NBDY (NoBody, non-annotated P-body dissociating polypeptide) is a 68-amino-acid, intrinsically disordered microprotein translated from a small open reading frame in the LINC01420 transcript, previously annotated as a long non-coding RNA. It is conserved among mammals but has no homology to larger proteins or known domains. NBDY binds directly to the enhancer of decapping EDC4, through a short motif (residues 22-41, with Leu30 and Trp34 essential) that contacts the EDC4 N-terminal WD40 region and, more weakly, its C-terminal self-association domain; its C-terminal proline-rich segment also binds the EVH1 domain of DCP1A. Through these contacts it associates with the cytoplasmic mRNA decapping complex and localizes to processing bodies (P-bodies). NBDY acts as a negative regulator of P-body assembly: raising its level disperses P-bodies, and its loss increases P-body number. Its phosphorylation at Thr40 during mitosis (downstream of CDK activity) and at Ser61 after EGF receptor signalling (by protein kinase C) is required for P-body dissolution under those conditions, and the unphosphorylated protein can form liquid condensates with RNA in vitro that phosphorylation dissolves. NBDY does not change the catalytic activity of the decapping complex in vitro, but its loss alters the half-lives of more than 1,400 transcripts. DCP2 substrates are stabilized or destabilized depending on their 5' UTRs, which requires the EDC4 interaction; other transcripts change through secondary effects on gene expression.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000932 P-body | IDA PMID:27918561 A human microprotein that interacts with the mRNA decapping ... | ACCEPT | Summary: FLAG-NoBody expressed at low levels co-localizes with the P-body marker EDC4, and endogenous NBDY was later shown to co-localize with P-bodies using a specific antibody (PMID:33059440). Reason: Direct imaging evidence, confirmed for the endogenous protein. The P-body is where NBDY's EDC4 partner and the decapping machinery concentrate, and where NBDY acts. This is the most specific appropriate cellular component. Supporting Evidence: PMID:27918561 Indeed, lentiviral transfection of FLAG-NoBody with low viral titers resulted in a population of cells (~10% of total) where NoBody co-localized with the P-body marker EDC4 (Fig. 4a). PMID:33059440 Endogenous NBDY co-purifies with components of the RNA decapping complex (Figure 1b) and colocalizes with P-bodies (Figure 2a and Figure S1a), as previously reported for epitope-tagged NBDY3. |
| GO:0000932 P-body | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping from the UniProt subcellular-location annotation "Cytoplasm, P-body", which is itself based on PMID:27918561. Reason: Consistent with, and redundant to, the experimental P-body annotation above. The underlying location is well supported for both tagged and endogenous NBDY. Supporting Evidence: PMID:27918561 This suggests that NoBody localizes to P-bodies at low expression levels. |
| GO:0000956 nuclear-transcribed mRNA catabolic process | IDA PMID:27918561 A human microprotein that interacts with the mRNA decapping ... | MODIFY | Summary: In PMID:27918561 the basis is a change in the steady-state level of one nonsense-mediated decay substrate (mutant p53 mRNA in Calu-6 cells) when NBDY levels change. NBDY knockdown decreased this mRNA and weak overexpression increased it. The authors say the decay rate was not measured, and they read the direction of the effect as negative regulation of decay. Later genome-wide work (PMID:33059440) showed that NBDY loss changes RNA half-lives in both directions. It also showed that NBDY does not affect the catalytic activity of the decapping complex. Reason: NBDY does not do any of the work of mRNA breakdown. It has no catalytic activity, and it does not change the decapping activity of the complex it binds in vitro (PMID:33059440). Its tethered-decay function is also dispensable: NBDY 22-41 disrupts P-bodies without altering tethered mRNA decay (PMID:40360209). The original evidence is a steady-state change, not a measured decay rate, and it points toward inhibition. The effect NBDY does have is regulatory and bidirectional: loss of NBDY stabilizes about 1,200 transcripts and destabilizes about 226, and the effect on DCP2 targets depends on the EDC4 interaction. GO:0043488 regulation of mRNA stability explicitly covers processes that both stabilize and destabilize mRNAs, so it describes NBDY's role accurately. Direct participation in the catabolic process does not. Proposed replacements: regulation of mRNA stability Supporting Evidence: PMID:27918561 These results demonstrate that the cellular concentration of NoBody is inversely linked to steady-state abundance of an NMD substrate in Calu-6 cells, though the effect of NoBody on the decay rate of this mRNA has not been established PMID:27918561 Our observation that loss of NoBody causes a decrease in the cellular levels of an NMD substrate is most consistent with negative regulation of mRNA decay in cells by NoBody PMID:33059440 NBDY KO dysregulates >1400 RNA half-lives, with 1200 cellular RNAs post-transcriptionally stabilized and 226 destabilized relative to WT PMID:33059440 We concluded that NBDY does not affect the in vitro enzymatic activity of the decapping complex PMID:40360209 However, despite its impact on P-body formation, NBDY 22–41 did not affect mRNA degradation under SMG7-induced conditions |
| GO:0005515 protein binding | IPI PMID:27918561 A human microprotein that interacts with the mRNA decapping ... | REMOVE | Summary: Binding to EDC4 (Q6P2E9). This is supported by co-immunoprecipitation, GST pull-down with purified proteins, and benzophenone photo-crosslinking, and is mapped to NBDY residues 22-41 (Leu30 and Trp34). GOA carries this row twice, from UniProt and IntAct. Reason: Bare protein binding carries no functional information, so the row is removed rather than rewritten. Removal does not mean the interaction is false. The interaction is direct, specific and functionally required: the EDC4-binding mutant W34A fails to rescue P-body number or decay rates of DCP2 substrates (PMID:33059440). But GO has no MF term for a non-catalytic protein that binds an enhancer of decapping and interferes with its self-association (PMID:40360209). The biology is captured by the P-body assembly regulation and mRNA stability process annotations. Supporting Evidence: PMID:27918561 Combined, these in-cell and in vitro experiments are consistent with a direct interaction between NoBody and EDC4, though we cannot exclude additional interactions with other proteins. PMID:27918561 Immunoprecipitation of FLAG-NoBody alanine mutants revealed two key residues, Leu30 and Trp34, that impair co-precipitation of NoBody with myc-EDC4 (Fig. 3d). PMID:36877681 The results confirm that the N-terminus of EDC4 is required for interaction with NBDY inside the cell. |
| GO:0005515 protein binding | IPI PMID:27918561 A human microprotein that interacts with the mRNA decapping ... | REMOVE | Summary: Binding to DCP1A (Q9NPI6), shown by reciprocal co-immunoprecipitation (IntAct curation). Later work mapped this contact to the DCP1A EVH1 domain and the NBDY C-terminal polyproline motif. Reason: Bare protein binding carries no functional information, so the row is removed. Removal does not mean the interaction is false. In the original study it may be at least partly indirect through EDC4, since depleting EDC4 abolished co-purification of the other decapping factors. It is also dispensable for NBDY's effect on DCP2-substrate decay (PMID:33059440), so no more informative MF term is supportable. Supporting Evidence: PMID:27918561 Reciprocal co-immunoprecipitation of FLAG-NoBody with myc-tagged EDC4 and Dcp1A confirmed its co-purification with these proteins (Fig. 2c, Supplementary Fig. 5). PMID:33059440 Independently, the DCP1A EVH1 domain interacts with the C-terminal polyproline repeats of NBDY (Figure S2), as expected based on the binding specificity of EVH1 domains30, though deletion of the polyproline motif does not affect RNA stability and therefore may not be necessary for NBDY function |
| GO:0010607 negative regulation of cytoplasmic mRNA processing body assembly | IDA PMID:27918561 A human microprotein that interacts with the mRNA decapping ... | ACCEPT | Summary: Raising NBDY levels disperses P-bodies without degrading their components, and this depends on the EDC4-binding region. siRNA knockdown increases P-bodies 1.5- to 2-fold. CRISPR knockout triples P-bodies, and re-expression rescues this (PMID:33059440). Phosphorylation of NBDY at Thr40 or Ser61 is required for P-body loss during mitosis and after EGF stimulation (PMID:34346674). NBDY 22-41 blocks EDC4-EDC4 self-association, which gives a mechanism (PMID:40360209). Reason: This is NBDY's best-supported and defining biological role. It is shown by gain- and loss-of-function, rescue, and point mutants in the EDC4-binding motif and at the phosphosites. A proposed mechanism, competition with EDC4 self-association, shows that NBDY acts directly on the assembly process. Core annotation. Supporting Evidence: PMID:27918561 P-body dispersal requires residues 31–41 of NoBody, indicating that the interaction between NoBody and EDC4 is involved (Fig. 4c). PMID:27918561 We observed a 1.5- to 2-fold increase in P-bodies per cell when NoBody was silenced (Fig. 4d,e). PMID:33059440 Consistent with previously reported data using siRNA3, endogenous P-body numbers increase by 3-fold in NBDY KO cells, which can be rescued by stably reintroducing the NBDY coding sequence on the KO background PMID:34346674 Expression of the nonphosphorylatable NBDY S61A mutant in the NBDY KO background abrogated EGF-dependent P-body disappearance PMID:40360209 A dose-dependent reduction in EDC4-EDC4 self-interaction was observed as NBDY 22–41 concentration increased (Fig. 3B), indicating that NBDY 22–41 effectively competes with EDC4, thereby disrupting its self-association. |
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Download this section (compressed HTML)Q: Should NBDY be annotated as part of the RNA decapping complex (GO:0098745)? It co-purifies with and binds EDC4 and DCP1A directly, but it does not change the complex's in vitro activity, and it may be a regulatory binder rather than a stoichiometric subunit.
Q: How does one EDC4-binding microprotein both stabilize and destabilize different DCP2 substrates, and is the 5' UTR-length dependence a direct effect on substrate access to DCP2?
Q: Is the in vitro NBDY-RNA coacervation (critical concentration about 150 uM) relevant at endogenous NBDY concentrations, or is phospho-regulation of P-bodies mediated entirely through EDC4?
Experiment: Measure decapping of specific DCP2 substrates (e.g. EPC2, GJC1) in vitro with purified decapping complex, with and without recombinant NBDY and full-length capped transcripts carrying native 5' UTRs.
Type: in vitro biochemistry
Experiment: Determine the stoichiometry of endogenous NBDY relative to EDC4 and DCP2 by quantitative proteomics, and test whether phospho-mutants (T40A, S61A) change EDC4 self-association in cells.
Type: quantitative proteomics / co-immunoprecipitation
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