IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay

  1. Kotaro Akaki
  2. Kosuke Ogata
  3. Yuhei Yamauchi
  4. Noriki Iwai
  5. Ka Man Tse
  6. Fabian Hia
  7. Atsushi Mochizuki
  8. Yasushi Ishihama
  9. Takashi Mino
  10. Osamu Takeuchi  Is a corresponding author
  1. Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Japan
  2. Graduate School of Biostudies, Kyoto University, Japan
  3. Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan
  4. Laboratory of Mathematical Biology, Institute for Frontier Life and Medical Sciences, Kyoto University, Japan

Abstract

Regnase-1 is an endoribonuclease crucial for controlling inflammation by degrading mRNAs encoding cytokines and inflammatory mediators in mammals. However, it is unclear how Regnase-1-mediated mRNA decay is controlled in interleukin (IL)-1β- or Toll-like receptor (TLR) ligand-stimulated cells. Here, by analyzing the Regnase-1 interactome, we found that IL-1β or TLR stimulus dynamically induced the formation of Regnase-1-β-transducin repeat-containing protein (βTRCP) complex. Importantly, we also uncovered a novel interaction between Regnase-1 and 14-3-3 in both mouse and human cells. In IL-1R/TLR-stimulated cells, the Regnase-1-14-3-3 interaction is mediated by IRAK1 through a previously uncharacterized C-terminal structural domain. Phosphorylation of Regnase-1 at S494 and S513 is critical for Regnase-1-14-3-3 interaction, while a different set of phosphorylation sites of Regnase-1 is known to be required for the recognition by βTRCP and proteasome-mediated degradation. We found that Regnase-1-14-3-3 and Regnase-1-βTRCP interactions are not sequential events. Rather, 14-3-3 protects Regnase-1 from βTRCP-mediated degradation. On the other hand, 14-3-3 abolishes Regnase-1-mediated mRNA decay by inhibiting Regnase-1-mRNA association. In addition, nuclear-cytoplasmic shuttling of Regnase-1 is abrogated by 14-3-3 interaction. Taken together, the results suggest that a novel inflammation-induced interaction of 14-3-3 with Regnase-1 stabilizes inflammatory mRNAs by sequestering Regnase-1 in the cytoplasm to prevent mRNA recognition.

Introduction

The expression of proinflammatory cytokines is the hallmark of innate immune responses against microbial infection. Whereas inflammatory responses are critical for the elimination of invading pathogens, excess and chronic inflammation can culminate in tissue destruction and autoimmune diseases. When innate immune cells encounter pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), they are sensed by pattern-recognition receptors such as Toll-like receptors (TLRs), triggering the transcription of inflammatory genes (Fitzgerald and Kagan, 2020; Takeuchi and Akira, 2010).

The expression of inflammatory genes is also controlled by post-transcriptional mechanisms to facilitate or limit inflammatory responses (Anderson, 2010; Carpenter et al., 2014; Turner and Díaz-Muñoz, 2018). Regnase-1 (also referred to as Mcpip1, Gene name: Zc3h12a), an RNase, is a critical regulator of inflammation. Regnase-1 binds to and degrades inflammatory mRNAs such as Il6 or Il12b by recognizing stem-loop structures present in the 3’ untranslated regions (Matsushita et al., 2009; Mino et al., 2015). Zc3h12a-deficient mice exhibit an autoimmune phenotype, indicating its importance as a negative regulator of inflammation (Matsushita et al., 2009; Uehata et al., 2013). Regnase-1 efficiently suppresses the expression of its target genes by degrading CBP80-bound mRNAs during the pioneer-round of translation by associating with ribosome and a helicase protein, UPF1 (Mino et al., 2015; Mino et al., 2019). CBP80 binds to newly synthesized mRNAs in the nucleus and is replaced by eIF4E after the pioneer round of translation following mRNA export from the nucleus (Maquat et al., 2010; Müller-McNicoll and Neugebauer, 2013). Thus, it is possible that Regnase-1 recognizes target mRNAs in the steps leading to the pioneer round of translation.

The stability of cytokine mRNAs is dynamically regulated in innate immune cells under inflammatory conditions (Carpenter et al., 2014; Hao and Baltimore, 2009; Turner and Díaz-Muñoz, 2018). Post-translational control of Regnase-1 in response to inflammatory stimuli contributes to extending half-lives of inflammatory mRNAs. Stimulation of cells with TLR-ligands, IL-1β, or IL-17 results in the activation of IκB kinases (IKKs), which phosphorylate Regnase-1 at S435 and S439, in addition to IκBα (Iwasaki et al., 2011; Kakiuchi et al., 2020; Nanki et al., 2020; Tanaka et al., 2019). Regnase-1, phosphorylated at S435 and S439 is subsequently recognized by βTRCP, one of the components of the SKP1-CUL1-F-box (SCF) complex, which induces K48-linked polyubiquitination of Regnase-1, followed by proteasome-mediated degradation (Iwasaki et al., 2011). On the other hand, these stimuli also induce transcription of Zc3h12a (Iwasaki et al., 2011). Consequently, the protein level of Regnase-1 drastically changes during these stimulations; Regnase-1 levels decrease immediately after the stimulation and then increase to levels higher than its pre-stimulation. However, the post-translational regulatory mechanism of Regnase-1 following inflammatory stimuli is still not fully elucidated.

14-3-3 family proteins are conserved among species and are known to form hetero- or homodimer (Aitken, 2006; Pennington et al., 2018). The 14-3-3 dimer binds to various phosphorylated proteins using its two phosphor-S/T binding pockets which recognize unique phospho-peptides (Muslin et al., 1996; Yaffe et al., 1997). Although 14-3-3 itself has no enzymatic activity, 14-3-3 is known to modulate the properties of target proteins, such as protein stability or localization (Aitken, 2006; Pennington et al., 2018).

In this study, we utilized an interactome-based approach to isolate Regnase-1 protein complexes and found that TLR-ligand, IL-1β, or IL-17 stimulation induces the formation of the Regnase-1-14-3-3 complex. The phosphorylation of Regnase-1 at S494 and S513 is responsible for binding with 14-3-3, which in turn stabilizes Regnase-1 protein by excluding βTRCP. However, 14-3-3-bound Regnase-1 is not functional because 14-3-3 prevents Regnase-1 from recognizing target mRNAs. In addition, we found that nuclear-cytoplasmic shuttling of Regnase-1 is inhibited by 14-3-3’s association with Regnase-1. Collectively, we identified a novel 14-3-3-mediated molecular mechanism which controls Regnase-1; a distinctly independent mechanism from βTRCP-mediated protein degradation of Regnase-1.

Results

Regnase-1 interactome analysis revealed dynamic recruitment of 14-3-3 upon stimulation

To comprehensively uncover Regnase-1-associating proteins in steady state and under inflammatory conditions, we stimulated HeLa cells expressing FLAG-HA-tagged Regnase-1 with or without IL-1β and immunoprecipitated Regnase-1 immediately after the treatment with a crosslinking reagent, Dithiobis(succinimidyl propionate) (DSP) (Figure 1A). Consistent with the previous reports, mass spectrometry analysis revealed that Regnase-1 interacted with translation-related proteins such as ribosomal proteins in unstimulated cells (Mino et al., 2015). Whereas IL-1β stimulation reduced the association between Regnase-1 and translation-related proteins, the stimulation strongly induced the association between Regnase-1 and SCF complex proteins such as βTRCP1/2, CUL1, and SKP1 (Iwasaki et al., 2011). In addition to these proteins, we identified 14-3-3 family proteins as novel Regnase-1-associating proteins under IL-1β-stimulated conditions (Figure 1B). Consistently, immunoprecipitation analysis revealed that endogenous Regnase-1 was co-precipitated with Myc-tagged 14-3-3ε as well as with HA-tagged βTRCP in HeLa cells in response to IL-1β stimulation (Figure 1C and Figure 1—figure supplement 1).

Figure 1 with 2 supplements see all
IL-1β or TLR1/2/4/7/8/9-ligand stimulation induces Regnase-1-14-3-3 interaction.

(A) Schematic illustration of the DSP-crosslinking workflow. (B) Protein-protein interaction of the Regnase-1 (Reg1)-associating proteins. Each node represents Regnase-1 associating protein. The proteins whose association with Regnase-1 is weakened or enhanced in IL-1β-stimulated cells are colored in blue or red, respectively. (C) Immunoblot analysis of immunoprecipitates (IP: Myc or IP: HA) and WCL (whole cell lysates) from HeLa cells transiently expressing Myc-14-3-3ε and HA-βTRCP1 stimulated with IL-1β (10 ng/ml) for indicated time. (D) Immunoblot analysis of immunoprecipitates (IP: Myc) and WCL from RAW264.7 or RAW264.7 stably expressing Myc-14-3-3ε stimulated with Pam3CSK4 (10 ng/ml), poly I:C (100 μg/ml), LPS (100 ng/ml), R848 (100 nM), or CpG DNA (1 μM) for 4 hr.

As the 14-3-3 family consists of seven paralogs in human and mouse (Aitken, 2006), we investigated the binding of these members to Regnase-1 via immunoprecipitation (Figure 1—figure supplement 2). Among seven of the 14-3-3 proteins, 14-3-3-β, γ, and ε strongly interacted with Regnase-1, while 14-3-3-ζ, η, and θ showed weak interaction. Interestingly, Regnase-1 failed to associate with 14-3-3-σ, the latter of which was reported to exclusively form a homodimer but not a heterodimer with other 14-3-3 isoforms (Verdoodt et al., 2006).

To investigate if stimulation with TLR ligands also induces Regnase-1-14-3-3 binding, we stimulated RAW267.4 macrophages stably expressing Myc-14-3-3ε with Pam3CSK4 (a ligand for TLR1/2), poly I:C (a ligand for TLR3), LPS (a ligand for TLR4), R848 (a ligand for TLR7/8), or CpG DNA (a ligand for TLR9), and immunoprecipitated 14-3-3ε with an anti-Myc antibody. The Regnase-1-14-3-3 interaction was induced by all TLR ligands tested except for poly I:C (Figure 1D). All TLRs other than TLR3 signal through MyD88, while TLR3 utilizes TRIF as an adaptor to trigger intracellular signaling (Fitzgerald and Kagan, 2020; O'Neill et al., 2013; Takeuchi and Akira, 2010). Considering that IL-1β signal is also dependent on MyD88 (Akira et al., 2006), MyD88-dependent, but not TRIF-dependent, signaling pathways trigger the Regnase-1-14-3-3 binding.

Collectively, these results demonstrate that IL-1R/TLR stimulation induces dynamic remodeling of the Regnase-1-associating protein complex from translation machineries to SCF complexes and/or 14-3-3 proteins.

Phosphorylation of Regnase-1 at S494 and S513 is necessary for Regnase-1-14-3-3 binding

Since 14-3-3 proteins are known to recognize phosphorylated proteins (Muslin et al., 1996), we investigated if 14-3-3-bound Regnase-1 is phosphorylated by inflammatory stimuli. SDS-PAGE analysis revealed that Regnase-1 band migration was slower in samples stimulated with IL-1β or TLR ligands (except for a TLR3 ligand, poly I:C) - a hallmark of Regnase-1 phosphorylation (Figures 1C–D and 2A, and Figure 2—figure supplement 1; Iwasaki et al., 2011; Tanaka et al., 2019). Indeed, the mobility change of Regnase-1 was abolished when the cell lysates were treated with λ-protein phosphatase (λPP) (Figure 2A–B). Furthermore, the Regnase-1 band in the 14-3-3-precipitate migrated slower; λPP treatment of the 14-3-3-precipitate abolished this phenomenon (Figure 2A–B). Thus, 14-3-3 specifically binds to phosphorylated Regnase-1.

Figure 2 with 8 supplements see all
IL-1β-induced phosphorylation of Regnase-1 at S494 and S513 is necessary for Regnase-1-14-3-3 binding.

(A) Immunoblot analysis of λPP-treated immunoprecipitates (IP: HA) and WCL from HeLa cells transiently expressing HA-14-3-3ε stimulated with IL-1β (10 ng/ml) for 4 hr. S.E.: short exposure, L.E.: long exposure. (B) The intensity of Regnase-1-bands in (A). (C) Quantitation of phosphosites on Regnase-1 in HeLa cells stimulated with or without IL-1β (10 ng/ml) for 4 hr. Each dot shows phosphosite quantitative ratio between IL-1β + and IL-1β -. Phosphosites with log2 ratio > one were colored with red. Black horizontal line shows Regnase-1 protein quantitative ratio derived from the average of non-phosphopeptide quantitative ratios, and its error bars show the standard deviation. (D) Immunoblot analysis of immunoprecipitates (IP: HA) and WCL from HeLa cells transiently expressing HA-14-3-3ε and FLAG-Regnase-1-WT or indicated mutants stimulated with IL-1β (10 ng/ml) for 4 hr. (E) Schematic illustration of Regnase-1 protein. The amino acid sequence including S494 and S513 shown in (F) is highlighted in green. NTD: N-terminal domain, ZF: Zinc finger domain, CTD: C-terminal domain. (F) The amino acid sequences including S494 and S513 of Regnase-1 from mouse and other indicated vertebrates. (G) Immunoblot analysis of immunoprecipitates (IP: Myc) and WCL from HeLa cells transiently expressing Myc-14-3-3ε, HA-IRAK1/2, and FLAG-Regnase-1-WT or indicated mutants. (H) Schematic illustration of IRAK1 protein. The amino acid sequence in CSD of IRAK1 and IRAK2 from mouse and other indicated vertebrates are also shown. DD: Death domain, CSD: C-terminal structural domain. (I) Immunoblot analysis of immunoprecipitates (IP: HA) and WCL from HeLa cells transiently expressing FLAG-Regnase-1-WT, HA-14-3-3ε, and Myc-IRAK1-WT or indicated mutants.

We next scrutinized Regnase-1 phosphorylation sites induced by IL-1β stimulation to identify phosphorylation sites critical for the Regnase-1-14-3-3 interaction. We purified FLAG-HA-Regnase-1 from HeLa cells stimulated with or without IL-1β and identified IL-1β-inducible phosphorylation sites by LC-MS/MS (Figure 2C and Figure 2—figure supplement 2). We found that the phosphorylation at S21, S61, S62, S362, S439, S470, S494, and S513 of Regnase-1 was increased in response to IL-1β stimulation. To identify Regnase-1 phosphorylation sites responsible for binding with 14-3-3, we mutated serine residues on Regnase-1 phosphorylation sites into alanine and probed its association with 14-3-3. Among the Regnase-1-SA mutants, S494A and S513A mutants failed to be co-precipitated with 14-3-3 (Figure 2D), indicating that phosphorylation at both of S494 and S513 is necessary for the Regnase-1-14-3-3 interaction. Both phosphorylation sites harbor a pSxP sequence, which shows similarity with a known 14-3-3-binding motif, RxxpSxP, mode 1 (Yaffe et al., 1997). Noteworthy, amino acid sequences surrounding S494 and S513 are highly conserved among many species (Figure 2E–F).

We next investigated the mechanism of how Regnase-1 phosphorylation is regulated by inflammatory stimuli. In response to IL-1β or TLR ligands stimulation, MyD88 associates with IRAK kinases, IRAK1 and IRAK2, via the death domain (Gottipati et al., 2008; Wesche et al., 1997). A part of C-terminal region of IRAKs in turn interacts with TRAF6 to activate NF-κB (Ye et al., 2002). It was shown that Irak1 and Irak2 double deficiency abolished the phosphorylation of Regnase-1 after LPS stimulation (Iwasaki et al., 2011). We found that gene depletion of IRAK1/2 using the CRISPR-Cas9 system in HeLa cells severely impaired the association between Regnase-1 and 14-3-3 as well as the phosphorylation of Regnase-1 in response to IL-1β stimulation (Figure 2—figure supplement 3). Reciprocally, overexpression of IRAK1 and IRAK2-induced Regnase-1-14-3-3 binding (Figure 2G). In contrast, the interaction between Regnase-1 and 14-3-3 was not induced by the expression of a kinase-inactive mutant (T209A) IRAK1 (Kollewe et al., 2004) or a deletion mutant lacking death domain (Δ1–103) of IRAK1, indicating that the Regnase-1-14-3-3 binding requires the IRAK1 kinase activity as well as recruitment to MyD88 (Figure 2H–I). Although the C-terminal 619–710 portion of IRAK1 was also required for Regnase-1-14-3-3 binding, point mutations in TRAF6 binding sites (E541/E584/E704A) (Ye et al., 2002) did not abolish the Regnase-1-14-3-3 binding (Figure 2H–I). In silico prediction suggested the presence of a C-terminal structural domain (CSD) in the 619–710 of IRAK1 (Figure 2—figure supplement 4). In the CSD of IRAK1, highly conserved amino acids, R663 and K665, are critical for the Regnase-1-14-3-3 binding (Figure 2H–I), suggesting that the CSD of IRAK1 controls Regnase-1-14-3-3 interaction irrespective of the recruitment of TRAF6. Of note, the R663/K665A mutant IRAK1 was capable of activating NF-κB (Figure 2—figure supplement 5), indicating that the IRAK1 C-terminal region has two distinct functions: NF-κB activation through TRAF6-binding sites and the induction of Regnase-1-14-3-3 interaction through the CSD.

S494 and S513 of Regnase-1 are also reported to be phosphorylated by overexpression of Act1 together with TANK-binding kinase 1 (TBK1) or IKK-i/ε, which mimics IL-17 signaling (Tanaka et al., 2019). We detected phosphorylation at S494 and S513 of Regnase-1 in IL-17A-stimulated cells as well as IL-1β-stimulated cells by LC-MS/MS (Figure 2—figure supplements 6 and 7). Furthermore, we found that IL-17A stimulation also induced Regnase-1-14-3-3 binding (Figure 2—figure supplement 8).

Collectively, these data demonstrate that the IRAK-dependent phosphorylation of Regnase-1 at S494 and S513 is necessary for the association between Regnase-1 and 14-3-3.

βTRCP binds to 14-3-3-free Regnase-1

MyD88-dependent signaling also induces IKK-mediated phosphorylation of Regnase-1 at S435 and S439, which allows recognition of Regnase-1 by βTRCP (Iwasaki et al., 2011). With this, we examined the relationship between the association of Regnase-1 to 14-3-3 and to βTRCP. We found that Regnase-1 harboring S435A and S439A mutations permitted the interaction with 14-3-3 but failed to recruit βTRCP (Figure 3A–B). Reciprocally, the S494A or S513A mutation of Regnase-1 did not inhibit the association between Regnase-1 and βTRCP (Figure 3B), indicating that the phosphorylation of Regnase-1 at S494 or S513 or the Regnase-1-14-3-3 binding is dispensable for the Regnase-1-βTRCP association. We next compared the phosphorylation status of βTRCP-bound and 14-3-3-bound Regnase-1. Since βTRCP-mediated polyubiquitination potentially alters the molecular weight of Regnase-1, we utilized a βTRCP mutant which is unable to induce polyubiquitination due to the lack of the F-box domain (βTRCP-ΔF). Interestingly, the SDS-PAGE analysis revealed that βTRCP-ΔF-bound Regnase-1 migrated faster than 14-3-3-bound Regnase-1 (Figure 3C–D), indicating that βTRCP likely binds to 14-3-3-free Regnase-1.

βTRCP binds to 14-3-3-free Regnase-1.

(A) Immunoblot analysis of immunoprecipitates (IP: Myc) and WCL from HeLa cells transiently expressing Myc-14-3-3ε and HA-Regnase-1-WT or indicated mutants stimulated with IL-1β (10 ng/ml) for 4 hr. (B) Immunoblot analysis of immunoprecipitates (IP: FLAG) and WCL from HeLa cells transiently expressing FLAG-βTRCP1 and HA-Regnase-1-WT or indicated mutants stimulated with IL-1β (10 ng/ml) for 4 hr. (C) Immunoblot analysis of immunoprecipitates (IP: FLAG or HA) and WCL from HeLa cells transiently expressing FLAG-βTRCP-ΔF and HA-14-3-3ε stimulated with IL-1β (10 ng/ml) for 4 hr. S.E.: short exposure, L.E.: long exposure. (D) The intensity of Regnase-1-bands in (C).

These results demonstrate that the binding of Regnase-1 to 14-3-3 and βTRCP occurs independently although IL-1β stimulation simultaneously induces phosphorylation of Regnase-1 at S494 and S513 as well as S435 and S439. In addition, 14-3-3 inhibits the Regnase-1-βTRCP binding.

The S513A mutation destabilizes Regnase-1 protein without affecting target mRNA abundance

To evaluate the functional roles of Regnase-1-14-3-3 interaction, we generated Zc3h12aS513A/S513A knock-in mice (Figure 4—figure supplement 1). Zc3h12aS513A/S513A mice did not show gross abnormality, nor did they exhibit alteration in the numbers of T, B cells or macrophages (data not shown). We stimulated mouse embryonic fibroblasts (MEFs) derived from Zc3h12aWT/WT and Zc3h12aS513A/S513A mice with IL-1β and checked Regnase-1 expression (Figure 4A). Immunoblot analysis revealed that Regnase-1 was degraded 30 min after stimulation in both WT and S513A mutant MEFs. Following this, Regnase-1 levels increased in WT MEFs at 2 and 4 hr after stimulation (Figure 4A). Notably, most of the newly synthesized Regnase-1 showed slow migration, consistent with the immunoprecipitation experiment using HeLa cells or RAW264.7 cells shown in Figure 1C and D. On the other hand, the slowly migrating Regnase-1 band did not appear in Zc3h12aS513A/S513A MEFs after IL-1β stimulation. Interestingly, the amount of Regnase-1 at lower bands, which are not the binding target of 14-3-3 (Figure 2A), was comparable between WT and Zc3h12aS513A/S513A at corresponding time points. Consequently, total Regnase-1 protein expression was severely reduced in Zc3h12aS513A/S513A MEFs compared with WT after IL-1β stimulation (Figure 4A). Similar results were also obtained when bone marrow-derived macrophages (BMDMs) and thioglycollate-elicited peritoneal exudate cells (PECs) derived from Zc3h12aWT/WT and Zc3h12aS513A/S513A mice were stimulated with LPS (Figure 4B–C). Nevertheless, Zc3h12a mRNA levels were comparable between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells (Figure 4D–F), suggesting that S513A mutation affects the protein stability of Regnase-1. To address this, we examined the kinetics of Regnase-1 degradation following LPS stimulation by treating cells with cycloheximide (CHX). Indeed, Regnase-1-S513A was more rapidly degraded than Regnase-1-WT in PECs after LPS stimulation (Figure 4—figure supplement 2). Furthermore, treatment of Zc3h12aS513A/S513A PECs with MG-132, a proteasome inhibitor, resulted in the increase of smearing in the band patterns of Regnase-1 in LPS-stimulated cells (Figure 4C), possibly due to the inhibition of degradation of polyubiquitinated Regnase-1. These data indicate that the phosphorylation of Regnase-1 at S513 stabilizes Regnase-1 protein after IL-1β or LPS stimulation by binding with 14-3-3.

Figure 4 with 5 supplements see all
The S513A mutation destabilizes Regnase-1 protein but does not affect target mRNA abundance.

(AC) Immunoblot analysis of Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml) (A), BMDMs stimulated with LPS (100 ng/ml) (B), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) (C) for indicated time. PECs were pretreated with MG-132 (5 μM) 2 hr before the stimulation. (D)-(F) mRNA expression of Zc3h12a and Il6 in Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml) for 4 hr (D), BMDMs stimulated with LPS (100 ng/ml) for 4 hr (E), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) for indicated time (F). (G)-(I) IL-6 secretion in Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml), IL-17A (50 ng/ml), or TNF (10 ng/ml) for 24 hr (G), BMDMs stimulated with Pam3CSK4 (1 or 10 ng/ml), poly I:C (10 or 100 μg/ml), LPS (10 or 100 ng/ml), R848 (10 or 100 nM), or CpG DNA (0.1 or 1 μM) for 24 hr (H), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml), R848 (100 nM), or IL-1β (10 ng/ml) for 24 hr (I). (J) Schematic representation of Model 1 in which 14-3-3-bound Regnase-1 does not have the function of degrading its target mRNAs. This model could explain the experimental observations. (K) Schematic representation of Model 2 in which 14-3-3-bound Regnase-1 maintains some ability to degrade its target mRNAs. This model is not consistent with the experimental observations. In (D)-(I), bars represent mean values of biological replicates (n = 3), and error bars represent standard deviation. Data is representative of two independent experiments, each with three biological replicates.

We next checked whether the altered Regnase-1 expression by the S513A mutation affects Regnase-1-mediated mRNA decay. Despite the huge difference in Regnase-1 expression, the expression of Il6, a transcript degraded by Regnase-1 (Figure 4—figure supplement 3), was comparable between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells (Figure 4D–I). In addition, the stability of Regnase-1 target mRNAs including Il6, Zc3h12a, and Nfkbiz was equivalent between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells (Figure 4—figure supplement 4). Furthermore, even when we analyzed gene expression profile comparing Zc3h12aWT/WT and Zc3h12aS513A/S513A macrophages by an RNA-seq analysis (Figure 4—figure supplement 5), we did not identify any differentially expressed genes (adj p<0.05) between Zc3h12aWT/WT and Zc3h12aS513A/S513A macrophages irrespective of the stimulation with LPS.

To examine the mechanisms underlying these observations, we developed two mathematical models based on our previous studies (see Materials and methods) (Iwasaki et al., 2011; Mino et al., 2019). The first model (Model 1) assumes that 14-3-3-bound Regnase-1 is unable to degrade its target mRNAs (Figure 4J). The second model (Model 2) assumes that Regnase-1 binding with 14-3-3 maintains its ability to degrade its target mRNAs to a certain extent (Figure 4K). Mathematical analysis showed that in Model 2, the abundance of the Il6 mRNAs should be different between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells under the condition that the amount of 14-3-3-free Regnase-1 protein (lower bands in Figure 4A–C) is comparable between them. Our observations that the abundance of the target mRNAs did not differ between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells in the late phase of stimulation is inconsistent with Model 2, suggesting that Regnase-1 is inactivated upon binding to 14-3-3.

These results imply that the phosphorylation at S513 and the following association with 14-3-3 nullifies Regnase-1’s ability in degrading target mRNAs, although it stabilizes and significantly upregulates the abundance of Regnase-1.

14-3-3 inactivates Regnase-1 by inhibiting Regnase-1-RNA binding

The mathematical analysis suggests that 14-3-3-bound Regnase-1 is inactive as the S513A mutation failed to affect Il6 expression in MEFs or macrophages. To examine if this comparable Il6 expression was due to increased degradation of Regnase-1-S513A protein via βTRCP, we further mutated βTRCP-recognition sites, S435 and S439, to alanine in Regnase-1-S513A (Regnase-1-S435/439/513A). As shown in Figure 5A, the Regnase-1-S435/439/513A mutant was more abundantly expressed than Regnase-1-S513A after IL-1β stimulation, indicating that Regnase-1-S513A is degraded via the association with βTRCP. It is noteworthy that most of Regnase-1-S435/439/513A showed fast migration, whereas the majority of Regnase-1-S435/439A migrated slowly in response to the stimulation. To verify if 14-3-3-bound Regnase-1 is functional or not, we assessed the target mRNA suppression activity of each mutant by checking the expression of Il6 co-transfected with Regnase-1. Regnase-1-S435/439/513A was more potent in suppressing Il6 expression compared to WT or other SA mutants, S513A and S435/439A, in response to IL-1β stimulation (Figure 5B). These results indicate that IL-1β stimulation regulates the activity of Regnase-1 by two independent mechanisms via 14-3-3 and βTRCP, respectively.

Figure 5 with 2 supplements see all
14-3-3 bound to phospho-S494 and S513 inactivates Regnase-1 by inhibiting Regnase-1-mRNA binding.

(A) Immunoblot analysis of ZC3H12A-KO HeLa cells transiently expressing Regnase-1-WT or indicated mutants. Cells were stimulated with IL-1β (10 ng/ml) for indicated time. (B) mRNA expression of Il6 in HeLa cells transiently expressing Regnase-1-WT or indicated mutants together with Il6. Cells were stimulated with IL-1β (10 ng/ml) for indicated time. (C) Immunoblot analysis of immunoprecipitates (IP: FLAG) and WCL from HeLa cells transiently expressing FLAG-Regnase-1-WT or indicated mutants. For the IL-1β stimulation, cells were stimulated with IL-1β (10 ng/ml) for 4 hr. L.C.: light chain. (D) Schematic illustration of Regnase-1 and the amino acid sequences of Regnase-1-WT, -ExoSx2, and ExoSAAAx2. NTD: N-terminal domain, ZF: Zinc finger domain, CTD: C-terminal domain. (E) mRNA expression of Il6 in HeLa cells transiently expressing Regnase-1-WT or indicated mutants together with Il6. (F) Secreted IL6 concentration in (E). (G) mRNA expression of Il6 in HeLa cells transiently expressing Regnase-1-WT and IRAK1-WT or indicated mutants together with Il6. (H) The amount of IL6 mRNAs immunoprecipitated with FLAG-Regnase-1-D141N or other indicated mutants in HeLa cells. In (B), (E)-(H), bars represent mean values of biological replicates (n = 3), and error bars represent standard deviation. p-Values were calculated using unpaired, two-sided t-test. Data is representative of two independent experiments, each with three biological replicates.

To further examine the mechanism of how 14-3-3 inactivates Regnase-1, we attempted to generate a Regnase-1 mutant which constitutively binds to 14-3-3 even without stimulation. We generated a phospho-mimic mutant of Regnase-1 (S494D/S513D). However, this mutant failed to bind 14-3-3 (Figure 5C), indicating that the phosphate moiety, but not negative charge, is recognized by 14-3-3. Then we utilized a sequence of Exoenzyme S (ExoS), which is a bacterial protein derived from Pseudomonas aeruginosa and is known to bind to 14-3-3 without phosphorylation (Fu et al., 1993; Karlberg et al., 2018; Masters et al., 1999). The 22 amino acids of Regnase-1 covering S494 and S513 were substituted with two ExoS (419-429) sequences (Figure 5D). As a control, we additionally mutated Regnase-1-ExoSx2 by substituting its core sequences for 14-3-3 binding (L426, D427, and L428) with alanine residues (Regnase-1-ExoSAAAx2) (Ottmann et al., 2007; Yasmin et al., 2006). We observed that Regnase-1-ExoSx2, but not Regnase-1-ExoSAAAx2, interacted with endogenous 14-3-3 without any stimulation (Figure 5C). Using these mutants, we investigated whether 14-3-3 binding alters the activity of Regnase-1 to suppress Il6 expression. Consistent with its 14-3-3 binding capacity, Regnase-1-ExoSx2, but not Regnase-1-ExoSAAAx2 and -S494D/S513D, lost the activity to inhibit Il6 expression (Figure 5E). Furthermore, the production of IL-6 protein was similarly inhibited depending on the capacity of Regnase-1 to bind 14-3-3 (Figure 5F). In addition, Regnase-1-mediated suppression of Il6 expression was impaired by the overexpression of IRAK1-WT and E541/E584/E704A mutants, both of which induce Regnase-1-14-3-3 association (Figure 5G). On the other hand, IRAK1 mutants that failed to induce the Regnase-1-14-3-3 association (T209A, Δ1–103, Δ619–710, and R663/K665A) did not affect the activity of Regnase-1.

We next examined how 14-3-3 inhibits the activity of Regnase-1 by investigating Regnase-1-mRNA binding activity using various Regnase-1 mutants in HeLa cells. To stabilize Regnase-1-RNA binding, we generated a nuclease inactive version of Regnase-1 by introducing the D141N mutation to each of Regnase-1 mutant (Matsushita et al., 2009; Figure 5—figure supplement 1). As shown in Figure 5H, forced interaction of Regnase-1-D141N with 14-3-3 by the ExoSx2 mutation in HeLa cells abrogated the binding with IL6 mRNA, whereas IL6 was co-precipitated with Regnase-1-D141N, -ExoSAAAx2-D141N and -S494D/S513D-D141N (Figure 5H). In addition to IL6, Regnase-1-ExoSx2 failed to bind to other reported target mRNAs such as NFKBIZ, PTGS2, and CXC chemokines (Figure 5—figure supplement 2). Collectively, these data demonstrate that 14-3-3 inhibits Regnase-1-mRNA binding, thereby abrogating Regnase-1-mediated mRNA degradation.

14-3-3 inhibits nuclear import of Regnase-1

We have previously shown that Regnase-1 interacts with CBP80-bound, but not eIF4E-bound, mRNAs (Mino et al., 2019), indicating that Regnase-1 degrades mRNAs immediately after the export from the nucleus to the cytoplasm (Maquat et al., 2010; Müller-McNicoll and Neugebauer, 2013). Although Regnase-1 mainly localizes in the cytoplasm (Mino et al., 2015), we hypothesized Regnase-1 shuttles between the nucleus and the cytoplasm to recognize its target mRNAs in association with their nuclear export. To test this hypothesis, we examined the subcellular localization of Regnase-1 following the treatment with Leptomycin B (LMB), which inhibits CRM1 (also known as Exportin-1)-mediated protein export from the nucleus to the cytoplasm (Yashiroda and Yoshida, 2003). Although Regnase-1 localized in the cytoplasm in the steady state condition, LMB treatment induced rapid accumulation of Regnase-1 in the nucleus within 30 min (Figure 6A). These results suggest that Regnase-1 dynamically changes its localization between the cytoplasm and the nucleus. Given that Regnase-1 dominantly localizes in the cytoplasm in the steady state conditions, the frequency of its nuclear export seems to be higher than its nuclear import.

14-3-3 inhibit nuclear-cytoplasmic shuttling of Regnase-1.

(A) Immunofluorescence analysis of HeLa cells transiently expressing FLAG-Regnase-1-WT treated with Leptomycin B (LMB) (10 ng/ml) for indicated time. (B) The result of NES prediction of Regnase-1 by LocNES. Higher score indicates higher probability. (C) Schematic illustration of Regnase-1. The amino acid sequence shown in (D) is highlighted in blue. NTD: N-terminal domain, ZF: Zinc finger domain, CTD: C-terminal domain. (D) The amino acid sequences including S435/S439 and NES of Regnase-1 from mouse and other indicated vertebrates. (E) Immunofluorescence analysis of HeLa cells transiently expressing FLAG-Regnase-1-WT or indicated mutants. (F) Immunofluorescence analysis of HeLa cells transiently expressing FLAG-Regnase-1-WT or indicated mutants treated with LMB (10 ng/ml) for 1 hr. (G) Model of 14-3-3- and βTRCP-mediated regulation of Regnase-1. In the steady state, Regnase-1 shuttles between the nucleus and the cytoplasm and degrades target mRNAs such as Il6. Under IL-1β or TLR-ligands stimulation, two different regulatory mechanisms suppress the activity of Regnase-1 not to disturb proper expression of inflammatory genes; βTRCP induces protein degradation of Regnase-1 and 14-3-3 inhibits nuclear-cytoplasmic shuttling and mRNA recognition of Regnase-1. In (A), (E), and (F), white scale bars indicate 20 μm.

CRM1 is known to recognize a nuclear export signal (NES) of a cargo protein for the protein export (Hutten and Kehlenbach, 2007). Thus, we investigated if Regnase-1 harbors a NES. In silico prediction deduced amino acids 433–447 of Regnase-1 as a potential NES with high probability (Xu et al., 2015; Figure 6B–D). Indeed, Regnase-1 lacking 422–451 spontaneously accumulated in the nucleus (Figure 6E). Since NESs are characterized by hydrophobic residues (la Cour et al., 2003), we also inspected which hydrophobic resides of Regnase-1 were important for the efficient nuclear export of Regnase-1. We found that L440, M444, L447, and W448 of Regnase-1 were critical for the nuclear export of Regnase-1 (Figure 5E). Noteworthy, all the residues are highly conserved among species (Figure 5D).

We next examined whether 14-3-3 binding controls the localization of Regnase-1. Interestingly, Regnase-1-ExoSx2 failed to accumulate in the nucleus even after LMB treatment while Regnase-1-WT and -ExoSAAAx2 accumulated in the nucleus by LMB treatment (Figure 6F). This result indicates that Regnase-1-ExoSx2 is unable to translocate into the nucleus like Regnase-1-WT. Taken together, 14-3-3 inhibits the nuclear import of Regnase-1 as well as its binding to target mRNAs.

Discussion

In the present study, we discovered that IL-1β and TLR stimulation dynamically changes protein-protein interaction of Regnase-1. Particularly, these stimuli trigger the interaction of Regnase-1 with 14-3-3 as well as βTRCP via phosphorylation at distinct amino acids. Whereas phosphorylation of Regnase-1 at S494 and S513 is recognized by 14-3-3, βTRCP associates with Regnase-1 phosphorylated at S435 and S439. We demonstrated that each Regnase-1-14-3-3 and Regnase-1-βTRCP binding occurs independently. Furthermore, 14-3-3 prevent Regnase-1-βTRCP binding, resulting in protein stabilization of 14-3-3-bound Regnase-1.

The amino acid sequence surrounding S494 and S513 of Regnase-1 is widely conserved among species. Particularly, both S494 and S513 harbor the pSxP sequence motif, which overlaps with a known 14-3-3 binding motif, RxxpSxP, mode 1 (Yaffe et al., 1997). It is quite plausible that the 14-3-3 dimer directly binds to phosphorylated S494 and S513 of Regnase-1 with the phospho-peptide binding groove. Interestingly, only 14-3-3σ, which forms a homodimer but not a heterodimer with other 14-3-3 isoforms (Verdoodt et al., 2006), failed to bind with Regnase-1 (Figure 1—figure supplement 2). This result might be a clue to elucidate the target specificity of each 14-3-3 paralog.

14-3-3 and βTRCP inhibit Regnase-1-mediated mRNA decay via distinct mechanisms; 14-3-3 prevents Regnase-1-mRNA binding while βTRCP induces protein degradation of Regnase-1. Analysis of Zc3h12aS513A/S513A mice revealed that 14-3-3-mediated abrogation of Regnase-1 can be compensated by the degradation of Regnase-1. The presence of this dual regulatory system underscores the importance of restricting the activity of Regnase-1 to ensure proper inflammatory gene expression when cells encounter PAMPs or DAMPs (Figure 6G).

Notably, exome sequence analysis of the colon samples from ulcerative colitis patients discovered mutations in the βTRCP binding site of Regnase-1 (Kakiuchi et al., 2020; Nanki et al., 2020). Furthermore, a previous report showed that Zc3h12aS435/S439A mutant mice were resistant to experimental autoimmune encephalomyelitis (EAE) (Tanaka et al., 2019). All these mutations abolish βTRCP-mediated degradation of Regnase-1. However, genetic association between the 14-3-3-binding site of Regnase-1 and inflammatory diseases has not been identified so far. This is possibly because of the compensation by βTRCP-mediated regulation, which we observed in Zc3h12aS513A/S513A mice and HeLa cells transiently expressing Regnase-1-S513A and S435/439/513A. Previous studies have shown that viral proteins or lncRNAs inhibit βTRCP-mediated protein degradation (Guo et al., 2020; Neidel et al., 2019; van Buuren et al., 2014; Yang et al., 2020). 14-3-3-mediated regulation of Regnase-1 may serve as a backup mechanism to control the activity of Regnase-1 when βTRCP-mediated protein degradation is dysregulated.

While βTRCP regulates the abundance of Regnase-1 through protein degradation, 14-3-3 regulates the activity of Regnase-1. We found that 14-3-3-bound Regnase-1 failed to associate with mRNAs, indicating that 14-3-3 prevents Regnase-1 from recognizing target mRNA. We have previously shown that an RNase domain and an adjacent zinc finger domain play an important role in Regnase-1-RNA binding (Yokogawa et al., 2016). However, the 14-3-3-binding site of Regnase-1 is in the C-terminal part of Regnase-1, which is distant from RNase and zinc finger domains. Therefore, 14-3-3 is unlikely to inhibit Regnase-1-mRNA binding by simple competition between 14-3-3 and mRNAs for the RNA binding domain of Regnase-1. We have previously reported that Regnase-1 interacts with CBP80-bound, but not eIF4E-bound, mRNAs, indicating that Regnase-1 recognizes its target mRNA before or immediately after the nuclear export of the mRNA (Mino et al., 2019). In this study, we found that Regnase-1 shuttles between the nucleus and the cytoplasm while 14-3-3-bound Regnase-1 cannot enter the nucleus. Thus, it is tempting to speculate that Regnase-1 recognizes mRNA in the nucleus and induce mRNA decay during pioneer rounds of translation immediately after the nuclear export (Maquat et al., 2010; Müller-McNicoll and Neugebauer, 2013). Nevertheless, further investigation is required to clarify the mechanisms of Regnase-1-mediated mRNA decay depending on its nuclear-cytoplasmic shuttling. In addition, further studies are also necessary to clarify the molecular mechanisms how 14-3-3 controls the nuclear-cytoplasmic shuttling of Regnase-1 as well as how it regulates Regnase-1-mRNA binding by exploiting systems beyond the ExoS sequence-mediated interaction with 14-3-3.

βTRCP is likely to recognize 14-3-3-free Regnase-1, indicating that 14-3-3 inhibits Regnase-1-βTRCP interaction. There are two possible mechanisms to explain this. One posits that 14-3-3 bound to phosphorylated S494 and S513 of Regnase-1 conceals βTRCP-binding site (pS435 and pS439), although the 14-3-3-binding site does not overlap with βTRCP-binding site completely. The other possible explanation is that 14-3-3-mediated inhibition of nuclear-cytoplasmic shuttling of Regnase-1 controls βTRCP-mediated Regnase-1 degradation. Indeed, βTRCP localizes not only in the cytoplasm, but also in the nucleus (Davis et al., 2002). It is plausible that 14-3-3 prevents Regnase-1-βTRCP interaction in the nucleus, by inhibiting nuclear-cytoplasmic shuttling of Regnase-1. Of note, the NES of Regnase-1 is located just adjacent to βTRCP-binding site (Figure 6C–D), implying possibility of competitive binding of βTRCP and CRM1 to Regnase-1.

Among the molecules involved in MyD88-dependent signaling, we found that IRAK1/2 are potent inducers of the interaction between Regnase-1 and 14-3-3, thereby abrogating Regnase-1-mediated mRNA decay. We also found that kinase-inactive IRAK1 failed to induce the Regnase-1-14-3-3 complex, suggesting that the kinase activity of IRAK1 is required for the phosphorylation of Regnase-1 at S494 and S513. However, previously identified substrate sequence motifs of IRAK1, pSxV, and KxxxpS (Sugiyama et al., 2019) do not match the sequence surrounding S494 and S513 of Regnase-1 (Figure 2F). Although the motif analysis does not exclude the possibility of direct phosphorylation of Regnase-1 by IRAK1, it is possible that kinases activated by IRAK1/2 phosphorylates Regnase-1 at S494 and S513.

IRAKs are involved in stabilization of inflammatory mRNAs as well as NF-κB activation (Flannery et al., 2011; Hartupee et al., 2008; Wan et al., 2009). A previous study showed that IRAK1-mediated mRNA stabilization does not require IRAK1-TRAF6 association (Hartupee et al., 2008). Interestingly, the IRAK1-TRAF6 association is also dispensable for the Regnase-1-14-3-3 binding. Instead, other evolutionarily conserved amino acids in the C-terminal structural domain (CSD) of IRAK1, R663, and K665, are critical for Regnase-1-14-3-3 binding. Considering 14-3-3-mediated inactivation of Regnase-1, it is plausible that the CSD of IRAK1 is the key for stabilization of inflammatory mRNAs.

In summary, Regnase-1 interactome analysis revealed dynamic 14-3-3-mediated regulation of Regnase-1 in response to IL-1β and TLR stimuli. Since recent studies identified Regnase-1 as a high-potential therapeutic target in various diseases (Kakiuchi et al., 2020; Nanki et al., 2020; Wei et al., 2019), our findings may help maximize the effect of Regnase-1 modulation or provide an alternative way to control the activity of Regnase-1.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Zc3h12aNAGene ID: 230738
Gene (Homo sapiens)ZC3H12ANAGene ID: 80149
Strain, strain background (Mus musculus)Zc3h12aWT/WTCLEA JapanC57BL/6C57BL/6JJcl
Strain, strain background (Mus musculus)Zc3h12a-/-https://doi.org/10.1038/nature07924
Strain, strain background (Mus musculus)Zc3h12aS513A/S513Athis papergenerated using CRISPR-Cas9 system
Sequence-based reagentDNA oligo (for pX330)this paperCACCGCGGCTCAGACCAGTACTCTCfor Zc3h12aS513A/S513A generation
Sequence-based reagentDNA oligo (for pX330)this paperAAACGAGAGTACTGGTCTGAGCCGCfor Zc3h12aS513A/S513A generation
Sequence-based reagentDonor single strand oligothis paperGAAGGACAGGAGTGGGTGGGGGTAATGGGTACGGCTCAGCCCAGTACTCTCTGGATGGGTAGGTGGGTGGCGGGGGCACAfor Zc3h12aS513A/S513A generation
Sequence-based reagentDNA oligo (for pX459-IRAK1KO)https://doi.org/10.1093/bioinformatics/btu743CACCGGTCTGGTCGCGCACGATCA
Sequence-based reagentDNA oligo (for pX459-IRAK1KO)https://doi.org/10.1093/bioinformatics/btu743AAACTGATCGTGCGCGACCAGACC
Sequence-based reagentDNA oligo (for pX459-IRAK2KO)https://doi.org/10.1038/nbt.3437CACCGAAAACCGCAAAATCAGCCAG
Sequence-based reagentDNA oligo (for pX459-IRAK2KO)https://doi.org/10.1038/nbt.3437AAACCTGGCTGATTTTGCGGTTTTC
AntibodyAnti-mouse-Regnase-1
(rabbit polyclonal)
MBL life sciencecustom antibody production
(1:1000)
AntibodyAnti-human-Regnase-1
(rabbit polyclonal)
Atlas AntibodiesCat # HPA032053(1:500)
AntibodyAnti-14-3-3 (pan)
(mouse monoclonal)
Santa Cruz BiotechnologyCat # sc-1657(1:1000)
AntibodyAnti-IκB-α
(rabbit polyclonal)
Santa Cruz BiotechnologyCat # sc-371(1:1000)
AntibodyAnti-IRAK1
(mouse monoclonal)
Santa Cruz BiotechnologyCat # sc-5288(1:500)
AntibodyAnti-FLAG
(mouse monoclonal)
SigmaCat # F3165(WB:1:2000, IF:1:5000)
AntibodyAnti-FLAG
(rabbit polyclonal)
SigmaCat # F7425(1:2000)
AntibodyAnti-HA
(mouse monoclonal)
SigmaCat # H3663(1:2000)
AntibodyAnti-HA
(rabbit polyclonal)
SigmaCat # H6908(1:2000)
AntibodyAnit-Myc
(mouse monoclonal)
SigmaCat # M4439(1:2000)
AntibodyAnti-Myc
(rabbit polyclonal)
SigmaCat # C3956(1:2000)
AntibodyAnti-β-Actin-HRP
(mouse monoclonal)
Santa Cruz BiotechnologyCat # sc-47778-HRP(1:2000)
AntibodyAnti-Mouse IgG-HRP F(ab')2
(sheep polyclonal)
cytivaCat # NA9310-1ML(1:5000)
AntibodyAnti-Rabbit IgG-HRP F(ab')2
(donkey polyclonal)
cytivaCat # NA9340-1ML(1:5000)
AntibodyF(ab')2-anti-Mouse IgG (H+L)-AF488
(Goat polyclonal)
InvitrogenCat # A11017(1:2000)
Recombinant DNA reagentpX330-U6-Chimeric_BB-CBh-hSpCas9AddgeneRRID:Addgene_42230
Recombinant DNA reagentpSpCas9(BB)-2A-Puro (PX459) V2.0AddgeneRRID:Addgene_62988
Recombinant DNA reagentpMD2.GAddgeneRRID:Addgene_12259
Recombinant DNA reagentpMDLg/pRREAddgeneRRID:Addgene_12251
Recombinant DNA reagentpRSV-RevAddgeneRRID:Addgene_12253
Recombinant DNA reagentpInducer20AddgeneRRID:Addgene_44012
Recombinant DNA reagentpInducer20-purothis paperNeoR of pInducer20 (Addgene_44012) was replaced with PuroR
Recombinant DNA reagentpFLAG-CMV2SigmaCat # E7033
Recombinant DNA reagentpEGFP-C1Clontech
Peptide, recombinant proteinFLAG PeptideSigmaCat # F3290
Peptide, recombinant proteinHA peptideMBL Life scienceCat # 3320HA tagged Protein PURIFICATION KIT
Peptide, recombinant proteinrecombinant human IL-1βR and D SystemsCat # 201-LB-005
Peptide, recombinant proteinrecombinant mouse IL-1βBioLegendCat # 575102
Peptide, recombinant proteinrecombinant human IL-17ABioLegendCat # 570502
Peptide, recombinant proteinrecombinant human TNFBioLegendCat # 570104
Commercial assay or kitDynabeads Protein GInvitrogenCat # 10004D
Commercial assay or kitLambda Protein PhosphataseNEBCat # P0753S
Commercial assay or kitSignal Enhancer HIKARInacalai tesqueCat # 02270-81
Commercial assay or kitImmobilon Forte Western HRP SubstrateMilliporeCat # WBLUF0500
Commercial assay or kitTRIzol ReagentInvitrogenCat # 15596018
Commercial assay or kitRNA Clean and Concentrator-5Zymo ResearchCat # R1014
Commercial assay or kitPowerUp SYBR Green Master MixApplied BiosystemsCat # A25742
Commercial assay or kitIL-6 Mouse Uncoated ELISA KitInvitrogenCat # 88-7064-88
Chemical compound, drugDSP (dithiobis(succinimidyl propionate))TCICat # D2473
Chemical compound, drugPam3CSK4InvivoGenCat # tlrl-pms
Chemical compound, drugpoly I:CcytivaCat # 27473201
Chemical compound, drugLPSInvivoGenCat # tlrl-smlps
Chemical compound, drugR848InvivoGenCat # tlrl-r848-5
Chemical compound, drugCpG DNAInvivoGenCat # tlrl-1668-1ODN 1668
Chemical compound, drugMG-132SigmaCat # 474790
Chemical compound, drugActinomycin DSigmaCat # A9415
Chemical compound, drugLeptomycin BSigmaCat # L2913

Mice

Zc3h12a-deficient mice have been described previously (Matsushita et al., 2009). Zc3h12aS513A/S513A knock-in mice were generated using CRISPR/Cas9-mediated genome-editing technology as previously described (Fujihara and Ikawa, 2014). Briefly, a pair of complementary DNA oligos was annealed and inserted into pX330 (Addgene plasmid # 42230) (Cong et al., 2013). The plasmid was injected together with the donor single strand oligo into fertilized eggs of C57BL/6J mice. Successful insertion was confirmed by direct sequencing.

All mice were grown under specific pathogen-free environments. All animal experiments were conducted in compliance with the guidelines of the Kyoto University animal experimentation committee (Approval number: MedKyo21057).

Reagents

Recombinant cytokines, TLR ligands, and chemical compounds were listed in the key resources table.

Cell culture

Request a detailed protocol

HeLa cells, HEK293T cells, RAW264.7 cells, and MEFs were maintained in DMEM (nacalai tesque) with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin (nacalai tesque), and 100 µM 2-Mercaptoethanol (nacalai tesque). Mycoplasma contamination was routinely tested and found negative.

For the preparation of bone marrow-derived macrophages (BMDMs), bone marrow cells were cultured in RPMI-1640 (nacalai tesque) with 10% FBS, 1% Penicillin/Streptomycin, 100 μM 2-mercaptoethanol, and 20 ng/ml of macrophage colony-stimulating factor (M-CSF) (BioLegend) for 6 days.

For the preparation of thioglycolate-elicited peritoneal exudate cells (PECs), mice were intraperitoneally injected with 2 ml of 4% (w/v) Brewer’s thioglycollate medium. 3.5 days after the injection, peritoneal macrophages were collected and cultured in RPMI-1640 with 10% FBS, 1% Penicillin/Streptomycin, and 100 μM 2-mercaptoethanol.

Plasmids

For the expression of FLAG-tagged proteins, pFLAG-CMV2 (Sigma) was used as a backbone. For the expression of HA- or Myc-tagged proteins, the FLAG sequence of pFLAG-CMV2 was replaced by HA- or Myc-sequence. Mouse Zc3h12a cDNA was inserted into these vectors as previously described (Matsushita et al., 2009). The coding sequences of 14-3-3 and βTRCP were amplified by using cDNAs derived from HeLa cell as templates and inserted into vectors above using In-Fusion HD Cloning Kit (Takara Bio). For Myc-IRAK1 expression vector, coding sequence of IRAK1 derived from HA-IRAK1 expression vector (Iwasaki et al., 2011) was used. For the mouse Il6 expression vector, the EGFP sequence in pEGFP-C1 was replaced with Il6 gene.

Deletions or point mutations were introduced using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) or In-Fusion HD Cloning Kit.

For the lentiviral packaging vectors, pInducer20 (Addgene plasmid #44012) (Meerbrey et al., 2011) was modified to generate pInducer20-puro. FLAG-HA-Regnase-1 sequence was inserted into pInducer20-puro using In-Fusion HD Cloning Kit.

For the Cas9 and gRNA expression plasmids (pX459-IRAK1 and pX459-IRAK2), pX459 (Addgene Plasmid #62988) was digested at BbsI sites, and the annealed oligo coding guide sequence (key resources table) was inserted.

Plasmid transfection

Request a detailed protocol

Plasmids were transfected to HeLa cells or HEK293T cells using Lipofectamine 2000 (Invitrogen) or PEI max (Polysciences) respectively according to manufacturer's instructions.

Generation of doxycycline-inducible FLAG-HA-Regnase-1-expressing HeLa cells

Request a detailed protocol

HeLa cells expressing FLAG-HA-Regnase-1 in a doxycycline-dependent manner were generated by lentiviral transduction. To produce lentivirus, HEK293T cells were transfected with pInducer20-puro-FLAG-HA-Regnase1 together with third generation lentiviral packaging vectors. 6 hr after the transfection, the medium was changed to fresh medium and then the cells were incubated at 37°C for 48 hr. After the incubation, the medium containing lentivirus was harvested and filtrated through 0.45 μm filter. HeLa cells were incubated with the virus-containing medium at 37°C for 24 hr, followed by 48-hr-incubation with fresh medium. The transduced cells were selected by 0.5 μg/ml of puromycin (InvivoGen). Single clones were picked and evaluated for their expression of FLAG-HA-Regnase-1 in a dox-dependent manner by immunoblotting.

Knockout of IRAK1 and IRAK2

Request a detailed protocol

HeLa cells were transfected with two pX459 plasmids which contains gRNA sequence for IRAK1 and IRAK2. As the negative control, empty pX459 plasmid was transfected. Forty-eight hr after the pX459 transfection, puromycin (2 μg/ml) was added to the medium. After 48 hr selection with puromycin, the same number of cells were seeded to new dishes and incubated in fresh media without antibiotics for 48 hr. Knockout efficiency was check by immunoblotting using WCL samples.

DSP-crosslinking

View detailed protocol

Doxycycline-inducible FLAG-HA-Regnase-1-expressing HeLa cells were treated with doxycycline (1 μg/ml, Sigma) and incubated at 37°C for 4 hr before the DSP-crosslinking. As a negative control, cells were incubated without doxycycline, and for the IL-1β-stimulated sample, cells were stimulated with human IL-1β (10 ng/ml, R and D Systems) 2 hr before the crosslinking. After the incubation, cells were washed twice with pre-warmed PBS, and then incubated in PBS containing 0.1 mM DSP (TCI) at 37°C for 30 min. After crosslinking, cells were washed once with pre-warmed PBS and incubated in STOP solution (PBS containing 1 M Tris-HCl pH 7.4) at room temperature for 15 min. Cells were then washed with ice-cold PBS twice, followed by cell lysis and immunoprecipitation.

Immunoprecipitation

View detailed protocol

Before immunoprecipitation, pre-washed Dynabeads Protein G (Invitrogen) were incubated with either anti-FLAG antibody (Sigma), anti-HA antibody (Sigma), or anti-Myc antibody (Sigma) at 4°C with rotation for 1 hr.

For DSP-crosslinked samples, cells were lysed in IP buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.5% (vol/vol) NP-40) with cOmplete Mini EDTA-free (Sigma), PhosSTOP (Sigma), and 200 U/ml of Benzonase (Millipore) and incubated on ice for 10 min. The lysates were centrifuged at 15,000 rpm for 5 min and the supernatants were incubated with anti-FLAG-antibody-bound Dynabeads at 4°C with rotation for 2 hr. The beads were then washed with IP buffer three times and incubated in FLAG-elution buffer (100 μg/ml FLAG peptides (Sigma), 50 mM Tris-HCl pH7.4, and 150 mM NaCl) at 4°C with rotation for 10 min twice. Eluted proteins were then immunoprecipitated using anti-HA-antibody-bound Dynabeads at 4°C with rotation for 2 hr. After the second immunoprecipitation, the beads were washed three times with IP buffer and the proteins were eluted in Urea elution buffer (8 M Urea and 50 mM Tris-HCl pH 8.0). The samples were stored at -80°C until trypsin digestion. Proteins were reduced with 10 mM dithiothreitol (Fujifilm Wako) for 30 min, alkylated with 50 mM iodoacetamide (Fujifilm Wako) for 30 min in the dark, diluted fourfold with 50 mM ammonium bicarbonate (ABC) buffer, and then trypsin digestion was performed. After overnight incubation, digestion was stopped by adding trifluoroacetic acid (TFA) (Fujifilm Wako) to a final concentration of 0.5%. The peptide mixture solution was desalted with SDB-XC StageTips (Rappsilber et al., 2007). The eluates were dried and resuspended in 200 mM 2-[4-2(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid (HEPES) pH 8.5, mixed with 0.1 mg of TMT10-plex labeling reagents (Thermo Fisher Scientific) dissolved in 5 μL acetonitrile (ACN), and incubated for 1 hr at room temperature. The reaction mixtures were quenched by adding hydroxylamine (Sigma) to give a final concentration of 0.33%. After 15 min incubation, the samples were acidified with trifluoroacetic acid, diluted to 5% ACN, and desalted using SDB-XC StageTips. Peptides were dried, resolved in 5 mM ABC buffer and fractionated with a C18-StageTip. Peptides were eluted with 5 mM ABC containing acetonitrile (12.5%, 15%, 17.5%, 20%, 22.5% and 80%) in step gradient manner. Totally six fractions were obtained and analyzed by LC/MS/MS.

For the identification of phosphorylation sites of Regnase-1, HeLa cells expressing FLAG-HA-Regnase-1 or FLAG-Regnase-1 were stimulated with IL-1β (10 ng/ml) or IL-17A (50 ng/ml) respectively for 4 hr. The cells were washed with ice-cold PBS twice and lysed in IP buffer with cOmplete Mini EDTA-free and PhosSTOP. Regnase-1 was immunoprecipitated using anti FLAG antibody as described above and eluted from Dynabeads in SDS sample buffer (50 mM Tris–HCl pH 6.8, 2% (wt/vol) SDS, 15% (vol/vol) 2-mercaptoethanol, 10% (vol/vol) glycerol and bromophenol blue), followed by incubation at 95°C for 5 min. Regnase-1was isolated by electrophoresis and the pieces of the gel containing Regnase-1 was stored at 4°C until trypsin digestion. The gels were de-stained for 30 min with 200 μL of 50 mM ABC / 50% ACN. Then the gels were dehydrated by the addition of 100% ACN. Proteins were reduced with 500 μL of 10 mM dithiothreitol / 50 mM ABC for 30 min, alkylated with 50 mM iodoacetamide / 50 mM ABC for 30 min in the dark. The gels were washed two times with 200 μL of 0.5% acetic acid / 50% methanol. After washing, gels were re-equilibrated with 50 mM ABC, and subsequently dehydrated by the addition of 100% ACN. 10 μL of trypsin solution (10 ng/μL in 50 mM ABC) was added to gel pieces and incubated for 5 min. Another 50 μL of 50 mM ABC buffer was added to gel samples and incubated at 37°C for overnight. After that, elastase (Promega) (150 ng/μL in water) was added to the final concentration of 7.5 ng/μL and incubated for 30 min at 37°C (Dau et al., 2020). Digestion was stopped by the addition of 5 μL of 10% TFA. The supernatants were recovered into fresh Eppendorf tubes, and two additional extraction steps were performed with 50% ACN / 0.1% TFA and 80% ACN / 0.1% TFA. The peptides in the supernatants were dried, resuspended in 0.1% TFA, and desalted using SDB-XC StageTips.

For detecting protein-protein binding, cells were lysed in IP Buffer with cOmplete Mini EDTA-free and PhosSTOP and immunoprecipitated as described above using indicated antibodies. The proteins were eluted in the mixture of IP Buffer and SDS sample buffer (2:1) and incubated at 95°C for 5 min.

For detecting protein-RNA binding, cells were lysed in IP Buffer with cOmplete Mini EDTA-free and RNaseOut (Invitrogen) and immunoprecipitated as described above using indicated antibodies. Some of the precipitates were eluted in the mixture of IP Buffer and SDS sample buffer (2:1) to elute proteins and the others were eluted in TRIzol Reagent (Invitrogen) for RNA isolation.

LC-MS/MS

Request a detailed protocol

LC/MS/MS analyses were performed with an Orbitrap Fusion Lumos (Thermo Fisher Scientific) connected to an Ultimate 3000 pump (Thermo Fisher Scientific) and an HTC-PAL autosampler (CTC analytics). Peptides were separated by a self-pulled needle column (150 mm length, 100 μm ID, 6 μm needle opening) packed with Reprosil-Pur 120 C18-AQ 3 μm reversed-phase material (Dr. Maisch GmbH), using a 20 min or 65 min gradient of 5–40% B (mobile phase A: 0.5% acetic acid, mobile phase B: 0.5% acetic acid / 80% acetonitrile) at a flow rate of 500 nL/min. The applied ESI voltage was 2.4 kV. For TMT-labeled samples, the following parameters were applied: MS scan range of 375–1500, MS1 orbitrap resolution of 120,000, quadrupole isolation window of 0.7, HCD (higher-energy collision dissociation) collision energy of 38%, MS2 orbitrap resolution of 50,000, AGC target value of 50000. For non-labeled samples, the following parameters were applied: MS scan range of 300–1500, MS1 orbitrap resolution of 120,000, quadrupole isolation window of 1.6, HCD collision energy of 30%, MS2 orbitrap resolution of 15,000, MS2 AGC target value of 50,000.

Database searching and data processing

Request a detailed protocol

For DSP-crosslinked samples, peptides were identified with Mascot version 2.6.1 (Matrix Science) against the sequence of Mouse Regnase-1 in addition to the human database from UniprotKB/Swiss-Prot release 2017/04 and with a precursor ion mass tolerance of 5 ppm and a product ion mass tolerance of 20 ppm. Carbamidomethyl (C), TMT6plex (K) and TMT6plex (N-term) were set as a fixed modification, oxidation (M) was allowed as a variable modification, and up to two missed cleavages are allowed with strict Trypsin/P specificity. Identified peptides were rejected if the Mascot score was below the 95% confidence limit based on the identity score of each peptide. The quantification of peptides was based on the TMT reporter ion intensities in MS2 spectra. Protein quantitative values were calculated by summing the corresponding peptide intensity values. Only proteins with at least two unique peptides were used for further analysis.

For the identification of phosphorylation sites of Regnase-1, peptides were identified with Mascot version 2.7.0 against the sequence of mouse Regnase-1 with a precursor ion mass tolerance of 5 ppm and a product ion mass tolerance of 20 ppm. Carbamidomethyl (C) was set as a fixed modification, oxidation (M) and phosphorylation (STY) were allowed as variable modifications, and up to two missed cleavages are allowed with semitrypsin specificity. Identified peptides were rejected if the Mascot score was below the 99% confidence limit based on the identity score of each peptide. The label-free quantification of peptides was based on the peak area in the extracted ion chromatograms using Skyline-daily software version 21.0.9.118 (MacLean et al., 2010). The peak area ratios between stimulated and non-stimulated samples were calculated, log-scaled, and normalized by the median. For quantitation of phosphosites, the peak area ratios of all monophosphopeptides containing the phosphosites of interest were averaged. Phosphosite localization was evaluated with a site-determining ion combination method based on the presence of site-determining y- or b-ions in the peak lists of the fragment ions, which supported the phosphosites unambiguously (Nakagami et al., 2010).

Protein-protein interaction network of the Regnase-1-associating proteins (Log2 fold change over negative control > 2) was analyzed using STRING database (Szklarczyk et al., 2019) and visualized in Cytoscape (Shannon et al., 2003). Keratins contaminated in the samples were omitted from the analysis.

λ-protein phosphatase (λPP) treatment

Request a detailed protocol

HeLa cells transiently expressing HA-14-3-3ε were stimulated with or without IL-1β (10 ng/ml) for 4 hr and lysed in IP Buffer. Some of the lysates were used in immunoprecipitation as described above. The proteins were eluted using 250 μg/ml of HA peptides as described above. The lysate and the precipitates were treated with Lambda Protein Phosphatase (NEB) according to manufacturer's instructions. For the λPP-negative samples, the same amount of IP Buffer was added instead of λPP.

Immunoblotting

Request a detailed protocol

Cells were lysed in IP Buffer or RIPA buffer (1% (vol/vol) NP-40, 0.1% (wt/vol) SDS, 1% (wt/vol) sodium deoxycholate, 150 mM NaCl, 20 mM Tris-HCl pH 8.0, and 10 mM EDTA) with cOmplete Mini EDTA-free and PhosSTOP. The lysates were incubated on ice for 5 min and centrifuged at 15,000 rpm for 5 min. The supernatants were mixed with SDS sample buffer (2:1) and incubated at 95°C for 5 min. SDS-PAGE was performed using e-PAGEL 7.5% or 5~20% (ATTO) and the proteins were transferred onto 0.2 μm pore size Immun-Blot PVDF membranes (Bio-Rad), followed by blocking with 5% skim milk. The antibodies used in immunoblotting were listed in the key resources table. Luminescence was detected with Amersham Imager 600 (cytiva) and the images were analyzed with Fiji (Schindelin et al., 2012).

RNA isolation and RT-qPCR

Request a detailed protocol

Cells were lysed in TRIzol Reagent, and the RNA was isolated according to manufacturer's instructions. For the isolation of the RNA precipitated with Regnase-1, RNA was isolated using RNA Clean and Concentrator-5 (Zymo Research). RNA was reverse transcribed by using ReverTra Ace (TOYOBO). cDNA was amplified by using PowerUp SYBR Green Master Mix (Applied Biosystems) and measured with StepOnePlus Real-Time PCR System (Applied Biosystems). To analyze mRNA expression, each RNA level was normalized with 18S or ACTB. The primers used in qPCR were listed in Supplementary file 1.

RNA sequencing

Request a detailed protocol

PECs were harvested from Zc3h12aWT/WT and Zc3h12aS513A/S513A mice as described above. PECs were stimulated with LPS (100 ng/ml) for indicated time and the RNA was collected and isolated using TRIzol Reagent. cDNA library was prepared using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) and sequenced on NextSeq 500 System (Illumina) according to the manufacturer’s instructions. Acquired data was analyzed using Galaxy (Afgan et al., 2018). Briefly, identified reads were mapped on the murine genome (mm10) using HISAT2 (paired end, unstranded) (Galaxy Version 2.1.0), and the mapped reads were counted using featureCounts (Galaxy Version 1.6.3).

ELISA

Request a detailed protocol

Cytokine concentration was measured by using IL-6 Mouse Uncoated ELISA Kit (Invitrogen) according to manufacturer's instructions. Luminescence was detected with iMark Microplate Reader (Bio-Rad).

Luciferase assay

Request a detailed protocol

5xNF-κB firefly luciferase reporter vector, Renilla luciferase vector, and IRAK1-expressing vector were transfected in HeLa cells and the luciferase activity was measured by using PicaGene Dual Sea Pansy Luminescence Kit (TOYO B-Net). NF-κB activation was calculated by normalizing Firefly luciferase activity with Renilla luciferase activity.

Mathematical model

Request a detailed protocol

We developed two dynamical models for the inflammation system regulated by Regnase-1 based on different assumptions of the functions of 14-3-3-bound Regnase-1.

Model 1

Request a detailed protocol

In the first model, we assumed that the 14-3-3-bound Regnase-1 does not have the function of degrading its target mRNAs (Figure 4J). The ordinary differential equations are given as follows:

(1.1) dx1dt=k1signalt-d1x1x3-d4x1
dx2dt=k2signalt-d2x2x3-d5x2
dx3dt=k3x2-(d3+d6signalt+d7signalt)x3+d9x4
dx4dt=d7signaltx3-(d8+d9)x4

where x1x2x3, and x4 is the abundance of Il6 mRNA, Zc3h12a mRNA, Reg1 Protein, and 14-3-3-bound Reg1 Protein, respectively; k1 and k2 is the transcription rate constant of Il6, and Zc3h12a, respectively; k3 is the translation rate constant of Zc3h12a; d1 and d2 is the Reg1-induced degradation rate constant of Il6 mRNA and Zc3h12a mRNA, respectively; d3, d4, and d5 is the Reg1-independent degradation rate constant of Reg1 protein, Il6 mRNA, and Zc3h12a mRNA, respectively; d6 is the ubiquitin-dependent degradation rate constant of Reg1 protein; d7 is the binding rate constant of Reg1 protein to 14-3-3; d8 is the natural degradation rate constant of 14-3-3-bound Reg1 protein; d9 is the dissociation rate constant of Reg1 from 14-3-3. signalt is the strength of TLR stimulation, which is given as the following form (Mino et al., 2019):

(1.2) signal(t)={sbase(if 0ttdelay),sinputsbasetraise(ttdelay)+sbase(if tdelayttdelay+traise),sinput(if tdelay+traisettdelay+traise+tpulse),(sinputsbase)×exp(t(tdelay+traise+tpulse)tdelay)+sinput(if t>tdelay+traise+tpulse)

Model 2

Request a detailed protocol

We also developed an alternative model in which the 14-3-3-bound Regnase-1 maintains functions of degrading its target mRNAs (Figure 4J). The ordinary differential equations are given as follows:

(1.3) dx^1dt=k1signal(t)d1x^1x^3d1x^1x^4d4x^1
dx^2dt=k2signal(t)d2x^2x^3d2x^2x^4d5x2
dx^3dt=k3x2(d3+d6signal(t)+d7signal(t))x^3+d9x^4
dx^4dt=d7signal(t)x^3(d8+d9)x^4

where x^1, x^2, x^3, and x^4 is the abundance of Il6 mRNA, Zc3h12a mRNA, Reg1 Protein, 14-3-3-bound Reg1 Protein, respectively; d1 and d2 is the 14-3-3-bound Reg1-induced degradation rate constant of Il6 mRNA and Zc3h12a mRNA, respectively. The other parameters are defined in the same way as Model 1.

To determine which model is consistent with the experimental observations, we focus on the experimental findings that there was no difference in the abundance of Il6 mRNA, Zc3h12a mRNA, and Reg1- protein (without 14-3-3 bound) between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells in the late phase of stimulation (Figure 4A,B,D and E). We will show that in Model 2 (Equation 1.3), the abundance of the Il6 mRNAs should be different between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells under the condition that amount of the 14-3-3-free Reg1 protein is comparable between them.

Analysis of the equilibrium

Lemma 1

Request a detailed protocol

For Zc3h12aWT/WT cells, there exists only one nonnegative (biologically meaningful) equilibrium of the system (Equation 1.3) if and only if d3+d6sinput+d7sinput-d7d9sinputd7sinput+d90. If d3+d6sinput+d7sinputd7d9sinputd7sinput+d9<0, there is no equilibrium. For Zc3h12aS513A/S513A cells, there always exists only one nonnegative (biologically meaningful) equilibrium.

Proof of lemma 1

Request a detailed protocol

Setting all the derivatives of Equation (1.3) equal to zero yields

(1.4) 0=k1sinputd1X^1WTX^3WTd1X^1WTX^4WTd4X^1WT
0=k2sinputd2X^2WTX^3WTd2X^2WTX^4WTd5X^2WT
0=k3X^2WT(d3+d6sinput+d7sinput)X^3WT+d9X^4WT
0=d7sinputX^3WT(d8+d9)X^4WT

where X^1WT, X^2WT, X^3WT, and X^4WT are fixed points of x^1, x^2, x^3, and x^4, respectively. Given that signaltsinput as t, we assume signaltsinput at the equilibrium.

It follows from Equation (1.4) that

(1.5a) (d2+d7sinputd8+d9d2)K(X^3WT)2+d5KX^3WTk2sinput=0
(1.5b) X^4WT=d7sinputd8+d9X^3WT
(1.5c) X^2WT=KX^3WT
(1.5d) X^1WT=k1sinputd1X^3WT+d1X^4WT+d4

where

K:=1k3d3+d6sinput+d7sinput-d7d9sinputd7sinput+d9

It is easy to see that the quadratic Equation (1.5a) has a nonnegative solution if K0, that is, d3+d6sinput+d7sinput-d7d9sinputd7sinput+d90. If K<0, Equation (1.5a) has no nonnegative solution. If X^3WT0, it follows from (Equation 1.5b), (Equation 1.5c), and (Equation 1.5d) that X^4WT, X^2WT, X^1WT0.

For Zc3h12aS513A/S513A cells, we assume that d7=d8=d9=0. Substituting this equation into Equation (1.4) yields

(1.6) 0=k1sinputd1X^1SAX^3SAd4X^1SA
0=k2sinputd2X^2SAX^3SAd5X^2SA
0=k3X^2SA(d3+d6sinput)X^3SA
0=X^4SA

where X^1SA, X^2SA, X^3SA, and X^4SA are fixed points of x^1, x^2, x^3, and x^4 in Zc3h12aS513A/S513A cells, respectively.

It follows from (1.6) that

(1.7a) d2k3d3+d6sinput(X^2SA)2+d5X^2SAk2sinput=0
(1.7b) X^3SA=k3d3+d6sinputX^2SA
(1.7c) X^1SA=k1sinputd1X^3SA+d4

It is easy to see that the quadratic Equation (1.7a) has a nonnegative solution. If X^2SA0, it follows from (Equation 1.7b) and (Equation 1.7c) that X^3SA X^1SA0.

Lemma 2. There exists only one nonnegative (biologically meaningful) equilibrium of the system (Equation 1.1) if and only if d3+d6sinput+d7sinput-d7d9sinputd7sinput+d90. If d3+d6sinput+d7sinputd7d9sinputd7sinput+d9<0, there is no equilibrium. For Zc3h12aS513A/S513A cells, there always exists only one nonnegative (biologically meaningful) equilibrium.

Proof of lemma2:

With d1=d2=0 in lemma 1, we get the same conclusion.

Consistency with the experiments

Request a detailed protocol

The experimental observation shows that there was no difference in the abundance of Reg1 protein between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells at the late phase of stimulation (Figure 4A–C), which implies

(1.8) X^3WTX^3SA

based on the alternative model (1.3).

From (Equation 1.4) and (Equation 1.6), we get

(1.9a) X^1WT=k1sinputd1X^3WT+d1X^4WT+d4
X^2WT=k2sinputd2X^3WT+d2X^4WT+d5
(1.9b) X^1SA=k1sinputd1X^3SA+d4
X^2SA=k2sinputd2X^3SA+d5

By (Equation 1.8), (Equation 1.9a), and (Equation 1.9b), we obtain

(1.10a) X^1WT<X^1SA
(1.10b) X^2WT<X^2SA

Equation 1.10a and Equation 1.10b implies that in Model 2, the abundance of Il6 and Zc3h12a mRNA in Zc3h12aWT/WT cells should be smaller than that in Zc3h12aS513A/S513A cells at the late phase under the condition that amount of the Reg1 protein is comparable (Equation 1.8) between these two cells. It contradicts experimental observation that the abundance of the Il6 and Zc3h12a mRNAs did not differ between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells (Figure 4D–I). Thus, Model 2 (Equation 1.3) is not consistent with the experimental findings.

In contrast, in Model 1 (Equation 1.1), we assume from experimental findings that

(1.11) X3WTX3SA

just like (Equation 1.8), where X3WT is the fixed point of x3 in Zc3h12aWT/WT cells and X3SA is the fixed point of x3 in Zc3h12aS513A/S513A cells based on the model (Equation 1.1).

By substituting d7=d8=d9=0 into (Equation 1.9a) and (Equation 1.9b), we obtain

(1.12a) X1WT=k1sinputd1X3WT+d4
X2WT=k2sinputd2X3WT+d5
(1.12b) X1SA=k1sinputd1X3SA+d4
X2SA=k2sinputd2X3SA+d5

where X1WT and X2WT are fixed points of x1 and x2, respectively in Zc3h12aWT/WT cells and X1SA and X2SA are fixed points of x1 and x2, respectively in Zc3h12aS513A/S513A cells.

By (Equation 1.11), (Equation 1.12a), and (Equation 1.12b), we obtain

(1.13a) X1WTX1SA
(1.13b) X2WTX2SA

In this case, (Equation 1.13a) and (Equation 1.13b) are in agreement with the experimental facts that that the abundance of the target mRNAs did not differ between Zc3h12aWT/WT and Zc3h12aS513A/S513A cells.

These mathematical analyses indicate that Model 1 (Equation 1.1), but not Model 2 (Equation 1.3), is consistent with the experimental findings.

Immunofluorescence

Request a detailed protocol

Cells were cultured on cover glass, fixed with 4%-Paraformaldehyde Phosphate Buffer Solution (nacalai tesque), and permeabilized with 0.5% (vol/vol) Triton X-100 (nacalai tesque) in PBS, followed by incubation in blocking solution (2% (vol/vol) goat serum (FUJIFILM Wako Pure Chemical) and 0.1% (wt/vol) gelatin in PBS). The antibodies used in Immunofluorescence were listed in the key resources table. DNA was stained with Hoechst 33342 (Invitrogen). Fluorescence was detected with TCS SPE (Leica). Acquired images were analyzed with Fiji (Schindelin et al., 2012).

Amino acid sequence analysis

Request a detailed protocol

Amino acid sequence of each protein was obtained from NCBI. The results of T-coffee alignment (Notredame et al., 2000) were visualized by using Jalview (Waterhouse et al., 2009). Secondary structure was predicted by using PSIPRED 4.0 (Buchan and Jones, 2019; Jones, 1999). NES prediction was performed by using LocNES (Xu et al., 2015).

Data availability

Mass spectrometry data (PXD026561) is available at https://repository.jpostdb.org/entry/JPST001201.

The following data sets were generated
    1. Takeuchi O
    (2021) JPOST
    ID PXD026561. Phosphorylation-dependent Regnase-1 interactome.
    1. Akaki K
    2. Hia F
    3. Kakiuchi N
    4. Ogawa S
    5. Takeuchi O
    (2021) NCBI Gene Expression Omnibus
    ID GSE180028. Transcriptome analysis of LPS stimulated PECs (WT and Zc3h12a S513A).

References

Decision letter

  1. Carla V Rothlin
    Senior Editor; Yale School of Medicine, United States
  2. Nahum Sonenberg
    Reviewing Editor; McGill University, Canada
  3. Georg Stoecklin
    Reviewer

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

This study describes a new mechanism by which Regnase-1 is inhibited upon immune activation, which mediates the efficient synthesis of inflammatory mediators whose mRNAs are normally degraded by Regnase-1. The study report a novel interaction of Regnase-1 with 14-3-3. This provides an alternative mechanism by which inflammatory mRNAs are upregulated by inhibiting degradation via Regnase-1.

Decision letter after peer review:

Thank you for submitting your article "IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Carla Rothlin as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Georg Stoecklin (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

Most of the reviewers' excellent suggestions are intended for future experiments, which are not required for this paper. However, some experiments lack necessary controls and others do not adequately support the conclusions. The comments listed before must be addressed, and some statements (see specific reviewers' comments) need to be changed or deleted:

1) Figure 1C. Input control on the same blots as the IPs are run ought to be shown, and include a negative control blot (e.g. actin) as well.

2) Figure 1D. It is important to show a longer exposure of the Regnase1 blots for both the input blot and the blot from the immunoprecipitation.

3) You show that IL-6 mRNA levels are similar in Regnase-1 WT and S513A mutant cells (Figure 4D-F). This notion is difficult to reconcile with the author's finding that 14-3-3 binding to Regnase-1 prevents it from binding to and inducing the degradation of IL-6 mRNA. This discrepancy should be discussed critically. Moreover, previous studies have shown that changes in mRNA stability are not necessarily reflected by changes in mRNA steady-state levels, since they can be buffered by altered transcription (see e.g. Singh et al., 2019; PMID: 31116665). Therefore, mRNA degradation rates should be measured directly by actinomycin D chase or similar experiments.

4) You claim "that 14-3-3 inhibits Regnase-1-mRNA binding, thereby abrogating Regnase-1-mediated mRNA degradation". However, this is only shown for IL-6 mRNA (Figure 5G). Since this is a key result of the study, you should also test other targets of Regnase-1 so as to generalize their finding.

5) Figure 5A. Expression levels of exogenous Regnase-1 mutants ought to be shown.

Reviewer #2 (Recommendations for the authors):

The data suggest that IRAK1/2 may directly phosphorylate Regnase-1 (Fig.2G-I), although the authors do not address this question either experimentally or in the discussion. Do the authors have evidence that Regnase-1 is a direct target of IRAK1/2? Minimally, the authors should discuss this point and assess whether the identified phosphorylation sites conform to consensus IRAK target motifs.

The evidence for mutually exclusive binding of Regnase-1 to bTRCP or 14-3-3 is rather indirect, through the analysis of Regnase-1 phosphorylation status and phosphomutants (Fig.3). This point could be strengthened by competition assays, in which expressing increasing amounts of one protein should weaken the interaction with the other.

Figure 2G: The figure legend states that HeLa cells were "stimulated with IL-1b (10 ng/ml) for 4 hours". Is this correct? The blot shows the faster migrating Regnase-1 band and no interaction with 14-3-3 unless IRAK1/2 is overexpressed, which corresponds to the situation in unstimulated cells.

Figure 4A-C: As a direct assessment of Regnase-1 protein stability, the authors could conduct cycloheximide chase assays in Wt/Wt and S513A/S513A cells.

Figure 4 and 5: The figure legends say "…bars represent mean values of biological replicates (n=3)… similar results were obtained from at least two independent experiments". It is unclear what is meant by these two different statements.

lines 189 and 195: should refer to Figure 3, not 2.

line 231: should refer to Figure 4D-I

Figure 4J-K: figure legend should say Regnase-1 instead of Reganse-1

Reviewer #3 (Recommendations for the authors):

Line 26: "both interactions occur in a mutually exclusive manner, underscoring the importance of modulating Regnase-1's activity"

I don't think the fact that the interactions are mutually exclusive underscores the importance of modulating Regnase activity. That Regnase-1 can be inactivated by two different, and perhaps partially redundant, mechanisms underscores the importance of modulating Regnase activity.

Line 138: "14-3-3 specifically binds to phosphorylated Regnase-1"

The authors concluded that 14-3-3 binds to phosphorylated S494 and S513 of Regnase-1 but direct binding between Regnase-1 and 14-3-3 and their binding sites are not shown. The authors would need to perform experiments to address this point, or otherwise rewrite the manuscript to correctly interpret the data.

In Figure 2A, the IL-1β-induced interaction between 14-3-3 and Reg1 is reduced by phosphatase treatment, but it still appears relatively strong. Therefore, it is not possible to say from this IP that 14-3-3 binds specifically to phospho-Reg1. Since the band shift is abolished by PP in Figure 2A, it looks like 14-3-3 may still bind to non-phosphorylated Reg1, but perhaps with lower affinity.

Line 190-191: "We next checked the phosphorylation status of βTRCP-bound and 14-3-3-bound Regnase-1"

This sentence seems to be in the wrong place. It appears to refer to Figure 3A/B, but it comes before 3C.

Line 195: "βTRCP-ΔF-bound Regnase-1 migrated faster than 14-3-3-bound Regnase-1 (Figure 2C-D)"

Please clarify why Figure2C and 2D are referenced here. In particular, Figure 2C shows residues which are phosphorylated in IL-1B-stimulated cells.

Line 211: "Regnase-1 showed slow migration and was able to associate with 14-3-3"

Based on the band shift in Figure 4A, it is a reasonable assumption that the newly synthesized Reg1 was phosphorylated and therefore it could bind to 14-3-3, but the figure does not show that it was associated with 14-3-3. This should be made clear.

Figure 5A, Expression levels of exogenous Regnase-1 mutants need to be shown. It is considered that the expression levels must be the same at initial condition.

Line 231: "Figure 3D-I" should refer to "Figure 4D-I"

Line 248: "These results suggest that the phosphorylation at S513 and the following association with 14-3-3 nullifies Regnase-1's ability in degrading target mRNAs"

From the data presented up to this point in the manuscript, it is not possible to say whether 14-3-3 binding or S513 phosphorylation alone prevents Reg1 from degrading Il6 mRNA. The following data presented in Figure 5 does address this.

Line 262: The experiments using Reg-1ExoSx2 are valid only if interacting site of 14-3-3 with Regnase-1 is the same as that with ExoS. The authors should clearly mention this point or show that 14-3-3 binds to Regnase-1 with its phosphopeptide binding groove.

Line 278: In Figure 5D, the difference between Il6 only and Il6+ExoSx2 looks like it could be significant. What is the P value?

Line 335-336: "Analysis of Regnase-1S513A/S513A mice revealed that 14-3-3-mediated abrogation of Regnase-1 can be compensated by the degradation of Regnase-1"

The data from the S513A mutants doesn't show that degradation compensates for 14-3-3-mediated block of Reg1 function. It shows that not all Reg1 is bound to 14-3-3, and only the non-14-3-3-bound Reg1 regulates mRNA stability.

The authors could say that the absence of 14-3-3-mediated abrogation of Reg1 in the S513A mutant cells may be compensated for by increased degradation. But to be sure of this, the authors should demonstrate that translation is not affected.

Line 347: "compensation by βTRCP-mediated regulation, which we observed in Regnase-1S513A/S513A mice"

Could the authors demonstrate that this is definitely βTRCP-mediated regulation? If the mutant βTRCP used in Figure 2 acts as a dominant-negative, perhaps overexpression in S513A mutant cells could show this?

Line 369-370: "βTRCP is likely to recognize 14-3-3-free Regnase-1, indicating that 14-3-3 inhibits Regnase-1-βTRCP interaction"

The wording of this sentence suggests that 14-3-3-Reg1 binding takes precedence over βTRCP-Reg1. Is there any reason to assume that this is true? If binding is mutually exclusive, as suggested by Figure 3C, could βTRCP also inhibit the 14-3-3-Reg1 interaction? The authors should clarify here whether the interaction of Reg1 with 14-3-3 is likely to be dominant over the interaction with βTRCP.

Figure 1C suggests that βTRCP-Reg1 binding peaks sooner after IL-1β stimulation than 14-3-3-Reg1 binding. Could this mean that βTRCP binds to Reg1 first, before 14-3-3?

Line 387: "evolutionally conserved" should be "evolutionarily conserved"

Reviewer #4 (Recommendations for the authors):

1. In Figure 1C, the co-immunoprecipitation Regnase1 with myc-tagged 14-3-3epsilon doesn't look exceptionally robust, especially when seeing the original blot provided as source data. It's currently hard to tell whether this represents a small amount of endogenous Regnase1 or a larger percentage co-precipitating. What would help to improve the strength of this figure is to include an input control on the same blots as the IPs are run, and to include a negative control blot (e.g. actin) as well. This will help to validate the specificity of the interaction.

2. For Figure 1D, are the authors able to show a longer exposure of the Regnase1 blots as well? For both the input blot and the blot from the immunoprecipitation. This would help to strengthen their conclusion that R848 and CpG DNA stimuli promote the interaction between 14-3-3 and Regnase1.

3. The authors conclude that the interaction of Regnase1 and 14-3-3 is mediated by IRAK-dependent phosphorylation. As these experiments were all conducted by overexpression of IRAK1 or 2, it would significantly strengthen their conclusion if they carried out some knockdown experiments where they deplete endogenous IRAK1/2. Does this abolish the 14-3-3 interaction with Regnase1 in IL-1B treated cells?

4. While I agree with the authors that their data support different phosphorylated serine residues supporting interactions with either 14-3-3 or TRCP, I don't think that their data specifically support mutually exclusive interactions between the two. A convincing competition experiment to test this may be to express both 14-3-3 and TRCP at different levels along with Regnase1. This would allow them to immunopreciptate a tagged Regnase 1 and see if, for example, increasing TRCP expression increases its association with Regnase1, while decreasing the association of 14-3-3 with Regnase1.

5. For Figure 5G, the authors should also test specific Serine-to-Alanine mutants to see if they impact RNA binding. This could be accomplished with a mutant that cannot bind TRCP (to keep it stable), and compare it to a mutant that cannot bind both TRCP and 14-3-3. This would allow the authors to make conclusions on a Regnase1 mutant that did not contain additional bacterial stretches to it, which could bind to proteins other than 14-3-3 that they don't know about.

6. Similarly, the final conclusion of the manuscript is that 14-3-3 binding to Regnase1 localizes to the nucleus. However, this is based on Regnase mutants that utilize bacterial stretches that bind 14-3-3 proteins (and potentially other proteins as well). To strengthen their data, the authors should utilize a Regnase mutant that cannot bind to TRCP and/or contains mutations of serine residues such that it cannot bind 14-3-3 proteins. These mutants could then be used to test if nuclear localization is impaired without adding additional bacterial protein stretches to it.

https://doi.org/10.7554/eLife.71966.sa1

Author response

Essential revisions:

Most of the reviewers' excellent suggestions are intended for future experiments, which are not required for this paper. However, some experiments lack necessary controls and others do not adequately support the conclusions. The comments listed before must be addressed, and some statements (see specific reviewers' comments) need to be changed or deleted:

We thank the reviewers and editors for valuable suggestions. We revised the manuscript accordingly as indicated in our responses to the reviewers’ comments below.

1) Figure 1C. Input control on the same blots as the IPs are run ought to be shown, and include a negative control blot (e.g. actin) as well.

We thank the reviewer for the advice. According to the reviewer’s suggestion, we loaded the input and IP samples used in Figure 1C on the same gel and blotted membranes using anti-Actin antibody as well as anti-Regnase-1 antibody (new Figure 1—figure supplement 1). Compared with input samples, only trace amounts of Actin bands were detected in every IP-sample including 14-3-3ε negative. This is probably because of non-specific binding between Actin and Dynabeads or protein G used for immunoprecipitation. In contrast, strong Regnase-1 bands were detected in IL-1β-stimulated (2 or 4 hours) HA-14-3-3ε transfected samples, but not in IL-1β-stimulated (4 hours) HA-14-3-3ε negative sample. Therefore, these results further support our finding that the binding between Regnase-1 and 14-3-3 specifically induced by IL-1β stimulation.

2) Figure 1D. It is important to show a longer exposure of the Regnase1 blots for both the input blot and the blot from the immunoprecipitation.

We thank the reviewer for the constructive criticism. Since we acquired the image with only one exposure time setting (16-bit), we adjusted the range of brightness to show the bands at two different brightness level (8-bit) (new Figure 1D). These data further confirm that the MyD88-dependent signaling activated downstream of TLR/IL-1R induces the interaction between Regnase-1 and 14-3-3.

3) You show that IL-6 mRNA levels are similar in Regnase-1 WT and S513A mutant cells (Figure 4D-F). This notion is difficult to reconcile with the author's finding that 14-3-3 binding to Regnase-1 prevents it from binding to and inducing the degradation of IL-6 mRNA. This discrepancy should be discussed critically. Moreover, previous studies have shown that changes in mRNA stability are not necessarily reflected by changes in mRNA steady-state levels, since they can be buffered by altered transcription (see e.g. Singh et al., 2019; PMID: 31116665). Therefore, mRNA degradation rates should be measured directly by actinomycin D chase or similar experiments.

We thank the reviewer for the suggestion. To check mRNA degradation rates, we stimulated MEFs with IL-1β for 4 hours, at which time point the protein level of Regnase-1 shows huge difference between Regnase-1WT/WT and Regnase-1S513A/S513A cells, and added Actinomycin D. We found that degradation rates of Regnase-1 target mRNAs such as Il6, Regnase-1, and Nfkbiz were comparable Regnase-1WT/WT and Regnase-1S513A/S513A MEFs (now shown as new Figure 4—figure supplement 4).

4). You claim "that 14-3-3 inhibits Regnase-1-mRNA binding, thereby abrogating Regnase-1-mediated mRNA degradation". However, this is only shown for IL-6 mRNA (Figure 5G). Since this is a key result of the study, you should also test other targets of Regnase-1 so as to generalize their finding.

We thank the reviewer for the constructive criticism. Since we previously identified binding targets of Regnase-1 in HeLa cells (Mino et al., 2015), we checked if ExoSx2 mutation also abrogates the binding between Regnase-1 and some of these target mRNAs including PTGS2, NFKBIZ, REGNASE-1, CXCL1, CXCL2, CXCL3, MAFA, and NFKBID. As shown in new Figure 5—figure supplement 2, Regnase-1-ExoSx2 failed to bind with all Regnase-1 target mRNAs tested, in addition to IL6 shown in Figure 5H. These results further confirm that 14-3-3 inhibits Regnase-1-mRNA binding.

5). Figure 5A. Expression levels of exogenous Regnase-1 mutants ought to be shown.

We thank the reviewer for the comment. Since the majority of overexpressed Regnase-1 are not affected by IL-1β-mediated phosphorylation (e.g., Figure 2D, 3A, and 3B), we reduced the amount of transfected Regnase-1 as much as possible in former Figure 5A (Figure 5B) in order to observe the effect of each SA mutation on the activity of Regnase-1. However, such low expressed Regnase-1 was undetectable in immunoblotting with tag antibody. Therefore, to check the expression level of low expressed Regnase-1, we performed immunoblot analysis using anti-Regnase-1 antibody, which has strong affinity to Regnase-1, using REGNASE-1 KO HeLa cells transfected with Regnase-1-WT and SA mutants (new Figure 5A). The expression level of each exogenous Regnase-1 was comparable without IL-1β stimulation. Nevertheless, the S513A mutant Regnase-1 showed decreased expression 2 or 4 hours after IL-1β stimulation, consistent with our observation in Regnase-1S513A/S513A MEFs (Figure 4A). On the other hand, S435/439/513A mutant showed higher expression compared to S513A mutant.

Reviewer #2 (Recommendations for the authors):

The data suggest that IRAK1/2 may directly phosphorylate Regnase-1 (Fig.2G-I), although the authors do not address this question either experimentally or in the discussion. Do the authors have evidence that Regnase-1 is a direct target of IRAK1/2? Minimally, the authors should discuss this point and assess whether the identified phosphorylation sites conform to consensus IRAK target motifs.

We thank the reviewer for the suggestion. A previous kinome study comprehensively identifying kinase substrates suggests that the sequence motifs of target phosphorylation site of IRAK1 is pSxV and KxxxpS (Sugiyama et al., 2019; PMID: 31324866). However, these sequences do not match the sequence at S494 and S513 of Regnase-1 (Figure 2F). We speculate other kinases are activated by IRAK1/2 and phosphorylate Regnase-1 at S494 and S513, although we cannot exclude the possibility that Regnase-1 is directly phosphorylated by IRAK1/2 at S494 and S513. We added this point in the Discussion section.

The evidence for mutually exclusive binding of Regnase-1 to bTRCP or 14-3-3 is rather indirect, through the analysis of Regnase-1 phosphorylation status and phosphomutants (Fig.3). This point could be strengthened by competition assays, in which expressing increasing amounts of one protein should weaken the interaction with the other.

We thank the reviewer for the criticism. As the other reviewers point out, the wording of "mutually exclusive" might mislead readers. As shown in Figure 3C and 3D, βTRCP recognizes 14-3-3-free Regnase-1 but not slowly migrating Regnase-1, which is the binding target of 14-3-3. In addition, Regnase-1-S513A mutant is unstable after IL-1β or LPS stimulation (Figure 4A, 4B, 4C, and 5A). These results suggest that 14-3-3 stabilizes Regnase-1 by preventing the formation of Regnase-1-βTRCP complex. However, we have not shown the data indicating βTRCP inhibits Regnase-1-14-3-3 association. We therefore corrected the sentences about the relationship between Regnase-1-14-3-3 complex and Regnase-1-βTRCP complex throughout the manuscript. These binding events occur independently and not sequentially, and 14-3-3 inhibits Regnase-1-βTRCP binding. We did not investigate whether βTRCP affects Regnase-1-14-3-3 interaction or not because once proteins (substrates of SCF complex) bind to βTRCP, they get ubiquitinated and degraded via proteasome system.We agree with the reviewer that the competition assay will help to clarify the detailed mechanism how 14-3-3 inhibits Regnase-1-βTRCP binding. However, we feel thein vitroassay is beyond the scope of this study. 14-3-3 mediated abrogation of the nuclear-cytoplasmic shuttling of Regnase-1 might be one of clues to answer this question.

Figure 2G: The figure legend states that HeLa cells were "stimulated with IL-1b (10 ng/ml) for 4 hours". Is this correct? The blot shows the faster migrating Regnase-1 band and no interaction with 14-3-3 unless IRAK1/2 is overexpressed, which corresponds to the situation in unstimulated cells.

We thank the reviewer for pointing out our mistake in the Figure legend. We did not stimulate cells in the experiment shown in Figure 2G. We corrected the legend for Figure 2G as follows: “(G) Immunoblot analysis of immunoprecipitates (IP: Myc) and WCL from HeLa cells transiently expressing Myc-14-3-3ε, HA-IRAK1/2, and FLAG-Regnase-1-WT or indicated mutants.”

Figure 4A-C: As a direct assessment of Regnase-1 protein stability, the authors could conduct cycloheximide chase assays in Wt/Wt and S513A/S513A cells.

We thank the reviewer for the advice. According to the reviewer’s advice, we assessed Regnase-1 protein stability by adding cycloheximide (CHX) (new Figure 4—figure supplement 2). Since both WT and S513A Regnase-1 degraded rapidly (30 minutes) after the stimulation with IL-1β or LPS (Figure 4A-C), we added CHX and LPS at the same time and collected cell lysates at earlier time points (10, 20, and 30 minutes after the stimulation). We found that Regnase-1 S513A mutant degraded more quickly than WT Regnase-1 in PECs in response to LPS stimulation (Figure 4—figure supplement 2). This is because phosphorylated WT Regnase-1 (upper bands), which was not observed in Regnase-1S513A/S513A, persisted after the stimulation. This result further supports our finding that S513A mutation destabilizes Regnase-1 protein.

Reviewer #3 (Recommendations for the authors):

Line 26: "both interactions occur in a mutually exclusive manner, underscoring the importance of modulating Regnase-1's activity"

I don't think the fact that the interactions are mutually exclusive underscores the importance of modulating Regnase activity. That Regnase-1 can be inactivated by two different, and perhaps partially redundant, mechanisms underscores the importance of modulating Regnase activity.

We thank the reviewer for the criticism. As the reviewer pointed out, the abstract lacks logic to some extent because of the order of sentences. According to the reviewer’s comment, we revised the abstract section.

Line 138: "14-3-3 specifically binds to phosphorylated Regnase-1"

The authors concluded that 14-3-3 binds to phosphorylated S494 and S513 of Regnase-1 but direct binding between Regnase-1 and 14-3-3 and their binding sites are not shown. The authors would need to perform experiments to address this point, or otherwise rewrite the manuscript to correctly interpret the data.

In Figure 2A, the IL-1β-induced interaction between 14-3-3 and Reg1 is reduced by phosphatase treatment, but it still appears relatively strong. Therefore, it is not possible to say from this IP that 14-3-3 binds specifically to phospho-Reg1. Since the band shift is abolished by PP in Figure 2A, it looks like 14-3-3 may still bind to non-phosphorylated Reg1, but perhaps with lower affinity.

We thank the reviewer for the advice. 14-3-3 is well known to directly bind to phosphorylated proteins and the binding motif of 14-3-3 partially overlaps with the sequences surrounding S494 and S513 of Regnase-1 (Figure 2F). Furthermore, the mutations in S494 and S513 abrogated the interaction between Regnase-1 and 14-3-3 (Figure 2D), indicating that the phosphorylation of both sites is critical for Regnase-1-14-3-3 interaction. These results strongly suggest that the 14-3-3 dimer directly recognizes phosphorylated Regnase-1 at both S494 and S513 at least under IL-1β-, MyD88-dependent TLR ligand-, or IL-17A-stimulation. We added this argument in the Discussion section.

In Figure 2A-B, we treated the samples with λPP after the immunoprecipitation with HA-14-3-3ε, indicating that Regnase-1 is supposed to be detected in the precipitated samples irrespective of the λPP treatment. Indeed, the amounts of Regnase-1 co-precipitated with 14-3-3ε was not altered by the λPP treatment (Figure 2A). However, the λPP treatment changed the mobility of Regnase-1 in the immunoblot analysis and made Regnase-1 migrate faster. These results demonstrate that 14-3-3 associates phosphorylated, but not unphosphorylated, Regnase-1.

Line 190-191: "We next checked the phosphorylation status of βTRCP-bound and 14-3-3-bound Regnase-1"

This sentence seems to be in the wrong place. It appears to refer to Figure 3A/B, but it comes before 3C.

We thank the reviewer for the advice. According to the reviewer’s suggestion, we revised the sentence as follows: "We next compared the phosphorylation status of βTRCP-bound and 14-3-3-bound Regnase-1."

Line 211: "Regnase-1 showed slow migration and was able to associate with 14-3-3"

Based on the band shift in Figure 4A, it is a reasonable assumption that the newly synthesized Reg1 was phosphorylated and therefore it could bind to 14-3-3, but the figure does not show that it was associated with 14-3-3. This should be made clear.

We thank the reviewer for the concern. To clarify the point raised by the reviewer, we revised the sentences as follows: " Notably, most of the newly synthesized Regnase-1 showed slow migration, consistent with the immunoprecipitation experiment using HeLa cells or RAW264.7 cells shown in Figure 1C and 1D… Interestingly, the amount of Regnase-1 at lower bands, which are not the binding target of 14-3-3 (Figure 2A), was comparable between WT and Regnase-1S513A/S513A at corresponding time points."

Figure 5A, Expression levels of exogenous Regnase-1 mutants need to be shown. It is considered that the expression levels must be the same at initial condition.

This comment was found in the Essential Revisions and was already addressed.

Line 248: "These results suggest that the phosphorylation at S513 and the following association with 14-3-3 nullifies Regnase-1's ability in degrading target mRNAs"

From the data presented up to this point in the manuscript, it is not possible to say whether 14-3-3 binding or S513 phosphorylation alone prevents Reg1 from degrading Il6 mRNA. The following data presented in Figure 5 does address this.

We thank the reviewer for the comment. We agree with the concern and corrected the sentences as follows:

"These results imply that the phosphorylation at S513 and the following association with 14-3-3 nullifies Regnase-1’s ability in degrading target mRNAs"

Line 262: The experiments using Reg-1ExoSx2 are valid only if interacting site of 14-3-3 with Regnase-1 is the same as that with ExoS. The authors should clearly mention this point or show that 14-3-3 binds to Regnase-1 with its phosphopeptide binding groove.

We thank the reviewer for the concern. As we mentioned above, it is quite plausible that the 14-3-3 dimer directly recognizes phosphorylated S494 and S513 of Regnase-1. In this regard, we believe that the experiments using Regnase-1-ExoSx2 are meaningful.

Line 278: In Figure 5D, the difference between Il6 only and Il6+ExoSx2 looks like it could be significant. What is the P value?

The P value between Il6 only and Il6+ExoSx2 is 0.00048. Since we overexpressed Regnase-1-ExoSx2, we speculate this difference is because some of 14-3-3-free ExoSx2 mutant functioned to suppress Il6 expression.

Line 335-336: "Analysis of Regnase-1S513A/S513A mice revealed that 14-3-3-mediated abrogation of Regnase-1 can be compensated by the degradation of Regnase-1"

The data from the S513A mutants doesn't show that degradation compensates for 14-3-3-mediated block of Reg1 function. It shows that not all Reg1 is bound to 14-3-3, and only the non-14-3-3-bound Reg1 regulates mRNA stability.

The authors could say that the absence of 14-3-3-mediated abrogation of Reg1 in the S513A mutant cells may be compensated for by increased degradation. But to be sure of this, the authors should demonstrate that translation is not affected.

We thank the reviewer for the concern. Reviewer #2 also suggests that we needed to make sure if S513A mutation affects protein stability of Regnase-1. According to the reviewers’ suggestion, we examined the stability of Regnase-1 by treating the cells with CHX. As shown in new Figure 4—figure supplement 2, we successfully show that Regnase-1-S513A is more unstable than WT after LPS and CHX treatment.

Line 347: "compensation by βTRCP-mediated regulation, which we observed in Regnase-1S513A/S513A mice"

Could the authors demonstrate that this is definitely βTRCP-mediated regulation? If the mutant βTRCP used in Figure 2 acts as a dominant-negative, perhaps overexpression in S513A mutant cells could show this?

We thank the reviewer for the concern. As shown in new Figure 5A, additional S435/439A mutation, which abrogates Regnase-1-βTRCP binding, stabilized Regnase-1-S513A protein after IL-1β stimulation. The result indicates that the degradation of Regnase-1-S513A in response to IL-1β stimulation is dependent on βTRCP. Therefore, we revised the sentence as follows: "…compensation by βTRCP-mediated regulation, which we observed in Regnase-1S513A/S513A mice and HeLa cells transiently expressing Regnase-1-S513A and S435/439/513A."

Line 369-370: "βTRCP is likely to recognize 14-3-3-free Regnase-1, indicating that 14-3-3 inhibits Regnase-1-βTRCP interaction"

The wording of this sentence suggests that 14-3-3-Reg1 binding takes precedence over βTRCP-Reg1. Is there any reason to assume that this is true? If binding is mutually exclusive, as suggested by Figure 3C, could βTRCP also inhibit the 14-3-3-Reg1 interaction? The authors should clarify here whether the interaction of Reg1 with 14-3-3 is likely to be dominant over the interaction with βTRCP.

Figure 1C suggests that βTRCP-Reg1 binding peaks sooner after IL-1β stimulation than 14-3-3-Reg1 binding. Could this mean that βTRCP binds to Reg1 first, before 14-3-3?

We thank the reviewer for the concern. As the reviewer pointed out, both Regnase-1-WT and S513A undergo βTRCP-mediated degradation within 30 minutes after IL-1β or LPS stimulation (Figure 4A-C) (Iwasaki et al., 2011). This is probably because of the higher activity of IKKs at the early time point compared to later time points (Iwasaki et al., 2011). We think that Regnase-1 can also associate with 14-3-3 at such an early time point because Regnase-1-WT showed strong phosphorylated band 30 minutes after LPS stimulation when protein degradation was inhibited by MG-132 (Figure 4C). Moreover, we found that Regnase-1 phosphorylation, which was not observed in Regnase-1S513A/S513A, occurred 10 minutes after LPS stimulation (new Figure 4—figure supplement 2). These data indicate that Regnase-1 can be phosphorylated at S513 at the early time point after IL-1β or LPS stimulation. Since 14-3-3 does not covalently bind to Regnase-1, it is plausible that Regnase-1-14-3-3 binding is a reversible reaction, while protein degradation of Regnase-1 is irreversible. We believe this is the reason why 14-3-3 failed to protect Regnase-1 from βTRCP-mediated protein degradation at the early time point.

Reviewer #2 also pointed out the wording, "mutually exclusive", might not appropriate. As we described in our response to the comment of Reviewer #2, we have corrected the wording.

Reviewer #4 (Recommendations for the authors):

Major points to be addressed:

1. In Figure 1C, the co-immunoprecipitation Regnase1 with myc-tagged 14-3-3epsilon doesn't look exceptionally robust, especially when seeing the original blot provided as source data. It's currently hard to tell whether this represents a small amount of endogenous Regnase1 or a larger percentage co-precipitating. What would help to improve the strength of this figure is to include an input control on the same blots as the IPs are run, and to include a negative control blot (e.g. actin) as well. This will help to validate the specificity of the interaction.

This comment was found in the Essential Revisions and was already addressed.

2. For Figure 1D, are the authors able to show a longer exposure of the Regnase1 blots as well? For both the input blot and the blot from the immunoprecipitation. This would help to strengthen their conclusion that R848 and CpG DNA stimuli promote the interaction between 14-3-3 and Regnase1.

This comment was found in the Essential Revisions and was already addressed.

3. The authors conclude that the interaction of Regnase1 and 14-3-3 is mediated by IRAK-dependent phosphorylation. As these experiments were all conducted by overexpression of IRAK1 or 2, it would significantly strengthen their conclusion if they carried out some knockdown experiments where they deplete endogenous IRAK1/2. Does this abolish the 14-3-3 interaction with Regnase1 in IL-1B treated cells?

We thank the reviewer for the suggestion. We have previously showed that double knockout of Irak1 and Irak2 abolished the LPS-induced phosphorylation of Regnase-1, resulting in the disappearance of slowly migrating Regnase-1 in immunoblot analysis (Iwasaki et al., 2011). To further investigate if IRAK1/2 are necessary for the association between Regnase-1 and 14-3-3 as well as the phosphorylation of Regnase-1, we utilized CRISPR-Cas9 system by introducing pX459 plasmids, which express Cas9 and gRNAs for IRAK1 and IRAK2 in HeLa cells, followed by the selection with puromycin. Then we transfected HA-14-3-3ε in the puromycin-resistant cells. We confirmed that the cells lack IRAK1 expression by immunoblot analysis, although antibodies detecting endogenous human IRAK2 were not available which precluded the evaluation of IRAK2 knockout efficiency. It is known that IL-1β stimulation induces various post translational modification of IRAK1, rendering the detection of IRAK1 unable by immunoblot analysis (Vollmer et al., 2017; PMID: 28512203). Consistently, we also did not detect the clear band of IRAK1 in IL-1β-stimulated control sample. Consistent with our previous finding using MEFs (Iwasaki et al., 2011), phosphorylation of Regnase-1 is diminished in cells transfected with pX459 for IRAK1/2 (Figure 2—figure supplement 3). Furthermore, immunoprecipitation with HA-14-3-3ε revealed that the association of Regnase-1 and 14-3-3 was also diminished by depletion of IRAK1/2 expression. These results indicate that IRAK1/2 (at least IRAK1 in HeLa cells) is necessary for Regnase-1-14-3-3 interaction.

4. While I agree with the authors that their data support different phosphorylated serine residues supporting interactions with either 14-3-3 or TRCP, I don't think that their data specifically support mutually exclusive interactions between the two. A convincing competition experiment to test this may be to express both 14-3-3 and TRCP at different levels along with Regnase1. This would allow them to immunopreciptate a tagged Regnase 1 and see if, for example, increasing TRCP expression increases its association with Regnase1, while decreasing the association of 14-3-3 with Regnase1.

We thank the reviewer for the suggestion. The other reviewers also pointed out the wording, "mutually exclusive". As we described in our response to the Reviewer #2, we corrected the wording.

5. For Figure 5G, the authors should also test specific Serine-to-Alanine mutants to see if they impact RNA binding. This could be accomplished with a mutant that cannot bind TRCP (to keep it stable), and compare it to a mutant that cannot bind both TRCP and 14-3-3. This would allow the authors to make conclusions on a Regnase1 mutant that did not contain additional bacterial stretches to it, which could bind to proteins other than 14-3-3 that they don't know about.

We thank the reviewer for raising the concern. We agree that introduction of the ExoS sequence may cause effects on Regnase-1 other than binding with 14-3-3. The difficulty of using the S513A mutant to evaluate the functional effect of 14-3-3 on Regnase-1 is that not all the overexpressed Regnase-1 is phosphorylated by IL-1β stimulation. Particularly, IL-1β-induced phosphorylation rate of overexpressed Regnase-1 is not so high (e.g., Figure 2D, 3A, and 3B). Since RNA-immunoprecipitation requires large amount of Regnase-1 to evaluate its RNA-binding capacity, we feel it difficult to finely detect the changes in the binding of Regnase-1 WT and S513A to mRNA in response to IL-1β stimulation. Thus, utilizing an ExoS mutant and its control, an ExoSAAA mutant, was the most practical way to elucidate the mechanism how 14-3-3 affects the function of Regnase-1, although we agree that further studies are required to conclude that 14-3-3 inhibits Regnase-1-RNA binding in vivo. We discuss the requirement of future studies in the Discussion section.

6. Similarly, the final conclusion of the manuscript is that 14-3-3 binding to Regnase1 localizes to the nucleus. However, this is based on Regnase mutants that utilize bacterial stretches that bind 14-3-3 proteins (and potentially other proteins as well). To strengthen their data, the authors should utilize a Regnase mutant that cannot bind to TRCP and/or contains mutations of serine residues such that it cannot bind 14-3-3 proteins. These mutants could then be used to test if nuclear localization is impaired without adding additional bacterial protein stretches to it.

We thank the reviewer for the criticism. Similar to the aforementioned response to comment #5, the difficulty in the use of Regnase-1-overexpressing cells is that IL-1β-induced phosphorylation rate of overexpressed Regnase-1 is not so high (e.g., Figure 2D, 3A, and 3B), thus it is hard to assess the IL-1β-induced changes in intracellular dynamics of Regnase-1 by using overexpression system. Therefore, we believe that the use of the ExoS system is the most practical way to assess the role of Regnase-1-14-3-3 binding, although further studies are necessary to elucidate the function and detailed mechanism of nuclear and cytoplasmic shuttling of Regnase-1 in vivo. We discussed these issues in the Discussion section.

https://doi.org/10.7554/eLife.71966.sa2

Article and author information

Author details

  1. Kotaro Akaki

    1. Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan
    2. Graduate School of Biostudies, Kyoto University, Kyoto, Japan
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0059-3291
  2. Kosuke Ogata

    Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
    Contribution
    Formal analysis, Investigation, Visualization, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0634-3990
  3. Yuhei Yamauchi

    Laboratory of Mathematical Biology, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
    Contribution
    Formal analysis, Visualization, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
  4. Noriki Iwai

    Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan
    Contribution
    Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Ka Man Tse

    Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan
    Contribution
    Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  6. Fabian Hia

    Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan
    Contribution
    Formal analysis, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7209-4312
  7. Atsushi Mochizuki

    Laboratory of Mathematical Biology, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
    Contribution
    Supervision, Writing - review and editing
    Competing interests
    No competing interests declared
  8. Yasushi Ishihama

    Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
    Contribution
    Supervision, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7714-203X
  9. Takashi Mino

    Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9562-008X
  10. Osamu Takeuchi

    Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing
    For correspondence
    otake@mfour.med.kyoto-u.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1260-6232

Funding

Japan Agency for Medical Research and Development (JP20gm4010002)

  • Osamu Takeuchi

Japan Society for the Promotion of Science (Core-to-Core Program)

  • Osamu Takeuchi

Japan Society for the Promotion of Science (221S0002)

  • Takashi Mino

Japan Society for the Promotion of Science (16H06279)

  • Takashi Mino

Japan Society for the Promotion of Science (18H05278)

  • Osamu Takeuchi

Japan Society for the Promotion of Science (19H03488)

  • Takashi Mino

Japan Agency for Medical Research and Development (21ae0121030)

  • Osamu Takeuchi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank S Ogawa and N Kakiuchi in Kyoto university for performing RNA sequencing and providing ZC3H12A-KO HeLa cells, Y Okumoto for secretarial assistance and lab members for helpful discussion. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI [18H05278]; AMED-FORCE [JP20gm4010002] from Japan Agency for Medical Research and Development and the JSPS through Core-to-Core Program. KA was supported by ‘Kibou Projects’ Scholarship for doctoral Students in Immunology. TM was funded by JSPS KAKENHI (19H03488), Grant-in-Aid for Scientific Research on Innovative Areas ‘Genome Science’ (221S0002 and 16H06279), Takeda Science Foundation, the Uehara Memorial Foundation, Shimizu Foundation for Immunology and Neuroscience, Naito Foundation, Senri Life Science Foundation, Nakajima Foundation, and Mochida Memorial Foundation for Medical and Pharmaceutical Research.

Ethics

Animal experimentation: All animal experiments were conducted in compliance with the guidelines of the Kyoto University animal experimentation committee. (Approval number: MedKyo21057).

Senior Editor

  1. Carla V Rothlin, Yale School of Medicine, United States

Reviewing Editor

  1. Nahum Sonenberg, McGill University, Canada

Reviewer

  1. Georg Stoecklin

Publication history

  1. Received: July 6, 2021
  2. Preprint posted: July 15, 2021 (view preprint)
  3. Accepted: October 8, 2021
  4. Accepted Manuscript published: October 12, 2021 (version 1)
  5. Accepted Manuscript updated: October 13, 2021 (version 2)
  6. Version of Record published: October 28, 2021 (version 3)

Copyright

© 2021, Akaki et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,463
    Page views
  • 285
    Downloads
  • 4
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Kotaro Akaki
  2. Kosuke Ogata
  3. Yuhei Yamauchi
  4. Noriki Iwai
  5. Ka Man Tse
  6. Fabian Hia
  7. Atsushi Mochizuki
  8. Yasushi Ishihama
  9. Takashi Mino
  10. Osamu Takeuchi
(2021)
IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay
eLife 10:e71966.
https://doi.org/10.7554/eLife.71966
  1. Further reading

Further reading

    1. Computational and Systems Biology
    2. Immunology and Inflammation
    Harry Kane, Nelson M LaMarche ... Lydia Lynch
    Research Article

    Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to non-peptide antigens, such as lipids. While the transcriptional profiles of naive, effector and memory adaptive T cells have been well studied, less is known about transcriptional regulation of different iNKT cell activation states. Here, using single cell RNA-sequencing, we performed longitudinal profiling of activated murine iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2 and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation, and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen experienced iNKT cells.

    1. Cancer Biology
    2. Immunology and Inflammation
    Lei Yang, Xichen Dong ... Zhenjun Wang
    Research Article

    Efficacy of immunotherapy is limited in patients with colorectal cancer (CRC) because high expression of tumor-derived transforming growth factor (TGF)-β pathway molecules and interferon (IFN)-stimulated genes (ISGs) promotes tumor immune evasion. Here, we identified a long noncoding RNA (lncRNA), VPS9D1-AS1, which was located in ribosomes and amplified TGF-β signaling and ISG expression. We show that high expression of VPS9D1-AS1 was negatively associated with T lymphocyte infiltration in two independent cohorts of CRC. VPS9D1-AS1 served as a scaffolding lncRNA by binding with ribosome protein S3 (RPS3) to increase the translation of TGF-β, TGFBR1, and SMAD1/5/9. VPS9D1-AS1 knockout downregulated OAS1, an ISG gene, which further reduced IFNAR1 levels in tumor cells. Conversely, tumor cells overexpressing VPS9D1-AS1 were resistant to CD8+ T cell killing and lowered IFNAR1 expression in CD8+ T cells. In a conditional overexpression mouse model, VPS9D1-AS1 enhanced tumorigenesis and suppressed the infiltration of CD8+ T cells. Treating tumor-bearing mice with antisense oligonucleotide drugs targeting VPS9D1-AS1 significantly suppressed tumor growth. Our findings indicate that the tumor-derived VPS9D1-AS1/TGF-β/ISG signaling cascade promotes tumor growth and enhances immune evasion and may thus serve as a potential therapeutic target for CRC.