IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay
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Evaluation Summary:
This is an important study that 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 interaction with 14-3-3 presented here was not known before. This provides an alternative mechanism by which inflammatory mRNAs are upregulated by inhibiting degradation via Regnase-1.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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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.
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Author Response:
Reviewer #1 (Public Review):
In this manuscript, Akaki et al. describe a new mechanism by which the activity of Regnase-1, an endonuclease that degrades mRNAs encoding inflammatory mediators, can be regulated. By determining the interactome of Regnase-1 in IL-1b or TLR-ligand stimulated cells, they found that Regnase-1 binds to bTRCP (as previously described) as well as to 14-3-3 proteins, which is novel. The authors further identify the phosphorylation sites on Regnase-1 that are required for the Regnase-1:14-3-3 interaction, and show that the interaction is mediated by the activity of IRAK1/2. By generating knock-in mice carrying a phosphodeficient mutant of Regnase-1, the authors demonstrate that the interaction with 14-3-3 blocks the ability of Regnase-1 to degrade its target mRNA IL-6, as it can no longer bind …
Author Response:
Reviewer #1 (Public Review):
In this manuscript, Akaki et al. describe a new mechanism by which the activity of Regnase-1, an endonuclease that degrades mRNAs encoding inflammatory mediators, can be regulated. By determining the interactome of Regnase-1 in IL-1b or TLR-ligand stimulated cells, they found that Regnase-1 binds to bTRCP (as previously described) as well as to 14-3-3 proteins, which is novel. The authors further identify the phosphorylation sites on Regnase-1 that are required for the Regnase-1:14-3-3 interaction, and show that the interaction is mediated by the activity of IRAK1/2. By generating knock-in mice carrying a phosphodeficient mutant of Regnase-1, the authors demonstrate that the interaction with 14-3-3 blocks the ability of Regnase-1 to degrade its target mRNA IL-6, as it can no longer bind to the target mRNA. Finally the authors show that binding to 14-3-3 prevents nucleocytoplasmic shuttling of Regnase-1 and therefore target mRNA recognition.
General comment:
This is an important study that describes a new mechanism by which Regnase-1 is inhibited upon immune activation, which mediates efficient synthesis of inflammatory mediators whose mRNAs are normally degraded by Regnase-1. The interaction with 14-3-3 presented here was not known before, and the authors describe the interaction and its consequences in great detail. In general, the study is well conducted and the results are both clear and convincing. The analysis of phosphodeficident Regnase-1 knock-in mice is a major strength of the study. However, there are some smaller points that the authors could address to further strengthen the manuscript, e.g. the mutually exclusive binding of Regnase-1 to bTRCP or 14-3-3, and the possibility that IRAK1/2 may directly phosphorylate Regnase-1. In addition, they should more directly measure the effect of phosphodeficient Regnase-1 on IL-6 mRNA decay, and generalize their observation that 14-3-3 binding prevents Regnase-1 mRNA binding and decay.
Specific comments:
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 the in vitro assay 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.
Reviewer #2 (Public Review):
The authors used immunoprecipitation followed by mass spectrometry to identify proteins interacting with Regnase-1 before and after stimulation with IL-1β. IL-1β treatment induced a previously unknown interaction between Regnase-1 and 14-3-3 proteins. 14-3-3 bound predominantly to phosphorylated Regnase-1 and specific phosphorylation sites were identified. 14-3-3 binding to Regnase-1 was mutually exclusive with βTRCP, binding of which is known to induce ubiquitination and degradation of Regnase-1. 14-3-3 binding prevented Regnase-1 degradation, but also inactivated it by blocking mRNA binding. 14-3-3 binding also prevented translocation of Regnase-1 from the cytoplasm to the nucleus. This study has identified a second mechanism by which Regnase function can be blocked to increase expression of inflammation-related mRNAs.
Overall, the authors' conclusions are supported by the data. The results of this study significantly advance the understanding of the regulation of Regnase-1 activity in inflammatory gene expression. The data are likely to be of interest to those investigating the intracellular signaling pathways that control gene expression in response to inflammation. The authors identified important sites for Regnase-1 regulation and created several mutant Regnase-1 constructs that will be of use to the research community. In addition, the transcriptomic and proteomic datasets generated in this study are likely to be of further benefit.
We thank the reviewer for evaluating out manuscript interesting.
Reviewer #3 (Public Review):
Here, Akaki and colleagues set out to identify how Regnase1 is regulated upon cells being stimulated with IL-1Beta or TLR ligand stimulation. To do this they stimulated cells and then carried out a proteomic analysis to identify proteins that specifically interact with Regnase1 in stimulated cells. They identified Rengase1 interacting with the Beta-transducin-repeat containing complex (TRCP), a previously published interaction, which leads to Regnase1 ubiquitination and degradation. Interestingly, they also identify 14-3-3 proteins. Based on other data, they conclude that TRCP and 14-3-3 interact with Regnase1 in a mutually exclusive manner. They go on to show that the interaction between 14-3-3 and Regnase1 is mediated in IL-1B/TLR-stimulated cells by IRAK1/2 through an uncharacterized C-terminal domain. Two phosphorylation sites (S494 and S513) regulate 14-3-3 interaction with Regnase1, while different sites are required for Regnase1 interaction with TRCP and proteosomal mediated degradation. Finally, they conclude based on their data that 14-3-3 binding to Regnase1 stabilizes Regnase1 but prevents nuclear-cytoplasmic shuttling of Regnase and also Regnase1-mRNA association.
The manuscript is interesting and presents another layer with respect to how Regnase-1 activity is regulated during the immune response. However, several points should be addressed in this reviewer's opinion that would help strengthen the manuscript.
We thank the reviewer for considering our manuscript interesting.
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Evaluation Summary:
This is an important study that 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 interaction with 14-3-3 presented here was not known before. This provides an alternative mechanism by which inflammatory mRNAs are upregulated by inhibiting degradation via Regnase-1.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Akaki et al. describe a new mechanism by which the activity of Regnase-1, an endonuclease that degrades mRNAs encoding inflammatory mediators, can be regulated. By determining the interactome of Regnase-1 in IL-1b or TLR-ligand stimulated cells, they found that Regnase-1 binds to bTRCP (as previously described) as well as to 14-3-3 proteins, which is novel. The authors further identify the phosphorylation sites on Regnase-1 that are required for the Regnase-1:14-3-3 interaction, and show that the interaction is mediated by the activity of IRAK1/2. By generating knock-in mice carrying a phosphodeficient mutant of Regnase-1, the authors demonstrate that the interaction with 14-3-3 blocks the ability of Regnase-1 to degrade its target mRNA IL-6, as it can no longer bind to the target mRNA. …
Reviewer #1 (Public Review):
In this manuscript, Akaki et al. describe a new mechanism by which the activity of Regnase-1, an endonuclease that degrades mRNAs encoding inflammatory mediators, can be regulated. By determining the interactome of Regnase-1 in IL-1b or TLR-ligand stimulated cells, they found that Regnase-1 binds to bTRCP (as previously described) as well as to 14-3-3 proteins, which is novel. The authors further identify the phosphorylation sites on Regnase-1 that are required for the Regnase-1:14-3-3 interaction, and show that the interaction is mediated by the activity of IRAK1/2. By generating knock-in mice carrying a phosphodeficient mutant of Regnase-1, the authors demonstrate that the interaction with 14-3-3 blocks the ability of Regnase-1 to degrade its target mRNA IL-6, as it can no longer bind to the target mRNA. Finally the authors show that binding to 14-3-3 prevents nucleocytoplasmic shuttling of Regnase-1 and therefore target mRNA recognition.
General comment:
This is an important study that describes a new mechanism by which Regnase-1 is inhibited upon immune activation, which mediates efficient synthesis of inflammatory mediators whose mRNAs are normally degraded by Regnase-1. The interaction with 14-3-3 presented here was not known before, and the authors describe the interaction and its consequences in great detail. In general, the study is well conducted and the results are both clear and convincing. The analysis of phosphodeficident Regnase-1 knock-in mice is a major strength of the study. However, there are some smaller points that the authors could address to further strengthen the manuscript, e.g. the mutually exclusive binding of Regnase-1 to bTRCP or 14-3-3, and the possibility that IRAK1/2 may directly phosphorylate Regnase-1. In addition, they should more directly measure the effect of phosphodeficient Regnase-1 on IL-6 mRNA decay, and generalize their observation that 14-3-3 binding prevents Regnase-1 mRNA binding and decay.
Specific comments:
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.
The authors show that IL-6 mRNA levels are similar in Regnase-1 WT and S513A mutant cells (Fig.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.
The authors 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 (Fig.5G). Since this is a key result of the study, the authors should also test other targets of Regnase-1 so as to generalize their finding.
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Reviewer #2 (Public Review):
The authors used immunoprecipitation followed by mass spectrometry to identify proteins interacting with Regnase-1 before and after stimulation with IL-1β. IL-1β treatment induced a previously unknown interaction between Regnase-1 and 14-3-3 proteins. 14-3-3 bound predominantly to phosphorylated Regnase-1 and specific phosphorylation sites were identified. 14-3-3 binding to Regnase-1 was mutually exclusive with βTRCP, binding of which is known to induce ubiquitination and degradation of Regnase-1. 14-3-3 binding prevented Regnase-1 degradation, but also inactivated it by blocking mRNA binding. 14-3-3 binding also prevented translocation of Regnase-1 from the cytoplasm to the nucleus. This study has identified a second mechanism by which Regnase function can be blocked to increase expression of …
Reviewer #2 (Public Review):
The authors used immunoprecipitation followed by mass spectrometry to identify proteins interacting with Regnase-1 before and after stimulation with IL-1β. IL-1β treatment induced a previously unknown interaction between Regnase-1 and 14-3-3 proteins. 14-3-3 bound predominantly to phosphorylated Regnase-1 and specific phosphorylation sites were identified. 14-3-3 binding to Regnase-1 was mutually exclusive with βTRCP, binding of which is known to induce ubiquitination and degradation of Regnase-1. 14-3-3 binding prevented Regnase-1 degradation, but also inactivated it by blocking mRNA binding. 14-3-3 binding also prevented translocation of Regnase-1 from the cytoplasm to the nucleus. This study has identified a second mechanism by which Regnase function can be blocked to increase expression of inflammation-related mRNAs.
Overall, the authors' conclusions are supported by the data. The results of this study significantly advance the understanding of the regulation of Regnase-1 activity in inflammatory gene expression. The data are likely to be of interest to those investigating the intracellular signaling pathways that control gene expression in response to inflammation. The authors identified important sites for Regnase-1 regulation and created several mutant Regnase-1 constructs that will be of use to the research community. In addition, the transcriptomic and proteomic datasets generated in this study are likely to be of further benefit.
-
Reviewer #3 (Public Review):
Here, Akaki and colleagues set out to identify how Regnase1 is regulated upon cells being stimulated with IL-1Beta or TLR ligand stimulation. To do this they stimulated cells and then carried out a proteomic analysis to identify proteins that specifically interact with Regnase1 in stimulated cells. They identified Rengase1 interacting with the Beta-transducin-repeat containing complex (TRCP), a previously published interaction, which leads to Regnase1 ubiquitination and degradation. Interestingly, they also identify 14-3-3 proteins. Based on other data, they conclude that TRCP and 14-3-3 interact with Regnase1 in a mutually exclusive manner. They go on to show that the interaction between 14-3-3 and Regnase1 is mediated in IL-1B/TLR-stimulated cells by IRAK1/2 through an uncharacterized C-terminal domain. …
Reviewer #3 (Public Review):
Here, Akaki and colleagues set out to identify how Regnase1 is regulated upon cells being stimulated with IL-1Beta or TLR ligand stimulation. To do this they stimulated cells and then carried out a proteomic analysis to identify proteins that specifically interact with Regnase1 in stimulated cells. They identified Rengase1 interacting with the Beta-transducin-repeat containing complex (TRCP), a previously published interaction, which leads to Regnase1 ubiquitination and degradation. Interestingly, they also identify 14-3-3 proteins. Based on other data, they conclude that TRCP and 14-3-3 interact with Regnase1 in a mutually exclusive manner. They go on to show that the interaction between 14-3-3 and Regnase1 is mediated in IL-1B/TLR-stimulated cells by IRAK1/2 through an uncharacterized C-terminal domain. Two phosphorylation sites (S494 and S513) regulate 14-3-3 interaction with Regnase1, while different sites are required for Regnase1 interaction with TRCP and proteosomal mediated degradation. Finally, they conclude based on their data that 14-3-3 binding to Regnase1 stabilizes Regnase1 but prevents nuclear-cytoplasmic shuttling of Regnase and also Regnase1-mRNA association.
The manuscript is interesting and presents another layer with respect to how Regnase-1 activity is regulated during the immune response. However, several points should be addressed in this reviewer's opinion that would help strengthen the manuscript.
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