorded for the sibling rows is an eaf1 morpholino, so which smad3a manipulation underlies this row cannot be checked. Deferring to the curator, but this is a developmental output of the signalling core, shared with smad2 and smad3b, and so non-core. Supporting Evidence: PMID:18025082 Thus, our data reveal that Nodal signaling and mesendoderm induction depend on Smad2/3 |
| 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 does not describe an activity of Smad3a beyond being bound and inhibited, so there is no evidence-backed, more informative replacement term. 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 enter the nucleus with SMAD4 to bind DNA; Smad3a occupies myf5 promoter elements in vivo. Supporting Evidence: PMID:21159776 However, two PCR products with 218 bp containing SBE1 and SBE2 were found in embryos injected with either pHis-Smad3a or pHis-Smad4 |
| GO:0005667 transcription regulator complex | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the MH1 domain. Reason: R-SMADs act in heteromeric transcription regulator complexes with SMAD4 and DNA-binding cofactors. The more specific heteromeric SMAD protein complex (IBA) is the better description. |
| GO:0005667 transcription regulator complex | TAS PMID:28887217 Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm... | ACCEPT | Summary: ZFIN TAS from the Eaf1/2 paper, which proposes that Eaf1/2 act inside TGF-beta transcriptional complexes. Reason: Consistent with the conserved R-SMAD mechanism. The paper is abstract-only here; the location is correct for smad3a regardless. Supporting Evidence: PMID:28887217 Furthermore, Eaf1/2 might co-localize and interact with TGF-Ξ² transcriptional factors in the transcriptional complex as repressors to target and suppress TGF-Ξ² signaling activity. |
| 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; the zebrafish study of myf5 regulation also describes cytoplasmic Smad2-Smad3a without Smad4. Supporting Evidence: PMID:21159776 Although there is some active Smad2Β·Smad3a in cytoplasm, Smad2Β·Smad3a is unable to enter the nucleus to activate the myf5 promoter without the assistance of Smad4 protein. |
| 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:0006355 regulation of DNA-templated transcription | IPI PMID:21159776 dickkopf-3-related gene regulates the expression of zebrafis... | ACCEPT | Summary: From the Dkk3r/myf5 study, where Smad3a with Smad4 activates the myf5 promoter. Reason: Correct. The data show activation of an RNA polymerase II promoter (myf5) by Smad3a with Smad4, which the IBA row to GO:0045944 (positive regulation of transcription by RNA polymerase II) captures more specifically; kept at this level for consistency with the InterPro row to the same term. Supporting Evidence: PMID:21159776 Although the ability of Smad2 and Smad3a to activate the myf5 promoter requires Smad4 to form a complex and enter the nucleus, Smad3b only plays a minor role in myf5 promoter activation. |
| GO:0007179 transforming growth factor beta receptor signaling pathway | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference at the SMAD2/3 node. smad3a itself (ZFIN:ZDB-GENE-000509-3) is among the descendant evidence, reflecting its own experimental annotation. Reason: Core process of an R-SMAD. The receptor-phosphorylated SSVS motif is conserved, and a Smad3-responsive reporter that responds to both paralogs is repressed by ALK4/ALK5 inhibitors. Supporting Evidence: PMID:25286120 Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. |
| GO:0007179 transforming growth factor beta receptor signaling pathway | IMP PMID:28887217 Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm... | ACCEPT | Summary: ZFIN IMP from the Eaf1/2 study using TGF-beta reporters. Reason: The paper is abstract-only here, so the smad3a manipulation cannot be seen. The process is the core of smad3a function and is independently supported (reporter, dominant-negative and constitutively active Smad3a data). Deferring to the curator. Supporting Evidence: PMID:25286120 Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. |
| GO:0007179 transforming growth factor beta receptor signaling pathway | IMP PMID:28887217 Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm... | ACCEPT | Summary: Same finding as the previous row; the WITH field records the eaf1 morpholino (ZDB-MRPHLNO-090820-4, MO1-eaf1 on ZFIN). Reason: The process is correct for smad3a; the genotype details are in the full text, which is not cached. Deferring to the curator. |
| GO:0007498 mesoderm development | IGI PMID:28687631 Clock1a affects mesoderm development and primitive hematopoi... | KEEP AS NON CORE | Summary: smad3a mRNA restores mesoderm marker expression reduced by clock1a knockdown. Reason: The genetic interaction is shown (rescue of clock1a morphants by smad3a mRNA), and Smad2/3 activity is required for mesendoderm induction. It is a developmental outcome of the signalling function, shared with smad2 and smad3b. Supporting Evidence: PMID:28687631 These effects were largely compromised by co-injection of smad3a- mRNA. PMID:18025082 Thus, our data reveal that Nodal signaling and mesendoderm induction depend on Smad2/3 |
| GO:0007501 mesodermal cell fate specification | IMP PMID:28887217 Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm... | KEEP AS NON CORE | Summary: ZFIN IMP from the Eaf1/2 study. Reason: Supported in principle by the Smad2/3 requirement for mesendoderm induction (PMID:18025082). The cached paper is abstract-only; deferring to the curator. Non-core developmental outcome. Supporting Evidence: PMID:18025082 Smad2/3-deficient cells transplanted into the blastodermal margin of wild-type hosts preferentially differentiated into ectodermal tissues rather than mesendodermal tissues, supporting the idea that response of cells to mesendoderm inducers requires Smad2/3 activities. |
| GO:0007501 mesodermal cell fate specification | IMP PMID:28887217 Transcriptional factors Eaf1/2 inhibit endoderm and mesoderm... | KEEP AS NON CORE | Summary: Same finding as the previous row, recorded with the eaf1 morpholino in WITH. Reason: As for the previous row. |
| GO:0009653 anatomical structure morphogenesis | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Very broad family-level PAINT process. Reason: SMAD signalling shapes many structures, so the term is not wrong, but it is uninformative and describes downstream outcomes rather than what smad3a does. |
| GO:0009880 embryonic pattern specification | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA transfer of embryonic patterning. Reason: Smad2/3 activity is needed for mesendoderm induction and neuroectoderm patterning in zebrafish (shown with dominant-negative constructs that block Smad2/3 generally). Plausible but non-core. Supporting Evidence: 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 the zebrafish embryo is driven mainly by BMP-SMAD1/5 signalling. No smad3a loss-of-function evidence shows a dorsal/ventral patterning defect. The organizer-gene induction reported for smad3b comes from overexpression. The ARBA rule is too broad for this R-SMAD. |
| GO:0030154 cell differentiation | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Broad family-level PAINT process. Reason: Correct in general (e.g. smad3a 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 the receptor-regulated SMAD of the TGF-beta/activin/nodal branch; its C-terminal SSVS motif is phosphorylated by TGF-beta type I receptors, and in zebrafish the Smad3 reporter is blocked by ALK4/ALK5 inhibitors. BMP signals regulate smad7 alongside Smad3 but that does not place Smad3a in the BMP pathway. No zebrafish evidence shows BMP receptors signalling through 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; smad3a is inside the node with conserved receptor-phosphorylation motif. |
| GO:0038092 nodal signaling pathway | TAS PMID:28687631 Clock1a affects mesoderm development and primitive hematopoi... | ACCEPT | Summary: ZFIN TAS from the Clock1a paper, which frames smad3a as the nodal effector in early mesoderm induction. Reason: Supported by the Clock1a study and by dominant-negative Smad3a blocking nodal-induced mesendoderm. Supporting Evidence: PMID:28687631 Here we show that Clock1a coordinated mesoderm development and primitive hematopoiesis in zebrafish embryos by directly up-regulating Nodal-Smad3 signaling. PMID:18025082 Overexpression of these mutants abolished mesendoderm induction by ectopic Nodal signaling in zebrafish embryos. |
| GO:0043565 sequence-specific DNA binding | IPI PMID:21159776 dickkopf-3-related gene regulates the expression of zebrafis... | ACCEPT | Summary: In vivo ChIP places His-tagged Smad3a on two SMAD-binding elements of the myf5 upstream region. Reason: Direct in vivo evidence of sequence-specific DNA binding by smad3a; the IBA to GO:0000978 captures the same function more specifically. Supporting Evidence: PMID:21159776 However, two PCR products with 218 bp containing SBE1 and SBE2 were found in embryos injected with either pHis-Smad3a or pHis-Smad4 |
| GO:0045944 positive regulation of transcription by RNA polymerase II | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference at the SMAD2/3 node. Reason: smad3a activates the myf5 promoter in embryos. Supporting Evidence: PMID:21159776 Using a luciferase assay to detect myf5 promoter activity when smad2 , smad3a , smad3b , or smad4 mRNAs were overexpressed, we found that excessive smad2 and smad3a showed enhanced myf5 promoter activity but not excessive smad3b or smad4 mRNA. |
| GO:0060215 primitive hemopoiesis | IGI PMID:28687631 Clock1a affects mesoderm development and primitive hematopoi... | KEEP AS NON CORE | Summary: smad3a mRNA restores scl expression reduced by clock1a knockdown. Reason: The genetic interaction is shown, and the acts_upstream_of_or_within qualifier correctly limits the claim. Primitive hemopoiesis follows from nodal-dependent mesoderm induction, so this is an indirect, non-core outcome. Supporting Evidence: PMID:28687631 These effects were largely compromised by co-injection of smad3a- mRNA. |
| 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: Binding of SMAD7 to R-SMADs is a conserved feedback mechanism, and zebrafish smad7 is itself a Smad3 target. No residue loss in the MH2 domain of smad3a (99.5% identical to human over MH2). 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: In zebrafish, Smad3a needs Smad4 to form a complex and enter the nucleus to activate myf5. Supporting Evidence: PMID:21159776 In addition, Smad2Β·Smad3a required the recruitment of Smad4 to enter the nucleus. |
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Download this section (compressed HTML)Q: Do smad3a single mutants have any phenotype, and is smad3b upregulated in smad3a PTC mutants (transcriptional adaptation)?
Q: Does the circadian, Clock1a-driven expression of smad3a (absent from smad3b) have a physiological consequence that smad3b cannot supply?
Q: Is the difference in myf5 promoter activation between Smad3a and Smad3b a real protein difference, e.g. linked to the diverged smad3b linker?
Experiment: Compare ventral aorta diameter in smad3a-/-, smad3b-/-, smad3a-/-;smad3b+/- and double mutants, including an RNA-less smad3a allele to exclude transcriptional adaptation.
Hypothesis: smad3a and smad3b are redundant for the ventral aorta phenotype.
Type: genetic interaction analysis
Experiment: Side-by-side SBE-reporter and target-gene assays of equal doses of Smad3a, Smad3b and a Smad3b linker-swap (smad3a linker) in MZ smad3a;smad3b embryos.
Hypothesis: Smad3a and Smad3b have equivalent transcriptional activity.
Type: cross-rescue
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