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. Author manuscript; available in PMC: 2022 Aug 18.
Published in final edited form as: J Am Chem Soc. 2021 Aug 4;143(32):12675–12687. doi: 10.1021/jacs.1c05386

Phosphorylation of a human microprotein promotes dissociation of biomolecular condensates

Zhenkun Na 1,2,$, Yang Luo 1,2,$, Danica S Cui 1, Alexandra Khitun 1,2, Stephanie Smelyansky 1,2,3, J Patrick Loria 1,3, Sarah A Slavoff 1,2,3,*
PMCID: PMC8564862  NIHMSID: NIHMS1748709  PMID: 34346674

Abstract

Proteogenomic identification of translated small open reading frames in human has revealed thousands of microproteins, or polypeptides of fewer than 100 amino acids, that were previously invisible to geneticists. Hundreds of microproteins have been shown to be essential for cell growth and proliferation, and many regulate macromolecular complexes. However, the vast majority of microproteins remain functionally uncharacterized, and many lack secondary structure and exhibit limited evolutionary conservation. One such intrinsically disordered microprotein is NBDY, a 68-amino acid component of membraneless organelles known as P-bodies. In this work, we show that NBDY can undergo liquid-liquid phase separation, a biophysical process thought to underlie the formation of membraneless organelles, in the presence of RNA in vitro. Phosphorylation of NBDY drives liquid phase remixing in vitro and macroscopic P-body dissociation in cells undergoing growth factor signaling and cell division. These results suggest that NBDY phosphorylation enables regulation of P-body dynamics during cell proliferation, and more broadly that intrinsically disordered microproteins may contribute to liquid-liquid phase separation and remixing behavior to affect cellular processes.

Graphical Abstract

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Introduction:

Small open reading frames (smORFs) encoding polypeptides of fewer than 100 amino acids have been largely excluded from eukaryotic genome annotations because they are challenging to differentiate from randomly-occurring, non-translated ORFs1. Recent advances in ribosome profiling and proteogenomics have enabled the discovery of thousands of expressed smORFs in the human genome2. A number of human “microproteins” encoded in smORFs (also referred to as small proteins or micropeptides3) have now been shown to have important cellular and biological functions4, but the vast majority remain uncharacterized. Indeed, whether microproteins are broadly functional - and how to even define the term function in the context of smORFs5 - remains in question. Multiple studies suggest that smORFs represent proto-genes, or instances of de novo gene birth, which have not yet been optimized for functionality by natural selection6–7. Supporting the proto-gene model, the products of short, evolutionarily young genes exhibit limited sequence homology to known protein domains8, and are enriched for amino acid compositions consistent with predicted intrinsic disorder9.

Intrinsically disordered protein regions can undergo weak, multivalent macromolecular interactions that drive liquid-liquid phase separation (LLPS) to form membraneless organelles10. We therefore propose that intrinsic disorder could be a feature, rather than a flaw, of microproteins, positioning them as a class of molecules that may collectively function in LLPS in cells. Supporting this hypothesis, several smORF-encoded microproteins have recently been shown to localize to the nucleolus11, cytoplasmic membraneless organelles12–13, and endoplasmic reticulum (ER) membrane contact sites14–15, all of which may represent phase separated regions16–18.

The NoBody (NBDY) microprotein localizes to cytoplasmic membraneless organelles called P-bodies via interaction with components of the mRNA decapping complex, which catalyzes the first committed step in 5′-to-3′ decay of deadenylated mRNAs12–13. P-bodies are conserved in all eukaryotes and have been reported to exhibit properties of phase-separated liquid droplets18. While 5′-to-3′ mRNA decay does not require microscopically visible P-bodies19 and can occur in the cytoplasm20–21, recent evidence suggests that the activity of the mRNA decapping enzyme DCP2 is exquisitely controlled, both positively and negatively, by conformational changes and protein-protein interactions in the liquid droplet phase22.

Inspired by the paradigmatic demonstration23 that positively charged peptides can form liquid droplets in the presence of RNA in vitro, and that phosphorylation of specific amino acid residues within these peptides drives liquid phase remixing of peptide-RNA coacervates, we propose that post-translational modifications of NBDY could control phase separation of P-body components. Importantly, such a phenomenon has been previously shown for several P-body proteins whose phosphorylation promotes P-body dissociation24–25. In this work, we demonstrate that NBDY phase separates in the presence of RNA under specific conditions in vitro, and that phosphorylation of NBDY dissociates these liquid droplets. We further show that NBDY is a master regulator of P-bodies in cells, taking phosphorylation inputs from multiple signaling pathways to promote P-body disappearance prior to cell division. These results demonstrate that phosphorylation switches the positively charged, intrinsically disordered microprotein NBDY from promoting to preventing LLPS both in vitro and in cells, and suggest that microproteins could more generally contribute to formation and regulation of liquid condensates in cells.

Results:

NBDY undergoes liquid-liquid phase separation in the presence of RNA in vitro

NBDY bears a predicted charge of +4 at physiological pH, with high local positive charge near its middle segment (residues 22–27). NBDY is also enriched in proline residues (23.5% of the amino acids) and is predicted to lack stable secondary structure10, 26. Because LLPS is associated with intrinsically disordered proteins10, 27 and can occur via complex coacervation (nonspecific association of oppositely charged polyanions), we hypothesized that NBDY may undergo LLPS in the presence of RNA in vitro - and that this process could be disrupted by phosphorylation23, 28–31. First, to experimentally examine intrinsic disorder in NBDY, we performed a 1H-15N HSQC NMR experiment with backbone-labeled NBDY. The narrow distribution of NBDY cross-peaks indicated the absence of a well-defined three-dimensional structure in solution (Figure S1)32.

We therefore investigated the ability of purified NBDY to form liquid droplets in the presence of RNA. Neither NBDY nor an unstructured polyuridine polymer (polyU) form liquid droplets alone in the presence of a molecular crowder, PEG (Figure 1A), but when mixed in the same buffer, NBDY and polyU RNA undergo LLPS (Figure 1A–B and Movie S1). The NBDY-RNA liquid droplets wetted the glass surface over time, further supporting their liquid-like state33 (Movie S1). Total RNA purified from HEK 293T also promoted LLPS of NBDY in vitro (Figure 1C). NBDY phase separation requires co-partitioning with RNA, as indicated by co-localization of fluorescently labeled polyU RNA and NBDY in liquid droplets (Figure 1D). Liquid droplet formation measured as an increase in turbidity depends on the concentration of NBDY protein, as expected, and the critical coacervate concentration23 of NBDY-polyU droplets is 150 μM under the tested condition (Figure 1E and Figure S2A). Addition of 100 mM NaCl decreased the turbidity to ~35%, and the coacervates dissolved at 200 mM NaCl, consistent with a requirement for charge-charge interactions (Figure 1F and Figure S2B). PEG, a molecular crowding reagent, was required for NBDY-RNA coacervate formation, similar to in vitro phase separation studies of other biomolecules like tau33. PEG additives of various molecular weights supported the formation of NBDY-RNA coacervates, though a slightly higher concentration of NBDY was required to achieve phase separation when PEG400 was used (Figure S2C, D). Taken together, these results are consistent with phase separation of NBDY in the presence of RNA in vitro via the electrostatic process of complex coacervation.

Figure 1|. Characterization of NBDY-RNA coacervates.

Figure 1|

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(a) TMR-NBDY or polyU RNA alone does not form phase separated liquid droplets. Transmission brightfield (left) and fluorescence images (right). (b,c) Transmission brightfield (left) and fluorescence images (right) show coacervate phase droplets formed by 0.4% w/v polyU RNA (polyuridylic acid) and 200 μM TMR-NBDY (b) or by 0.5 μg/μL HEK 293T RNA and 200 μM TMR-NBDY (c). (d) 0.5 μg/μL FITC-labelled U40 RNA and 200 μM TMR-NBDY colocalize in coacervate droplets. Buffer: 25 mM HEPES, pH 7.4, 0.5 mM sodium azide, 0.5% w/v Tryptone, 10% PEG3350 at 30 °C. Scale bars, 10 μm. (e,f) Turbidity plot of NBDY-polyU coacervates at increasing concentration of NBDY (e) or NaCl (f). 0.4% w/v polyU was used in all cases, and 200 μM NBDY was used in (f). Error bars in turbidity plots represent the standard deviation of three independent trials. Buffer: 25 mM HEPES, pH 7.4, 0.5 mM sodium azide, 0.5% w/v Tryptone, 10% PEG3350 at 30 °C.

NBDY phosphorylation is required for P-body disappearance during mitosis

Cellular P-bodies contain hundreds of proteins and thousands of RNAs34, and thus are far more complex and likely have different biophysical properties than the reductionist NBDY-RNA complex coacervation system that we examined in vitro. Nonetheless, we hypothesized that NBDY may contribute to and regulate P-body formation and maintenance in cells. In particular, we hypothesized that phosphorylation of NBDY in cells could perturb local charge density to drive P-body dissociation. P-bodies dissociate during mitosis, when DCP1A and other P-body proteins are phosphorylated24, 35. We therefore examined NBDY phosphorylation during mitosis. Phosphoproteomic analysis of NBDY immunopurified from cells treated with the G2/M checkpoint inhibitor nocodazole revealed phosphorylation at residue T40 (pT40, Figure 2A–B and Table S1). We generated a NBDY T40 phospho-specific antibody, which was demonstrated to specifically bind NBDY isolated from nocodazole-treated cells (that is, NBDY pT40), but not a non-phosphorylatable point mutant of NBDY immunopurified under the same condition. This antibody furthermore very weakly recognizes NBDY isolated from cells (1) in the absence of nocodazole treatment and (2) after phosphatase treatment subsequent to isolation from nocodazole-treated cells, confirming its specificity for NBDY pT40 (Figure 2C). We then quantified NBDY phosphorylation using Phos-tag SDS-PAGE followed by western blotting with NBDY and NBDY pT40-specific antibodies (Figure 2D). Under these conditions, NBDY phosphorylation at T40 reaches 50% occupancy (Figure 2D). We next defined the signaling pathway upstream of NBDY phosphorylation at T40. Since cyclin-dependent kinases (CDKs) regulate the G2/M transition36, we hypothesized that NBDY would be phosphorylated by, or downstream of, CDKs activity. Consistent with this hypothesis, the CDKs-selective inhibitor AT751937 specifically prevented NBDY phosphorylation under nocodazole treatment, while the control compound wortmannin, an inhibitor of phosphoinositide 3-kinases (PI3K), did not (Figure 2E). NBDY is therefore phosphorylated at T40 at the G2/M checkpoint downstream of CDKs activity (Figure 2F).

Figure 2|. NBDY is phosphorylated at T40 downstream of cyclin-dependent kinases (CDKs) at the G2/M checkpoint.

Figure 2|

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(a) Mitosis-specific NBDY phosphorylation site (red). (b) NBDY phosphorylation at T40 (pT40) after nocodazole treatment identified by LC-MS/MS. Red peaks, y-ions; yellow peaks, b-ions. c) NBDY pT40 antibody validation. Immunopurified samples were prepared from HEK 293T cells transiently expressing WT or T40A FLAG-NBDY with or without nocodazole treatment. λPPase indicates treatment with λ-phosphatase before SDS–PAGE. (d) Phos-tag PAGE and western blotting validation of NBDY pT40. Immunopurified samples were prepared from HEK 293T cells transiently expressing FLAG-NBDY with or without nocodazole treatment. β-Actin, loading control. (e) Phosphorylation of NBDY is regulated by CDKs. Cells expressing FLAG-NBDY were treated with nocodazole in the presence of a CDKs inhibitor (AT7519) or a PI3K inhibitor (wortmannin) as a negative control before IP and western blotting. (f) Timing of NBDY phosphorylation at T40 downstream of CDKs at the G2/M checkpoint of the cell cycle.

Given the prior observation that single site phosphorylation of model peptides bearing four arginine residues was sufficient to promote liquid phase remixing of peptide-RNA coacervates23, we hypothesized that NBDY T40 phosphorylation could perturb phase separation into P-bodies. To test the involvement of NBDY pT40 in mitotic P-body loss, we stably expressed the nonphosphorylatable mutant NBDY T40A in a NBDY CRISPR/Cas9 knockout (KO) HEK 293T cell line13 and compared P-body dynamics in these cells to cells expressing wild-type (WT) NBDY - which can be phosphorylated - on the same KO background (Figure S3A). Remarkably, while WT NBDY-expressing cells recapitulated previously reported mitotic P-body disassembly after 12 h of nocodazole treatment, the T40A mutant-expressing cells exhibited no detectable decrease in P-bodies under the same conditions (Figure 3). Phosphorylation of NBDY at T40 by CDKs at the mitotic checkpoint is therefore necessary for P-body disappearance.

Figure 3|. NBDY phosphorylation at T40 is required for P-body disappearance at the G2/M checkpoint.

Figure 3|

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P-body disappearance during mitosis requires NBDY phosphorylation at T40. P-body numbers in nocodazole or vehicle-treated cells expressing NBDY or NBDY T40A were imaged via immunofluorescence. Yellow, anti-DCP1A immunofluorescence; blue, DAPI. Six fields of view (>180 cells) were used to quantitate average P-bodies per cell in each condition (b). Data represent mean values from each field of view ± s.e.m, and significance was evaluated with one-way ANOVA. ***P < 0.001, Dunnett’s test. Scale bars, 20 μm.

NBDY phosphorylation is required for P-body disappearance during epidermal growth factor receptor (EGFR) signaling

Because NBDY phosphorylation occurs during cell division, we reasoned that growth factor signaling might also lead to NBDY modification and P-body loss. Treatment of HEK 293T cells with epidermal growth factor (EGF) led to phosphorylation of NBDY at S61 (pS61), as detected by phosphoproteomic analysis of immunopurified NBDY (Figure 4A–B and Table S1). Resolution with Phos-Tag SDS-PAGE and immunoblotting with a NBDY pS61-specific antibody showed ~65% NBDY phosphorylation stoichiometry in the presence of EGF (Figure 4C–D). NBDY phosphorylation appeared within 5 min of EGF treatment, consistent with the timescale of EGFR activation38 (Figure S3B). As little as 1 ng/ml EGF induced detectable NBDY pS61 (Figure S3C). In order to exclude confounding effects from supraphysiologic EGF concentrations, we hypothesized that cancer-associated activating mutations in EGFR could increase endogenous NBDY pS61 without the need for growth factor addition. We therefore compared four non-small cell lung carcinoma lines: one expressing wild-type EGFR (A549), two with different activating EGFR mutations (HCC827 and H3255), and one activating mutation in EGFR combined with T790M conferring first-generation EGFR inhibitor resistance (H1975)39. We found that the level of NBDY pS61 correlated with activation of EGFR without significantly changing NBDY expression (Figure 4E). Importantly, treatment of these cells with erlotinib, a first-generation EGFR inhibitor, abrogated NBDY pS61 in all cells except resistant H1975, confirming that NBDY phosphorylation requires EGFR signaling.

Figure 4|. NBDY is phosphorylated at S61 downstream of protein kinase C (PKC) during EGFR signaling.

Figure 4|

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(a) NBDY phosphorylation site observed during EGFR signaling (red). (b) EGF-dependent NBDY phosphorylation site identified by LC-MS/MS. Red peaks, y-ions; yellow peaks, b-ions. (c) Validation of anti-NBDY pS61 antibody. Extracts were prepared from HEK 293T cells transiently expressing FLAG-NBDY or FLAG-NBDY S61A with or without EGF treatment. λPPase indicates treatment with λ-phosphatase before SDS–PAGE. (d) Phos-tag PAGE and Western blotting validation of NBDY pS61. Transiently expressed FLAG-NBDY was immunopurified from HEK 293T cells with or without EGF treatment. β-Actin, loading control. (e) Endogenous NBDY pS61 was assayed in lung cancer cells with wild-type EGFR (A549), activating EGFR mutation (HCC827 and H3255), or activating EGFR mutation and erlotinib resistance (H1975) in the presence of vehicle or erlotinib. (f) NBDY pS61 is regulated by PKC. Extracts were prepared from FLAG-NBDY expressing cells treated with vehicle, or with EGF in the presence of vehicle, EGFR inhibitor (AG1478) or PKC inhibitor (BIMII, bisindolylmaleimide II) followed by immunopurification and western blotting. (g) In vitro kinase assay. Purified NBDY was incubated with purified control kinases (GRK2, GSK3β) or PKCα in the presence of ATP, then subjected to Western blotting. (h) Model: NBDY is phosphorylated at S61 downstream of PKC during EGFR signaling.

We then determined the kinase responsible for NBDY phosphorylation at S61. EGFR signals through a complex network of intermediates including ERK (extracellular signal-regulated kinase), MAPK (mitogen-activated protein kinase), PI3K (phosphoinositide 3-kinase)-AKT (protein kinase B), and PLCγ1 (phospholipase C gamma 1)-PKC (protein kinase C)35,36. Inhibitors of the ERK/MAPK and PI3K-AKT pathways, either alone or in combination, had no effect on EGF-dependent NBDY pS61 (Figure S4), while inhibition of PKC with bisindolylmaleimide II decreased EGF-dependent pS61 to the same extent as EGFR inhibition with AG1478 (Figure 4F). Furthermore, purified PKCα can phosphorylate NBDY at S61 in vitro (Figure 4G), while control kinases cannot. It is therefore possible that PKC phosphorylates NBDY at S61 downstream of EGFR via a previously described PLCγ pathway40–41 (Figure 4H).

We hypothesized that phosphorylation of NBDY during EGFR signaling could also drive P-body dissociation. However, unlike mitosis, the effect of EGF on P-bodies had not been previously reported. Remarkably, we found that EGF treatment at both 20 ng/mL and 100 ng/mL led to near complete P-body loss in wild-type HEK 293T over 30 min (Figure 5A–C). Expression of the nonphosphorylatable NBDY S61A mutant in the NBDY KO background abrogated EGF-dependent P-body disappearance, while the phosphomimetic NBDY S61D mutant supported EGF-dependent P-body loss identical to the expression of the WT NBDY coding sequence (Figure 6A–C). NBDY pS61 is necessary but not sufficient for P-body disappearance, because NBDY S61D rescue cells exhibit wild-type P-body numbers in the absence of EGF treatment (Figure 6B). EGF-dependent P-body loss is not observed in NBDY KO cells, similar to the S61A mutant (Figure S5A and Figure 6C). Interactions of the decapping complex components DCP1A, EDC4 and NBDY are not perturbed by EGF treatment (Figure S5B), suggesting that NBDY phosphorylation is required for dissociation of P-bodies but does not disrupt individual decapping complexes. Finally, the observed dissociation is not a consequence of crosstalk from the T40 phosphorylation, as we showed that these two phosphosites are mutually exclusive under the experimental conditions employed (Figure S6). We therefore propose that phosphorylated NBDY is required for dissociation of P-bodies into sub-microscopic decapping complexes during both mitosis and EGF signaling, but altering the charge on NBDY in the absence of EGFR signaling is not sufficient for this effect. Rather, we propose that NBDY phosphorylation tips the balance of electrostatic repulsion within a network of P-body proteins that are also phosphorylated downstream of EGFR activation35.

Figure 5|. P-body disappearance during EGF stimulation.

Figure 5|

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(a,b) EGF (20 ng/ml) induces P-body disassembly. Six fields of view (>180 cells) were used to quantitate average P-bodies per cell at each timepoint (b). Data represent mean ± s.e.m, and significance was evaluated with one-way ANOVA. ***P < 0.001, ****P < 0.0001, Dunnett’s test. (c) EGF concentration dependence for P-body dissociation. Six fields of view (>180 cells) were used to quantitate P-bodies per cell at each EGF concentration. Data represent mean (P-bodies per cell calculated from each field of view) ± s.e.m, and significance was evaluated with one-way ANOVA. ****P < 0.0001, Dunnett’s test.

Figure 6|. NBDY phosphorylation at S61 is required for P-body disappearance during EGFR signaling.

Figure 6|

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P-bodies were visualized in NBDY rescue, NBDY KO, NBDY S61A rescue and NBDY S61D rescue cell lines after treatment with EGF or vehicle. Yellow, anti-DCP1A immunofluorescence; blue, DAPI. Six fields of view (>180 cells) were used to quantitate average P-bodies per cell in untreated (b) or EGF-treated cells at indicated time points (c). Data represent mean values ± s.e.m, and significance was evaluated with one-way ANOVA. n.s., not significant; *P < 0.05; ****P < 0.0001, Dunnett’s test. Scale bars, 20 μm.

NBDY phosphorylation drives dissociation of NBDY/RNA coacervates in vitro

To provide further support for our electrostatic model of P-body dissociation in a simple model system, we examined whether NBDY phosphorylation could dissociate NBDY-RNA coacervates in vitro. While this system lacks the complex network of P-body components and is unlikely to recapitulate their biophysical properties under cellular conditions (see Discussion), it is analogous to a previously reported phosphorylation-switchable peptide-RNA coacervate system23 and reports on whether alteration of the overall NBDY charge by phosphorylation is sufficient to disrupt its electrostatic interactions with RNA in artificial condensates. Since PKC phosphorylates NBDY at S61 downstream of EGFR and in vitro, we used PKC to achieve NBDY phosphorylation in coacervates. Addition of PKC to the in vitro NBDY-RNA LLPS system dissociates liquid droplets in a time-dependent manner (Figure 7A and Movie S2). As previously reported23, mixing fluorescently labeled NBDY, RNA and PKC in LLPS buffer containing PKC reaction components and ATP initially led to formation of liquid droplets that fell to the coverslip surface by 5 minutes, followed by remixing of the liquid phase that was essentially complete by 30 minutes (Movie S2). Similar results were obtained with unlabeled NBDY using turbidity measurements (Figure 7B, red trace). Importantly, liquid droplets formed by purified nonphosphorylatable NBDY S61A and RNA were refractory to kinase activity, as demonstrated by high turbidity in the presence of PKC (Figure 7B, blue trace). Finally, the S61D mutant failed to form liquid droplets even in the absence of PKC (Figure 7B, green trace). Similarly, the NBDY T40D mutation did not support NBDY-RNA coacervates that otherwise formed with wild-type NBDY or T40A mutant in vitro (Figure S7). Therefore, phosphorylation of NBDY at a single amino acid is sufficient to promote liquid phase remixing in vitro, likely as a result of decreased overall or local positive charge.

Figure 7|. Phosphorylation at S61 by PKC in vitro dissociates NBDY-RNA liquid droplets.

Figure 7|

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(a) NBDY-polyU coacervate dissolution by PKC activity. Top row: transmission brightfield images. Bottom row: TMR-NBDY fluorescence at the same time points. Buffer: 25 mM HEPES, pH 7.4, 20 μM CaCl2, 50 mM NaCl, 10 mM DTT, 100mM MgCl2, 0.5 mg/ml phosphatidylserine, 100 μM DTT, 0.5 mM sodium azide, 0.5% w/v Tryptone, 10% PEG3350 at 30 °C. 0.4% w/v polyU, 200 ng PKCα and 200 μM NBDY were used. Scale bars, 10 μm. (b) Solution turbidity as a function of time for purified wild-type (red), S61A NBDY (blue) or S61D NBDY (green) protein in the presence of PKC. Error bars represent the standard deviation of three independent trials. Buffer: 25 mM HEPES, pH 7.4, 20 μM CaCl2, 50 mM NaCl, 10 mM DTT, 100mM MgCl2, 0.5 mg/ml phosphatidylserine, 100 μM DTT, 0.5 mM sodium azide, 0.5% w/v Tryptone, 10% PEG3350 at 30 °C. 0.4% w/v polyU, 200 ng PKCα and 200 μM wild-type or mutant NBDY were used.

NBDY phosphorylation regulates cell proliferation

The observation that NBDY phosphorylation is required for P-body dissociation into submicroscopic decapping complexes during growth factor signaling and mitosis suggests that this process might be important for cell proliferation. We therefore asked whether cell proliferation is altered in cells that cannot phosphorylate NBDY and, therefore, cannot dissociate their P-bodies during cell division. We examined proliferation of cell lines expressing wild-type NBDY, which can be phosphorylated, or the phosphorylation-deficient mutants NBDY T40A or NBDY S61A, on the NBDY KO background. Seven days after plating equal numbers of cells from each line, we measured colony formation and observed a small but statistically significant decrease in proliferation in the mutant cell lines as compared to wild type (Figure 8). It follows that the absence of individual phosphorylation sites on NBDY can reproducibly decrease cell proliferation by ~10%, and, by extension, that NBDY phosphorylation-driven remixing of P-body components into the bulk cytoplasm contributes measurably to the rate of cell proliferation.

Figure 8|. Phosphorylation of NBDY regulates cell proliferation.

Figure 8|

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(a) Crystal violet staining of the NBDY rescue, NBDY T40A rescue and NBDY S61A rescue cell lines on day 7. (b) Relative cell proliferation of wild-type NBDY, NBDY T40A and NBDY S61A rescue cell line on day 7 (N = 3 biological replicates). Data represent mean values ± s.e.m., and significance was evaluated with two-tailed t-test. *P<0.05.

Discussion

Intrinsically disordered, post-translationally modified microproteins have the potential to promote and dynamically regulate liquid-like phase separated membraneless organelle formation. In this work, we demonstrated this principle in the context of the well-characterized microprotein NBDY. In a reductionist system, NBDY forms liquid droplets in the presence of RNA and molecular crowding reagents that can be dissociated by NBDY phosphorylation. We note that these artificial, in vitro liquid droplets are fundamentally dissimilar to P-bodies in several ways and are unlikely to form via RNA-NBDY coacervation inside cells. First, P-bodies contain hundreds of proteins and thousands of RNAs34, all of which contribute to their formation and biophysical properties. Second, the cellular concentration of NBDY is 72 nM13, far lower than that required to establish phase-separated NBDY-RNA coacervates in vitro, though its concentration within P-bodies has not been directly measured. Furthermore, artificial NBDY-RNA droplets undergo liquid phase remixing in vitro at lower salt concentrations than are found in the cell. Despite the fact that NBDY-RNA coacervates probably do not form in cells, they enable us to conclude that the overall charge of NBDY is near an inflection point for complex coacervate formation with RNA under the experimental conditions used, since installation of a single negative charge on NBDY drives their dissociation (in the case of phosphorylation) or prevents their formation (in the case of NBDY mutants). We propose that phosphorylation of NBDY in vitro disrupts the charge-charge interactions with RNA that are responsible for complex coacervation. We further speculate that, inside cells, NBDY may not interact directly with RNA, but rather cooperates with other mRNA decapping factors to promote phase separation and P-body formation, for example by binding to both DCP1A and EDC4 via independent linear binding motifs13. A similar principle has been demonstrated for yeast Dcp2/Dcp1/Edc3 condensates, as the critical concentration for the liquid-liquid phase separation of Dcp2/Dcp1 complex is lowered by 20-fold in the presence of Edc3 in vitro22. Along these lines, we propose a different mechanism of phospho-NBDY-mediated dissociation of P-bodies in cells, whereby negative charge on NBDY repels other phosphorylated P-body proteins to disrupt the network of protein-protein and protein-RNA interactions required for cellular LLPS (see below). Thus, the P-body phase transition is finely electrostatically tuned to the combined phosphorylation states of NBDY and other P-body proteins, allowing a single phosphorylation site on NBDY to tip the system across the phase boundary. Though we propose different molecular mechanisms for liquid phase remixing in vitro and in cells, both rely exquisitely on the charge state of NBDY.

While phase separation in P-bodies is described by a complex phase diagram dependent on a network of RNA-RNA, RNA-protein, and protein-protein interactions42, the current study demonstrates that NBDY phosphorylation is an essential driver of P-body dynamics during cell growth and division. Though NBDY is phosphorylated at different, mutually exclusive positions during mitosis and EGFR signaling under the conditions used in this study, both NBDY phosphorylation sites have a similar functional outcome: they are necessary, but not sufficient, for P-body disappearance and contribute to cell proliferation. We propose that NBDY phosphorylation is a required component of a network of multiple P-body protein phosphorylation sites that are required for P-body dissociation. DCP1A and EDC4 are phosphorylated during mitosis, and hyperphosphorylation of DCP1A is required for the complete dissociation of P-bodies24. Along with the observation that P-body dissociation does not occur efficiently in NBDY KO cells, these data are consistent with a model in which NBDY phosphorylation is the switch that drives electrostatic repulsion in the context of phosphorylation of multiple P-body proteins during mitosis and EGFR signaling, promoting liquid phase remixing of the components of macroscopic P-bodies (Figure 9). We note that the complete “phosphorylation code” for P-body disassembly is not yet fully understood. For example, Dcp2 phosphorylation was shown to have little effect on P-body assembly in yeast. Therefore, not all mitosis-dependent phosphorylation events of decapping complex subunits are essential to P-body dissociation, but this process does require the cooperation of multiple phosphorylation sites. Noneetheless, the data are consistent with a model in which NBDY is a master regulator of P-body dissociation that integrates inputs from multiple signaling pathways and cooperates with phosphorylation sites, likely on multiple additional P-body proteins, to promote cell proliferation. We note that this model is not at odds with previous reports demonstrating that NBDY KO increases the basal number of P-bodies in cells, because NBDY deletion primarily alters the RNA content of cells, promoting phase separation via increased RNA-RNA and RNA-protein interactions in P-bodies13.

Figure 9|.

Figure 9|

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Phosphorylated NBDY regulates P-body disassembly downstream of signaling pathways regulating cell proliferation: a model.

Remarkably, NBDY phosphorylation is upregulated in some cancer cells and increases the rate of cell proliferation. Substantial prior literature has linked P-bodies with the cell cycle. For example, a recent study by Bearss et al., demonstrated that, like NBDY, EDC3 phosphorylation is upregulated in many cancer types, and that the phosphorylation-null mutant of EDC3 decreases proliferation and invasion, probably via increased decay of integrin mRNAs25. Interestingly, roles for P-bodies during mitosis have also been reported in plants43 and yeast44. Specifically, in yeast, daughter cells that do not receive P-bodies during cell division show significantly smaller sizes and slower growth rate. In plants, timely degradation of messages specific to certain cell cycle phases is critical to progression of the cell cycle, which is correlated with P-body integrity. These findings implicate P-body dynamics in mitosis broadly in eukaryotes and suggest that NBDY phosphorylation is a mammal-specific12 regulatory mechanism that drives cell division during mitogenic signaling and cell division by promoting P-body dissociation. NBDY-controlled LLPS therefore likely has important physiological consequences in healthy cells as well as in cancer.

However, while the proliferative phenotype associated with NBDY-mediated LLPS regulation is clear, the molecular mechanism linking P-body dynamics to cell division remains unresolved. A number of recent studies have begun to elucidate the biological significance of P-bodies and their dynamics during cell division. P-bodies have been described as either venues for active mRNA decay or as storage sites for translationally repressed mRNA. While their cellular function remains under investigation, some recent studies suggest a hybrid model in which phase separation licenses specific P-body-enriched transcripts for decapping22. This model is consistent with our recent observation that NBDY expression levels alter the specificity of mRNA decapping13. Disrupting P-bodies alone, however, does not appear to affect the rate of mRNA decay19. Alternatively, phosphorylation of key components of P-bodies has been suggested to protect mRNAs from degradation during mitosis24. It is therefore possible that phosphorylation of NBDY and other P-body proteins repels mRNA, therefore simultaneously stabilizing specific transcripts by preventing its association with decapping enzymes and disrupting the molecular interactions critical to P-body integrity. Importantly, in the hybrid model wherein some mRNAs are stored in the P-body while others are decapped and degraded, this dissociation could hypothetically lead to complex and differential outcomes for the stability of specific transcripts. Alternatively, mRNA trafficking, rather than processing, may be altered by regulated LLPS to cause the observed phenotype. It has been previously proposed that disassembly of membraneless compartments during mitosis is required to ensure equal distribution of proteins and RNAs between two daughter cells45, suggesting that phosphorylation could promote this redistribution without altering mRNA processing. To differentiate these proposed models, experimental determination of the effect of phosphorylation and P-body dissociation on global mRNA stability will be critical.

In conclusion, these results demonstrate that a 7-kDa microprotein can integrate inputs from different signaling pathways to control the dynamics of a membraneless organelle, and motivate the need to further understand the modifications and interactions of thousands of yet-uncharacterized protein products of smORFs in human cells.

Experimental Section

Data analysis

Two-tailed and equal-variance Student’s t test, Mann Whitney U test, Kruskal-Wallis test and analysis of variance (ANOVA) with Dunnett’s test were performed using Excel, GraphPad Prism 7 or R. Statistical significance was defined as P value <0.05. Equal variance between samples was established using an F-test. P-body counting was performed using ImageJ as previously described46.

Recombinant expression, purification and fluorophore labeling of human NBDY

His-tagged human NBDY and NBDY S61A in pET28a were expressed in E. coli BL21(DE3) cells and purified via His-tag affinity chromatography exactly as previously described13. For fluorescence microscopy of liquid-liquid phase separated droplets, purified NBDY was labeled with tetramethylrhodamine-5-maleimide (Sigma). NBDY protein was prepared in phosphate buffered saline (PBS) buffer (pH 7.4) at a concentration of 1 mg/ml. 1 μg of tetramethylrhodamine (TMR)-5-maleimide (Thermo Scientific, Cat. T6027) was dissolved in DMSO, and added to the NBDY solution. The labeling reaction was allowed to proceed for up to two hours at room temperature in the dark. The reaction was stopped by addition of dithiothreitol (DTT) to a final concentration of 4 mM. Unconjugated dye was removed using a Sephadex G-25 column in PBS buffer. Labeled NBDY was mixed with non-labeled NBDY at 10% concentration (0.1 mg/ml TMR labeled NBDY+ 0.9 mg/ml NBDY) to give TMR-NBDY.

Turbidity measurements

Samples were prepared by mixing all components from stock solutions. Polyuridylic acid potassium salt (polyU, MW = 600–1000 kDa, Cat. P9528) and FITC-labeled 40-mer uridine oligonucleotides (FITC-U40) were purchased from Sigma-Aldrich and reconstituted in DEPC-treated water. Total cellular RNA was purified from HEK 293T cells grown in 15 cm dishes using Trizol (Invitrogen) following the manufacturer’s instructions, followed by DNase I (NEB) treatment, and dissolved in DEPC water. For liquid droplet formation, TMR-NBDY or NBDY was mixed with RNA (polyU, FITC-U40, or total cellular RNA) at the indicated concentrations in LLPS buffer (25 mM HEPES, pH 7.4, 0.5 mM sodium azide, 0.5% w/v Tryptone, 10% PEG 3350) at 30 °C. For the PKC assay, purified NBDY, NBDY S61A, NBDY S61D, NBDY T40A, or NBDY T40D (200 μM) was added to RNA (0.4% w/v polyU RNA) and kinase (PKCα) in a total volume of 100 μL in LLPS buffer with additional components required for PKCα activity (20 μM CaCl2, 10 mM DTT, 100mM MgCl2, 0.5 mg/ml phosphatidylserine, 100 μM DTT) at 30°C. Turbidity measurements were made in real time using a GENESYS 10S UV-vis spectrophotometer (Thermo Scientific) at a wavelength of 500 nm.

Confocal imaging of liquid droplets

Samples were prepared by mixing all components from stock solutions in LLPS buffer at the indicated concentrations and spotted on no.1.5 glass coverslips (Fisher Scientific). Confocal images of the suspension were acquired at room temperature immediately for samples without kinase using a Leica TCS SP8 with PL APO 63x/1.40 oil objective with CORR CS. For phosphorylation assays, 200 μM TMR-NBDY (or mutant), 0.4% w/v polyU RNA, kinase and required assay components were mixed in LLPS buffer as above, then spotted on coverslips. Images were acquired on an INUBG2AF-GSI2 (Tokai Hit) temperature-controlled stage held at 30°C.

NMR experiments

His-tagged human NDBY in pET28a was expressed in E. coli. BL21(DE3) cells grown in M9 minimal medium supplemented with 15NH4Cl (1.0g/L) purchased from Cambridge Isotope Laboratories (Cat. NLM-467–1). NBDY was purified on Talon metal affinity resin (Takara Bio) followed by cleavage with PreScission protease (Sigma-Aldrich, Cat. GE27-0843-01). NBDY was subsequently further purified by size exclusion chromatography using Superdex 75 (GE Healthcare) and eluted into NMR buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 7% D2O and 1 mM TCEP). For NMR experiments, NBDY was concentrated to 1 mM using Amicon Ultra-15 concentrators (4 kDa molecular weight cut off).

NMR experiments were performed on Varian 600 MHz spectrometers at 293 K. Two-dimensional transverse relaxation optimized spectroscopy 1H15N single quantum coherence spectra47 were collected with 4096 data points in the direct dimension, 128 t1 increments and spectral widths of 12000Hz and 2500Hz in the direct and indirect dimensions, respectively. Additionally, the 1H transmitter and 15N offsets are set to water resonance and 120 ppm respectively. Data were processed with NMRPipe and analyzed in nmrFAM-SPARKY48.

Cell culture

HEK 293T cells were amplified from early-stage stocks prepared from cells purchased from ATCC. HEK 293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Corning, Cat. 10–013-CV) supplemented with 10% (vol/vol) fetal bovine serum (FBS, Sigma Aldrich, Cat. F4135–500ML) and 100 U/mL penicillin-streptomycin (Sigma Aldrich, Cat. P4333–100ML). HEK 293T cells bearing NBDY genomic deletion (NBDY KO) were previously described13. A549, HCC827, H1975 and H3255 cells were a gift from Craig Crews (Yale University) and were cultured in RPMI-1640 (Corning, Cat. 10–040-CV) medium containing 10% FBS and 100 U/mL penicillin-streptomycin. The ATCC Universal mycoplasma detection kit was used to confirm mycoplasma-free status of all cell lines.

Antibodies, Peptides and Proteins

Primary antibodies used for Western blotting and/or immunofluorescence were: mouse monoclonal anti-FLAG (Sigma, F3165); rabbit polyclonal anti-EDC4 (Sigma, SAB4200114); rabbit polyclonal anti-DCP2 (Novus Biologicals, NBP1–41070); mouse monoclonal anti-beta actin (Invitrogen, BA3R); rabbit polyclonal anti-DCP1A (Sigma, D5444); rabbit monoclonal anti-DCP1A Alexa Fluor 647 (Abcam, ab209946); rabbit monoclonal anti-EGFR (Abcam, ab40815; rabbit monoclonal anti-EGFR (phospho Y1068) (Abcam, ab40815); mouse monoclonal anti-AKT (Cell Signaling technology, 2966S); rabbit monoclonal anti-AKT (phospho Ser473) (Cell Signaling technology, 4060T); rabbit monoclonal anti-ERK1/2 (Cell Signaling technology, 4695T); rabbit monoclonal anti-ERK1/2 (Cell Signaling technology, 4695T); rabbit monoclonal anti-ERK1 (phospho T202)+ERK2 (phospho T185) (Abcam, ab201015); rabbit polyclonal anti-NBDY (Covance, YU-342); rabbit polyclonal anti-NBDY(phospho T40) (Covance, YU-352); rabbit polyclonal anti-NBDY(phospho S61) (Covance, YU-344). Secondary antibodies for Western blotting were: goat anti-rabbit peroxidase conjugate (Rockland, 611-103-122; Merck, AP132P) and goat anti-mouse peroxidase conjugate (Rockland, 610–1319). Secondary antibody for immunofluorescence was goat anti-rabbit Alexa Fluor 568 (Life Technologies, A-11011).

Synthetic peptides used to generate antibodies against NBDY, NBDY(phospho T40) and NBDY(phospho S61) were as follows: NBDY (amino acids 8–28 of NBDY), phosphorylated Thr40 (amino acids 35–45 of NBDY), and phosphorylated Ser61 (amino acids 54–64 of NBDY). All peptides were synthesized and used as an immunogen in rabbits. All peptides and antibodies were generated by Covance Inc. (Princeton, New Jersey, USA).

Lentivirus production and infection

Lentivirus was produced essentially as previously reported49. HEK 293T cells were grown in 10-cm culture dishes to 60% confluency, then co-transfected using Lipofectamine 2000 (Thermo Scientific, 11668019) with pLJM1 encoding the transgene to be stably expressed, along with accessory plasmids pMD2.G and psPAX2, and growth media was replaced after 5 h. Forty-eight hours later, lentivirus-containing media was harvested, and cell debris was removed by passing through a 0.45-μm filter prior to aliquoting and flash freezing.

Stable cell lines were generated by treating HEK293T or HEK293T NBDY KO cells, grown in 6-well plates to 75% confluency, with ~20 μL of the appropriate lentivirus-containing media. A polyclonal population of stable cells were selected with 2 ng/ml puromycin (Sigma, Cat. P9620), and, forty-eight hours later, cells were harvested and transgene expression was confirmed by Western blotting.

NBDY immunoprecipitation and phosphoproteomics

To detect NBDY phosphorylation in cells undergoing mitosis, 70% confluent HEK 293T cells in a 10-cm dish released from 2 mM thymidine block for twenty-four hours were transfected with FLAG-tagged NBDY in pcDNA3 with Opti-MEM medium. Transfections were carried out using Lipofectamine 2000 (Thermo Fisher, 11668019) and 10 μg DNA per 10 cm dish of cells. After 6 hours, Opti-MEM medium was removed, and nocodazole (Sigma, Cat. M1404) was added to a final concentration of 100 ng/ml. To assess NBDY phosphorylation during epidermal growth factor stimulation, 70% confluent HEK 293T cells in a 10 cm dish were transfected with FLAG-tagged NBDY in pcDNA3, and 24 hours later were treated with 20 ng/ml EGF (Sigma, Cat. E9644) for 30 min. NBDY was subsequently immunopurified from both experimental sets of cells prior to phosphoproteomic analysis following a previously published protocol. Briefly, cells were harvested and lysed on ice using 400 μL Tris-buffered saline (TBS) with 1% Triton X-100 supplemented with Roche Complete protease inhibitor cocktail tablets. After 3×FLAG peptide (Sigma, Cat. F4799) elution of FLAG-NBDY from anti-FLAG agarose beads (clone M2, Sigma, Cat. A2220) in TBS, the entire eluate from each sample was loaded on an SDS–PAGE gel, separated, visualized with Coomassie stain and imaged.

Protein-containing gel slices were digested with trypsin as previously described14. The resulting peptide mixtures were extracted from the gel and dried, subjected to ethyl acetate extraction to remove residual detergent, and re-suspended in 15 μl of 3:8 70% formic acid: 0.1% TFA. A 10 μL aliquot of each sample was injected onto a pre-packed column attached to a nanoAcquity UPLC (Waters) in-line with an LTQ Orbitrap Velos (Thermo Scientific). Samples were trapped for 15 min with a flow rate of 2 μl/min on a 100-micron ID trap column packed for 5 cm with 5 μm Magic C18 AQ beads (Waters), and peptides were chromatographed on a 20 cm 75-micron ID analytical column (New Objective) packed in-house with 3 μm Magic C18 AQ beads (Waters). A NanoAcquity pump (Waters) eluted peptides at 300 nl/min flow rate as follows: Buffer A: water/0.1% formic acid; Buffer B: MeCN/0.1% formic acid; Gradient: 0% to 95% buffer B over 90 minutes. Tandem mass spectrometry (MS) was performed as follows: The full MS was collected over the range of 298–1750 m/z with a resolution of 30,000. MS/MS data were collected using a top 10 high-collisional energy dissociation method in data-dependent mode with a normalized collision energy of 33.0 eV and a 2.0 m/z isolation window. The first mass was 100 m/z in fixed mode. MS/MS resolution was 7500 and dynamic exclusion was 60 s.

Mass spectra were analyzed using Mascot (version 2.5.0.1). We searched a database comprising human Uniprot proteins (version 2019) with the NBDY protein sequence appended. Phosphorylation of Ser, Thr and Tyr, oxidation of methionine and N-terminal acetylation were set as variable modifications. The MS1 mass error was set to 20 ppm, and MS/MS peak error was set at a maximum of 0.6 Da. Up to two missed cleavages were permitted. The maximum false discovery rate (FDR) was set to 0.01 for peptide and protein identifications. The minimum peptide length was set at five amino acids.

Western blotting

For validation of NBDY phosphorylation sites detected by mass spectrometry, FLAG-NBDY immunoprecipitants were prepared as above and resolved on 15% SDS-PAGE gel containing 25 μM of Phos-tag acrylamide (Wako, Cat. AAL-107) and 125 μM MnCl2. After electrophoresis, Phos-tag acrylamide gels were washed with transfer buffer containing 10 mM EDTA for 30 min with gentle shaking and then with transfer buffer without EDTA for 30 min according to the manufacturer’s protocol. Immunoblots were blocked with 3% BSA in TBS-T for 1 hour at room temperature (RT), then probed with both anti-NBDY (Covance, YU-342), anti-NBDY(phospho T40) (Covance, YU-352) and anti-NBDY(phosphorS61) (Covance, YU-344) overnight at 4 °C. Control cell lysates were treated with 4 U/μl of Lambda phosphatase (NEB, P0753S) at 30 °C for 30 minutes before immunoprecipitation and Phos-tag SDS-PAGE and immunoblotting analysis.

For validation of NBDY phosphorylation downstream of CDKs and EGFR, proteins were transferred to nitrocellulose membranes followed by a standard Western blotting protocol12. Briefly, immunoblots were blocked with 3% BSA in TBS-T for 1 hour at RT and probed with primary antibodies against pathway (phospho)proteins in 3% BSA in TBS-T overnight at 4 °C. The membrane was washed three times with TBS-T, probed with secondary antibodies in 3% BSA in TBS-T for 1 hour at RT, and washed three times with TBS-T before development with Clarity ECL Western Blotting Substrate (Bio-Rad, Cat. 1705060) and imaging.

Kinase inhibition assay

To identify the kinase that phosphorylates NBDY during mitosis, HEK 293T cells released from 2 mM thymidine block for twenty-four hours were transfected with FLAG-tagged NBDY in pcDNA3 with Opti-MEM medium. Six hours later, Opti-MEM was removed, and a CDK inhibitor (1 μM AT7519 (Selleck Chemicals, Cat. S1524)) or PI3K inhibitor (1 μM wortamanin (Selleck Chemicals, Cat. S2758)) was added with 100 ng/ml nocodazole in complete media. Cells were harvested and suspended in 1 mL lysis buffer (Tris-buffered saline (TBS) with 1% Triton X-100 and Roche Complete protease inhibitor cocktail tablets). Then cells were sonicated (50% intensity, 5 s pulse with 25 s rest, 5×, MICROSON XL 2000) on ice followed by centrifugation at 21,130 × g, 4 °C, 10 min. The proteins were transferred to nitrocellulose membranes followed by a standard Western blotting protocol12 as described above.

To identify the kinase that phosphorylates NBDY during EGF stimulation, HEK 293T cells transfected with FLAG-tagged NBDY were treated with kinase inhibitors targeting EGFR (10 μM AG1478 (Selleck Chemicals, Cat. S2728)), MEK (5 μM U0126 (Selleck Chemicals, Cat. S1102)), PI3K (1 μM wortamannin (Selleck Chemicals, Cat. S2758)), or CDK (1 μM AT7519 (Selleck Chemicals, Cat. S1524)) for 4 hours; an inhibitor targeting PKC (10 μM Bisindolylmaleimide II (Cayman Chemicals, Cat. 11020)) was applied to cells for 8 hours. Following kinase inhibitor or vehicle treatment, cells were stimulated by adding 20 ng/ml EGF for 30 min. Cells were harvested and lysed, and proteins were subjected to a standard Western blotting protocol12 as described above.

In vitro kinase assay

In vitro assays of NBDY phosphorylation at S61 by purified kinases were performed essentially as described previously50. Briefly, 1 μg purified NBDY was incubated with PKCα (200 ng; Thermo Fisher, Cat. A41891) in a total assay volume of 20 μl of buffer containing 20 mM HEPES, pH 7.4, 20 μM CaCl2, 10 mM DTT, 100 mM MgCl2, 100 μM ATP, 0.6 mg/ml phosphatidylserine and 0.06 mg/mL diacylglycerol. Grk2 (200 ng; Thermo Fisher, Cat. PV3361) and GSK3β (200 ng; Thermo Fisher, Cat. PV3365) were incubated with 1 μg purified NBDY in a total assay volume of 20 μL of buffer containing 20 mM HEPES, pH 7.4, 1 mM DTT,15 mM MgCl2, and 100 μM ATP. After incubation for 30 min at 30 °C, reactions were stopped by addition of SDS loading buffer and proteins were separated by SDS-PAGE. Phosphorylation of NBDY was determined by Western blotting with rabbit polyclonal anti-NBDY(phospho S61) (Covance, YU-344), using rabbit polyclonal anti-NBDY (Covance, YU-342) antibody as a loading control.

Immunoprecipitation of NBDY from lung cancer cell lines

For agarose bead antibody conjugation, 100 μL NHS-activated agarose slurry (Thermo Scientific, Cat. 26200) was washed twice with PBS buffer (pH 7.4). Subsequently 30 μL rabbit polyclonal anti-NBDY (Covance, YU-342) (1 mg/ml) in PBS buffer was added, and the resulting mixture was incubated on ice for one hour. Unreacted NHS-beads were quenched with 1 M ethanolamine (pH 7.4) for another 15 minutes. The resulting resin was washed with PBS (5x) and stored in PBS on ice at 4°C for 12 hours. A549, HCC827, H1975 and H3255 were grown to 60–70% confluence in 15-cm dishes (treated with 5 μM erlotinib (Sigma, Cat. SML2156) or vehicle for 24 hours) were harvested and lysed using 400 μl lysis buffer (TBS with 1% Triton X-100 and Roche Complete protease inhibitor cocktail tablets). Cells were lysed on ice for 20 min followed by centrifugation at 14,000 r.p.m., 4°C, 15 min. A 50 μl aliquot of anti-NBDY agarose beads was suspended in the cell lysate supernatant. Bead suspensions were rotated at 4°C for 1 hour and washed 3 times with TBS-T. Elution was carried out by boiling the beads in 30 μL of 3× protein loading buffer (0.15 M Tris-HCl, pH 8.0, 15% glycerol, 50 mg/mL SDS, 50 mg/mL DTT, 50 μg/mL bromophenol blue). The protein was boiled for 10 minutes, diluted to 1×. Beads were removed by centrifugation, and the entire supernatant was loaded on a 15% SDS–PAGE gel. Western blotting with rabbit polyclonal anti-NBDY(phospho S61) (Covance, YU-344), using rabbit polyclonal anti-NBDY (Covance, YU-342) antibody as a loading control.

P-Body imaging

HEK 293T cells were grown on fibronectin-coated glass coverslips (AmScope, CS-R18–100, 18 mm diameter round microscope glass cover slides) in a 12-well plate to 70% confluency. Cells were fixed with 10% neutral buffered formalin (Fisher Scientific), permeabilized with methanol at −20 °C, and blocked with blocking buffer (3% BSA in PBS) for 1 h at room temperature. Cells were stained with rabbit polyclonal anti-NBDY (Covance, YU-342) at a 1:100 dilution (volume: volume) in blocking buffer overnight at 4 °, followed by 3 consecutive washes with PBS. Goat anti-rabbit Alexa Fluor 568 (Life Technologies, A-11011) was subsequently applied at a 1:1000 dilution in blocking buffer for 1 to 4 hours at room temperature in the dark, followed by 5 times with PBS washes. Anti-DCP1A Alexa Fluor 647(Abcam, ab209946) was then added at 1:1000 dilution and incubated for 1 hour, followed by 3x PBS washes. For quantifying P-bodies, cells were stained with rabbit anti-DCP1A (Sigma-Aldrich, D5444) at a 1:1000 dilution in blocking buffer overnight at 4°, followed by 3x PBS washes and incubation with goat anti-rabbit Alexa Fluor 568. Cells were post-fixed with 10% buffered formalin, nuclei were stained with DAPI (EMD Millipore, Cat. 268298, 1:20000 dilution in 1x PBS), and imaging was performed on a laser scanning confocal microscope (Leica TCS SP8) with PL (field planarity) APO (apochromatic) 63x/1.40 oil, CS2 and PL APO 100x/1.44 oil, CORR (correction collar) CS (confocal scanning).

Crystal violet staining

NBDY rescue, NBDY T40A rescue and NBDY S61A rescue cells were plated at 2×104 cells/plate in 6-well plates (triplicate) with fresh culture medium, and the medium was replaced every 3 days. After seven days, cells were fixed in 10% formalin for 15 min at room temperature. After washing with ddH2O, cells were stained with 0.1% crystal violet in methanol for 30 min at room temperature in the dark, followed with three ddH2O washes and dried. The cells were then immersed in 1 mL 10% acetic acid with shaking for 20 min. 20 μL of the solution was combined with 80 μL ddH2O in a 96-well plate, and the optical density at 590 nm was monitored with SynergyTM HT.

Safety statement

The third-generation lentivirus system was utilized for establishing the stable cell lines for improved safety51. All lentivirus experiments were performed in a designated laminar flow biological safety hood with posted biohazard label. All consumables in contact with virus or virus-containing cells were decontaminated with 10% bleach and discarded in BL2 waste. Generation of aerosols was stringently avoided. No other unusually high or unexpected chemical or biological safety hazards were encountered.

Supplementary Material

Supporting Information

Supporting Figures 1–5, additional data including 1H15N-HSQC spectrum of NBDY, fluorescence and widefield microscopy, Western blotting

Table S1

Table S1, Phosphoproteomic analysis of partially purified NBDY during mitosis and EGFR signaling

Movie S1

Movie S1, Timelapse fluorescence imaging of NBDY-polyU liquid droplets

Download video file (4.1MB, mov)
Movie S2

Movie S2, Timelapse fluorescence microscopy of NBDY-RNA liquid droplets in the presence of PKC

Download video file (7.6MB, mov)

Acknowledgments

We thank all members of the Slavoff lab, Ken Loh, Mark Lemmon, Ben Turk and Jesse Rinehart for helpful discussions. This work was supported by the Searle Scholars Program, an Odyssey Award from the Richard and Susan Smith Family Foundation, NIH R01GM122984, and Yale University West Campus start-up funds (to S.A.S.). A. K. (5T32GM06754 3-12) was in part supported by an NIH Predoctoral Training Grant.

Footnotes

SUPPORTING INFORMATION

The Supporting Information is available free of charge at https://pubs.acs.org

Accession Codes

Phosphoproteomic data are available from the PRIDE repository under accession number PXD026098 and may be accessed using username reviewer_pxd026098@ebi.ac.uk and password n40NqB88.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

Supporting Figures 1–5, additional data including 1H15N-HSQC spectrum of NBDY, fluorescence and widefield microscopy, Western blotting

NIHMS1748709-supplement-Supporting_Information.docx (10MB, docx)
Table S1

Table S1, Phosphoproteomic analysis of partially purified NBDY during mitosis and EGFR signaling

NIHMS1748709-supplement-Table_S1.xlsx (4.5MB, xlsx)
Movie S1

Movie S1, Timelapse fluorescence imaging of NBDY-polyU liquid droplets

Download video file (4.1MB, mov)
Movie S2

Movie S2, Timelapse fluorescence microscopy of NBDY-RNA liquid droplets in the presence of PKC

Download video file (7.6MB, mov)

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