TUNAR encodes pTUNAR (also called BNLN, beta cell and neural cell-regulin), a 48-amino-acid microprotein translated from a transcript long annotated as the long non-coding RNA TUNAR (TUNA, LINC00617; megamind in zebrafish). The peptide is highly conserved across vertebrates and carries a single C-terminal transmembrane helix that anchors it in the endoplasmic reticulum membrane. There it binds sarco/endoplasmic reticulum Ca2+-ATPases: SERCA3 (ATP2A3) in pancreatic beta cells, where the interaction increases at high glucose, and SERCA2 (ATP2A2) in neural cells. Its sequence lacks the xLFxxF motif of the regulin family (phospholamban, sarcolipin, myoregulin, endoregulin) and is unrelated to DWORF, so it appears to be an independently evolved SERCA-binding micropeptide. Altering its level changes ER and cytosolic Ca2+ handling, but the direction differs by cell type. In glucose-stimulated beta cells, overexpression lowers ER Ca2+, raises cytosolic Ca2+, reduces ER stress signalling and enhances glucose-stimulated insulin secretion. In mouse neural cells, overexpression enlarges the ER Ca2+ store and speeds cytosolic Ca2+ clearance, consistent with SERCA2 activation. The mRNA is enriched in insulin-producing beta cells of the islet and in the central nervous system. Loss of the peptide in mouse embryonic stem cells promotes neural differentiation, and its overexpression reduces neurite formation, so pTUNAR acts as a Ca2+-dependent brake on neural differentiation. Whether it activates or inhibits SERCA pump activity directly has not been measured.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005783 endoplasmic reticulum | IDA PMID:34513312 A putative long noncoding RNA-encoded micropeptide maintains... | ACCEPT | Summary: Tagged human BNLN/pTUNAR localizes mainly to the ER in INS-1 beta cells, co-staining with KDEL-RFP. Reason: Direct imaging evidence; the protein is a single-pass membrane protein of the ER. ER membrane (GO:0005789) is the more specific location and is also annotated. Supporting Evidence: PMID:34513312 these data show that BNLN is primarily located at the ER of pancreatic Ξ² cells |
| GO:0005783 endoplasmic reticulum | ISS GO_REF:0000024 | ACCEPT | Summary: Transfer from mouse pTUNAR (A0A1B0GQX2), which co-localizes with SERCA2 in the ER in brain and NIH3T3 cells. Reason: Consistent with the direct human-protein data (PMID:34513312) and with the conserved C-terminal transmembrane helix. Supporting Evidence: PMID:35036403 pTUNAR is a transmembrane protein that localizes in the endoplasmic reticulum |
| GO:0005789 endoplasmic reticulum membrane | EXP PMID:34513312 A putative long noncoding RNA-encoded micropeptide maintains... | ACCEPT | Summary: ER membrane location of the single-pass transmembrane micropeptide, based on ER co-localization of tagged BNLN and its conserved C-terminal TM domain. Reason: Correct and the most specific supported location; core location. Supporting Evidence: PMID:34513312 BNLN harbors a single-pass transmembrane domain at its C terminus PMID:34513312 these data show that BNLN is primarily located at the ER of pancreatic Ξ² cells |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic mapping from UniProt subcellular location (Endoplasmic reticulum membrane), itself grounded in PMID:34513312. Reason: Correct; duplicates the experimental ER membrane annotation. |
| GO:0032469 endoplasmic reticulum calcium ion homeostasis | IDA PMID:34513312 A putative long noncoding RNA-encoded micropeptide maintains... | ACCEPT | Summary: Overexpression of BNLN lowered ER Ca2+ (ER-GCaMP6-150 biosensor) in glucose-stimulated INS-1 cells; BNLN binds SERCA3. Reason: The ER Ca2+ effect is measured directly and the peptide sits in the ER membrane bound to the ER Ca2+ pump, so it plausibly acts in, rather than merely upstream of, ER Ca2+ homeostasis. The mouse neuronal study (PMID:35036403) also finds altered ER Ca2+ store, although in the opposite direction, so the term (direction-neutral) is the appropriate level. Core process. Supporting Evidence: PMID:34513312 Overexpression of BNLN lowered [Ca2+]ER level in the high glucose (17 mM) condition PMID:35036403 pTUNAR overexpression increases the calcium stored in the ER and accelerates calcium clearance from the cytoplasm |
| GO:0035774 positive regulation of insulin secretion involved in cellular response to glucose stimulus | IDA PMID:34513312 A putative long noncoding RNA-encoded micropeptide maintains... | KEEP AS NON CORE | Summary: BNLN overexpression increased glucose-stimulated insulin secretion in INS-1 cells and in mouse and human islets; an ATG-deleted construct had no effect and Tunar shRNA knockdown reduced GSIS in mouse islets. Reason: Well-supported phenotype, and the ATG-deletion control ties it to the peptide rather than the RNA, but it is a downstream physiological consequence of altered beta-cell Ca2+ handling rather than something the peptide itself executes. Retained as non-core; the core role is at the level of ER Ca2+ homeostasis/SERCA binding. Supporting Evidence: PMID:34513312 To verify that this effect was mediated by the translated micropeptide and not the RNA transcript, we transfected INS-1 cells with the 2nd ATG-deletion BNLN construct (which does not produce the micropeptide) and found that this construct did not affect GSIS PMID:34513312 suppression of BNLN decreased insulin secretion compared to control |
| GO:0045665 negative regulation of neuron differentiation | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Transfer from mouse pTUNAR: start-codon knockout mESCs (lncRNA level unchanged) show enhanced neural differentiation in teratomas, embryoid bodies and neural differentiation protocols; overexpression impairs neuronal differentiation. Reason: Good loss- and gain-of-function evidence in mouse, with a knockout design that separates peptide from RNA. The effect is attributed to altered intracellular Ca2+ signalling, so it is a downstream developmental consequence rather than the core molecular role. The 48-aa sequence and TM domain are highly conserved, so transfer to human is reasonable. Supporting Evidence: PMID:35036403 pTUNAR deficiency in mouse embryonic stem cells improves their differentiation potential towards neural lineage both in vitro and in vivo |
| GO:0048812 neuron projection morphogenesis | ISS GO_REF:0000024 | MODIFY | Summary: Transfer from mouse pTUNAR, whose overexpression reduced the number of neurites in N2A cells and in cortical neurons in vivo. Reason: The source data show that pTUNAR restrains neurite formation; it does not show that pTUNAR carries out neuron projection morphogenesis. The observed effect is better captured by negative regulation of neuron projection development (GO:0010977). Evidence is overexpression-only, so the replacement should be regarded as non-core. Proposed replacements: negative regulation of neuron projection development Supporting Evidence: PMID:35036403 pTUNAR overexpression in different models of neural differentiation impairs the formation of neurites |
| GO:0051117 ATPase binding | IPI PMID:34513312 A putative long noncoding RNA-encoded micropeptide maintains... | ACCEPT | Summary: BNLN binds SERCA3 (ATP2A3, Q93084): identified by pull-down/mass spectrometry in INS-1 cells, confirmed by co-IP in HEK293 cells and by proximity ligation in INS-1 cells, with the interaction enhanced by high glucose. Reason: Multiple orthogonal interaction assays; together with the mouse SERCA2 co-IP (PMID:35036403) this establishes TUNAR as a SERCA-binding ER membrane micropeptide. A direction-specific regulator term (ATPase inhibitor activity GO:0042030, as for ERLN; or transporter activator activity GO:0141109, as for STRIT1/DWORF) is not supported because the two studies infer opposite effects (possible SERCA3 inhibition in beta cells, possible SERCA2 activation in neurons) and neither measured pump activity. ATPase binding is therefore the most informative defensible MF and is kept as core. Supporting Evidence: PMID:34513312 We further validated this interaction by co-immunoprecipitation (coIP)-western blot analysis in HEK293 cells with BNLN and SERCA3 overexpression PMID:34513312 whether this is primarily the result of reduction of SERCA3 enzyme activity requires further investigation |
| GO:0051117 ATPase binding | ISS GO_REF:0000024 | ACCEPT | Summary: Transfer from mouse pTUNAR, which co-immunoprecipitates with SERCA2 (ATP2A2) in NIH3T3 cells. Reason: Consistent with the direct human-peptide SERCA3 interaction data. Core MF. Supporting Evidence: PMID:35036403 by co-immunoprecipitation experiments we could demonstrate that pTUNAR and SERCA2 physically interact |
| GO:0051480 regulation of cytosolic calcium ion concentration | ISS GO_REF:0000024 | ACCEPT | Summary: Transfer from mouse pTUNAR, whose overexpression accelerated cytosolic Ca2+ clearance in ESC-derived neurons. Reason: Both studies show that the peptide changes cytosolic Ca2+ (reduced in neurons, increased in glucose-stimulated beta cells). The direction-neutral term fits this context-dependent behaviour and is a direct consequence of its action on SERCA-mediated ER Ca2+ uptake. Supporting Evidence: PMID:35036403 pTUNAR modulates intracellular calcium dynamics increasing the efficiency by which calcium is removed from the cytoplasm, probably through its interaction with SERCA2 PMID:34513312 Overexpression of BNLN increased [Ca2+]c in INS-1 cells under both basal and high-glucose conditions |
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Download this section (compressed HTML)Q: Does pTUNAR/BNLN change SERCA pump kinetics directly, and in which direction? Beta-cell data (SERCA3; lower ER Ca2+) suggest inhibition, whereas neural data (SERCA2; faster cytosolic Ca2+ clearance) suggest activation. The answer decides whether GO:0042030 ATPase inhibitor activity, GO:0141109 transporter activator activity, or only the direction-neutral GO:1901894 regulation of ATPase-coupled calcium transmembrane transporter activity should be added, in line with ERLN and STRIT1 (DWORF).
Q: Is the opposite sign of the Ca2+ effect explained by SERCA isoform (SERCA3 vs SERCA2b) or by competition with other SERCA regulins expressed in each cell type (e.g. endoregulin with SERCA3)?
Q: Is the extracellular-vesicle pool of pTUNAR (seen for the overexpressed mouse peptide) physiologically relevant, or a by-product of overexpression?
Experiment: Co-reconstitute purified synthetic pTUNAR with SERCA2b or SERCA3a in proteoliposomes, or co-express them in HEK293 microsomes, and measure Ca2+-dependent ATPase activity (Vmax and KCa), with phospholamban and DWORF as inhibitor and activator controls. Add FRET or crosslinking to test whether pTUNAR occupies the regulin groove.
Hypothesis: pTUNAR directly alters SERCA2b and SERCA3 Ca2+-ATPase kinetics.
Experiment: Mutate the pTUNAR start codon in mouse beta cells or human stem-cell-derived beta cells, leaving the lncRNA intact as in the mESC knockout, and measure ER and cytosolic Ca2+ oscillations, ER stress markers and glucose-stimulated insulin secretion.
Hypothesis: Endogenous pTUNAR is required for normal beta-cell Ca2+ oscillations and GSIS.
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