TINCR encodes an 87-amino-acid ubiquitin-like microprotein (also called TUBL or pTINCR) translated from the main open reading frame of the TINCR transcript on chromosome 19, which was long classified as the long noncoding RNA "terminal differentiation-induced ncRNA". The protein is a single beta-grasp ubiquitin-like domain with a C-terminal SUMO-interacting motif; it is conserved in mammals, has been detected in the cornified stratum corneum and in other epithelia, and is induced by keratinocyte differentiation and by UV-induced DNA damage in a TP53-dependent manner. Endogenous protein is found in the nucleus and at cell-cell junctions, and overexpressed protein is also cytoplasmic. It binds SUMO1 and SUMO2/3 non-covalently through its SIM and associates with the Rho GTPase CDC42, increasing CDC42 SUMOylation and GTP loading, which promotes actin remodelling, junction formation and epithelial (keratinocyte) differentiation. Loss of the protein delays skin wound closure in mice and accelerates UVB-induced squamous cell carcinoma, and its expression suppresses growth of squamous carcinoma cells; in normal keratinocytes it has instead been reported to promote cell-cycle progression.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | EXP PMID:36369429 pTINCR microprotein promotes epithelial differentiation and ... | ACCEPT | Summary: Endogenous pTINCR detected by immunofluorescence mainly in the nucleus (and at cell-cell junctions) in HaCaT and MCF7 cells; nuclear/cytoplasmic fractionation was also performed. Reason: Direct observation of the endogenous protein in human epithelial cells. Nuclear function is not yet defined, but the location is well supported. Supporting Evidence: PMID:36369429 Endogenous pTINCR microprotein was detected in these cell lines localized mainly in the nucleus and at the cell-to-cell junctions |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping of the experimental nuclear localization (PMID:36369429). Reason: Mapping of a curated location that is supported by direct immunofluorescence of the endogenous protein. Supporting Evidence: PMID:36369429 Endogenous pTINCR microprotein was detected in these cell lines localized mainly in the nucleus and at the cell-to-cell junctions |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping of a cytoplasmic location (by similarity to mouse). Overexpressed FLAG-TUBL is diffusely cytoplasmic, Boix et al. also report a cytoplasmic pool, and tumour IHC is cytoplasmic. Reason: Consistent with several independent observations, although endogenous protein in HaCaT/MCF7 is mainly nuclear and junctional. Supporting Evidence: PMID:34351912 Immunofluorescence microscopic analysis revealed that FLAG epitope–tagged TUBL was diffusely distributed throughout the cytoplasm when expressed in HeLa cells PMID:36369429 in addition to its localization at cell-to-cell contacts and in the cytoplasm, pTINCR is also found in the nucleus |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | ACCEPT | Summary: Transfer from the mouse ortholog (A0A1B0GRQ3) based on Nita et al. (FLAG-TUBL immunofluorescence). Reason: The ortholog is highly conserved and the human protein itself has been reported in the cytoplasm (PMID:36369429). Supporting Evidence: PMID:34351912 Immunofluorescence microscopic analysis revealed that FLAG epitope–tagged TUBL was diffusely distributed throughout the cytoplasm when expressed in HeLa cells |
| GO:0010838 positive regulation of keratinocyte proliferation | IMP PMID:34351912 A ubiquitin-like protein encoded by the "noncoding" RNA TINC... | KEEP AS NON CORE | Summary: In normal human epidermal keratinocytes, siRNA depletion of TINCR (and with it TUBL) reduced S-phase cells and TUBL overexpression increased them; in mouse keratinocytes an ORF frameshift that preserves RNA structure abolished the pro-proliferative effect, and frameshift mice showed reduced keratinocyte proliferation during wound repair. Reason: The experiments are sound and protein-specific (frameshift controls), so the annotation stands. It is kept as non-core because the effect is context dependent and without a molecular mechanism: in transformed epithelial cells and squamous carcinoma the same protein decreases proliferation (PMID:36369429, PMID:36899004), and the best-characterised molecular activity of the protein (SUMO binding and CDC42 activation) is linked to differentiation rather than proliferation. Supporting Evidence: PMID:34351912 Expression of WT or SM forms of TINCR resulted in a marked increase in the percentage of cells in S phase of the cell cycle, whereas expression of WT_del or SM_del forms had no such effect PMID:34351912 In addition, we found that the proportion of cells in S phase of the cell cycle was markedly decreased for NHEKs transfected with the siRNAs targeting both TINCR and TUBL compared with those transfected with control siRNAs PMID:36369429 In addition, pTINCR reinforces cell-to-cell junctions and decreases proliferation. |
| GO:0070161 anchoring junction | EXP PMID:36369429 pTINCR microprotein promotes epithelial differentiation and ... | ACCEPT | Summary: Endogenous pTINCR is found at cell-cell junctions in HaCaT and MCF7 cells, and its overexpression promotes adherens and tight junction formation. Reason: The junctional staining is direct. The paper does not show colocalisation with a specific junction type, so the general anchoring junction term is an appropriate level. Supporting Evidence: PMID:36369429 Endogenous pTINCR microprotein was detected in these cell lines localized mainly in the nucleus and at the cell-to-cell junctions |
| GO:0070161 anchoring junction | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (Cell junction) of the experimental observation in PMID:36369429. Reason: Mapping of a curated, experimentally supported location. Supporting Evidence: PMID:36369429 Endogenous pTINCR microprotein was detected in these cell lines localized mainly in the nucleus and at the cell-to-cell junctions |
| GO:0090303 positive regulation of wound healing | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Transfer from mouse, where ORF-frameshift (TUBL-deficient) mice show delayed skin wound closure associated with reduced keratinocyte proliferation. Reason: The mouse phenotype is protein-specific and the ortholog is conserved, so the transfer is reasonable. It is a tissue-level outcome downstream of the keratinocyte effects, the delay is transient (days 4-10), and there is no human wound-healing evidence, so it is non-core. Supporting Evidence: PMID:34351912 Tubl–/–mice manifested delayed wound closure from days 4 to 10 postinjury compared with WT control animals |
| GO:0032183 SUMO binding | IDA PMID:36369429 pTINCR microprotein promotes epithelial differentiation and ... | NEW | Summary: GST pull-down shows that pTINCR binds SUMO1 and SUMO2/3 non-covalently, and the binding is lost when the SUMO-interacting motif (VLLL79-82AAAA) is mutated. The SIM mutant is less stable, does not activate CDC42 and does not reproduce pTINCR's cellular effects. Reason: Direct, motif-dependent binding of the human protein, and the motif is required for its function, so this is the most specific molecular activity that can be stated. It is an intrinsic activity of TINCR, not inferred from a complex. No existing annotation covers a molecular function. Supporting Evidence: PMID:36369429 GST-pull down assays confirmed that pTINCR binds to SUMO1 and SUMO2/3 in a non-covalent manner and that the interaction is lost in the pTINCR-SIMmut PMID:36369429 Interestingly, mutating the pTINCR SIM-domain or treating with a SUMOylation inhibitor impair the ability of pTINCR to activate CDC42 |
| GO:0030858 positive regulation of epithelial cell differentiation | IMP PMID:36369429 pTINCR microprotein promotes epithelial differentiation and ... | NEW | Summary: Start-codon knockout of pTINCR (lncRNA preserved) in HaCaT keratinocytes and MCF7 cells blocks calcium- and DNA-damage-induced differentiation, partially rescued by a synthetic ORF; overexpression induces differentiation markers, dependent on CDC42. Reason: Participation test: this is a regulation term, and pTINCR is itself the regulator, acting through binding SUMO and CDC42 and increasing CDC42 activation; the effect was separated from the RNA with a start-codon mutant and a recoded ORF. A keratinocyte-specific child (GO:0045618) was not chosen because MCF7 mammary cells were also used. Note that Nita et al. (PMID:34351912) saw no differentiation effect with two of three TINCR siRNAs in NHEKs, so the finding comes from one group. Supporting Evidence: PMID:36369429 By contrast, pTINCR-KO cells failed to activate CDC42 and to differentiate (measured by the expression of INVOLUCRIN) upon exposure to differentiation conditions, despite having a functional TINCR lncRNA PMID:36369429 Remarkably, this effect was partially reverted upon pTINCR re-expression in pTINCR-KO cells (pTINCR-KO + OE), demonstrating that it is pTINCR microprotein and not the lncRNA that is responsible for CDC42 activation during differentiation. |
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Download this section (compressed HTML)Q: How does a non-catalytic SUMO-binding ubiquitin-like protein increase CDC42 SUMOylation? Does it recruit a SUMO E2/E3 to CDC42, protect SUMO-CDC42 from SENPs, or act as a SUMO-loaded adaptor? The answer decides whether an adaptor or enzyme-regulator MF term is appropriate.
Q: Why does the protein promote proliferation in normal keratinocytes (PMID:34351912) but suppress proliferation in transformed epithelial and squamous carcinoma cells (PMID:36369429, PMID:36899004)?
Q: Is the CDC42 interaction direct? Only co-immunoprecipitation has been shown, so a small GTPase binding MF is not yet asserted.
Experiment: Binding assays with purified pTINCR (wild type and SIM mutant), SUMO2 and CDC42 in GDP and GTP states, plus in vitro SUMOylation of CDC42 with and without pTINCR.
Hypothesis: pTINCR binds CDC42 directly and SUMO-dependently.
Experiment: Compare start-codon knockout and SIM mutant knock-in in primary human keratinocytes, measuring cell-cycle progression, CDC42-GTP and differentiation markers in the same cells.
Hypothesis: The differentiation and proliferation phenotypes reflect one pathway acting in different cell states.
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