The IκB kinase complex is a regulator of mRNA stability
2018; Springer Nature; Volume: 37; Issue: 24 Linguagem: Inglês
10.15252/embj.201798658
ISSN1460-2075
AutoresNadine Mikuda, Marina Kolesnichenko, Patrick Beaudette, Oliver Popp, Bora Uyar, Wei Sun, Ahmet Buğra Tufan, Björn Perder, Altuna Akalin, Wei Chen, Philipp Mertins, Gunnar Dittmar, M. Hinz, Claus Scheidereit,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle22 November 2018free access Source DataTransparent process The IκB kinase complex is a regulator of mRNA stability Nadine Mikuda Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Marina Kolesnichenko Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Patrick Beaudette Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Oliver Popp Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Bora Uyar orcid.org/0000-0002-3170-4890 Bioinformatics/Mathematical Modelling Platform, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Wei Sun orcid.org/0000-0001-9607-1361 Laboratory for Functional Genomics and Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Ahmet Bugra Tufan Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Björn Perder Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Altuna Akalin Bioinformatics/Mathematical Modelling Platform, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Wei Chen orcid.org/0000-0003-3263-1627 Laboratory for Functional Genomics and Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Philipp Mertins Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Gunnar Dittmar orcid.org/0000-0003-3647-8623 Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Michael Hinz Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Claus Scheidereit Corresponding Author [email protected] orcid.org/0000-0002-0446-6129 Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Nadine Mikuda Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Marina Kolesnichenko Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Patrick Beaudette Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Oliver Popp Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Bora Uyar orcid.org/0000-0002-3170-4890 Bioinformatics/Mathematical Modelling Platform, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Wei Sun orcid.org/0000-0001-9607-1361 Laboratory for Functional Genomics and Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Ahmet Bugra Tufan Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Björn Perder Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Altuna Akalin Bioinformatics/Mathematical Modelling Platform, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Wei Chen orcid.org/0000-0003-3263-1627 Laboratory for Functional Genomics and Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Philipp Mertins Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Gunnar Dittmar orcid.org/0000-0003-3647-8623 Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Michael Hinz Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Claus Scheidereit Corresponding Author [email protected] orcid.org/0000-0002-0446-6129 Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Author Information Nadine Mikuda1,‡, Marina Kolesnichenko1,‡, Patrick Beaudette2, Oliver Popp2, Bora Uyar3, Wei Sun4,†, Ahmet Bugra Tufan1, Björn Perder1, Altuna Akalin3, Wei Chen4,†, Philipp Mertins2, Gunnar Dittmar2,†, Michael Hinz1 and Claus Scheidereit *,1 1Signal Transduction Laboratory, Max Delbrück Center for Molecular Medicine, Berlin, Germany 2Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany 3Bioinformatics/Mathematical Modelling Platform, Max Delbrück Center for Molecular Medicine, Berlin, Germany 4Laboratory for Functional Genomics and Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany †Present address: Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China †Present address: Luxemburg Institute of Health, Strassen, Luxemburg ‡These authors contributed equally to this work *Corresponding author. Tel: +49 3094063816; E-mail: [email protected] EMBO J (2018)37:e98658https://doi.org/10.15252/embj.201798658 See also: ND Perkins (December 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The IκB kinase (IKK) is considered to control gene expression primarily through activation of the transcription factor NF-κB. However, we show here that IKK additionally regulates gene expression on post-transcriptional level. IKK interacted with several mRNA-binding proteins, including a Processing (P) body scaffold protein, termed enhancer of decapping 4 (EDC4). IKK bound to and phosphorylated EDC4 in a stimulus-sensitive manner, leading to co-recruitment of P body components, mRNA decapping proteins 1a and 2 (DCP1a and DCP2) and to an increase in P body numbers. Using RNA sequencing, we identified scores of transcripts whose stability was regulated via the IKK-EDC4 axis. Strikingly, in the absence of stimulus, IKK-EDC4 promoted destabilization of pro-inflammatory cytokines and regulators of apoptosis. Our findings expand the reach of IKK beyond its canonical role as a regulator of transcription. Synopsis The IκB kinase – best known to control transcription via NF-κB activation – can bind mRNA decay factors and affect the formation of P bodies, revealing a new post-transcriptional axis in inflammatory signalling. DNA damage, TNFα or IL-1β stimulate an interaction between the IKK complex and mRNA decapping activator EDC4. IKK phosphorylates EDC4 to drive recruitment of decapping factors and an increase in P body numbers. The IKK-EDC4 axis regulates stability of hundreds of mRNAs, including NF-κB-dependent and -independent transcripts. In the absence of external stimuli, IKK-EDC4 promotes degradation of mRNAs encoding of pro-inflammatory cytokines and regulators of apoptosis. Introduction The IKK complex is a key mediator of cellular response to numerous stimuli, including DNA damage, bacterial and viral antigens, cytokines and oxidative stress (Hayden & Ghosh, 2008; Oeckinghaus et al, 2011; Hinz & Scheidereit, 2014). It consists of the catalytic subunits, IKKα and IKKβ, and the regulatory subunit IKKγ (NEMO). IKK is believed to exert its function primarily through activation of the downstream transcription factor, NF-κB. In response to stimulus, IKK phosphorylates IκB proteins, leading to their proteolysis and liberation of NF-κB. The latter drives the expression of numerous genes regulating cell proliferation and differentiation, apoptosis and inflammation (Karin & Ben-Neriah, 2000; Hayden & Ghosh, 2008). Constituting the largest fraction of transcriptional targets of NF-κB are inflammatory cytokines and chemokines that are key modulators of the immune response. Strength and duration of their expression are further regulated by mRNA stability, determined by intrinsic cis elements, frequently located in the 3′ untranslated region (UTR), and by recruited trans factors, including RNA-binding proteins (RBPs; Hao & Baltimore, 2009; Schoenberg & Maquat, 2012). Many mammalian mRNAs contain AU-rich elements (AREs) that generally render the transcript less stable and more prone to decay. RNA-binding proteins can promote rapid degradation of their target mRNA in either the 3′ to 5′ or in the 5′ to 3′ direction. The latter requires the removal of the 5′ cap structure by DCP1a and DCP2, followed by exonucleolytic digestion by exoribonuclease 1 (XRN1; Parker & Sheth, 2007). EDC4 is essential for the decapping process as it provides a scaffold for DCP1a, DCP2 and XRN1 (Jonas & Izaurralde, 2013; Chang et al, 2014). In addition, EDC4 is required for the assembly of the RBPs into higher order complexes, known as processing bodies (P bodies; Yu et al, 2005; Decker et al, 2007; Parker & Sheth, 2007). P bodies belong to a group of non-membrane-bound organelles, containing both proteins and RNA (Braun et al, 2012). mRNAs might be targeted to P bodies for degradation (Brengues et al, 2005; Teixeira et al, 2005). Other studies have shown that mRNAs are stored in P bodies and can be released to enter translation (Brengues et al, 2005; Bhattacharyya et al, 2006) and that a vast majority is protected from 5′ decay (Kedersha et al, 2005). In addition, a recent genome-wide study revealed that decay or stabilization of P-body-associated mRNAs is controlled in a context-dependent manner and in response to stress (Teixeira et al, 2005). These multiple functions of P bodies may thus strongly impact the timing and amplitude of cellular responses to exogenous stimuli. Here, we show a direct link between IKK and change in RNA stability of scores of transcripts. We identified the P body scaffold protein EDC4 as a novel interaction partner of the IKK complex. IKKβ phosphorylated EDC4 when activated by TNFα, IL-1β, or genotoxic or oxidative stress. Phosphorylation of EDC4 was crucial for its interaction with other RBPs, and subsequent formation of higher order complexes, reflected in an increase in detectable P bodies. RNA-Seq analysis revealed post-transcriptional regulation of hundreds of mRNAs by the IKK-EDC4 axis in response to stress. Formerly, IKK-dependent gene regulation was regarded to occur via activation of the transcription factor NF-κB. Our data provide evidence for a global mechanism and an extensive NF-κB-independent function of the IKK complex in the regulation of mRNA stability. Results IKK complex interacts with P body scaffold protein EDC4 We identified novel interaction partners of IKKγ by co-immunoprecipitation from SILAC-labelled human osteosarcoma cells treated with ionizing irradiation (IR; Fig 1A, Appendix Fig S1A and Table EV1). Gene Ontology (GO) term analysis and clustering of the proteins with increased association with IKKγ after IR stimulation showed an expected enrichment for the biological processes "response to DNA damage stimulus", "post-translational protein modification", "regulation of apoptosis", but interestingly, also for "mRNA metabolic process" (Appendix Fig S1B). The latter group comprised numerous RNA-binding proteins, exoribonuclease 3′-5′ (DIS3), RNase inhibitor 1 (RNH1), and P body proteins Poly(RC) binding protein 1 (PCBP1), and EDC4, among others (Appendix Fig S1A). EDC4 is essential for P body formation and was therefore selected for further analysis. Figure 1. EDC4 interacts with IKK in a stimulus-sensitive manner Schematic diagram of the SILAC screen. Immunoprecipitation of endogenous IKKγ from cytoplasmic cell lysates of unstimulated (ut) or irradiated U2-OS cells followed by Western blot (WB) of IKKγ and EDC4. Immunoprecipitation of endogenous EDC4 or IKKβ from unstimulated (ut), irradiated (20 Gy; 45 min post-stimulus) or TNFα-treated (10 ng/ml; 15 min) U2-OS cells, WB of EDC4 or IKKβ. Fold changes of co-precipitated proteins are indicated. Results are representative for three experiments. Proximity ligation assay (PLA) of unstimulated (ut), irradiated (20 Gy: 45 min post-stimulus) or TNFα-treated (10 ng/ml; 45 min post-stimulus) U2-OS cells; IKKβ-EDC4 interaction (red), nucleus (blue). As a negative control, PLA was performed without primary IKKβ antibody. Scale bar: 25 μm. Bottom panel: relative quantitation of independent experiments (n = 2) by ImageJ software, at least 500 cells per experiment ± s.d. unpaired t-test, *P < 0.05. Co-expression of full-length FLAG-IKKγ with HA-tagged EDC4 sub-regions, wild-type HA-IKKα (positive control) or empty vector (negative control) in HEK293 cells, followed by immunoprecipitation of FLAG-IKKγ and WB of FLAG and HA. Immunoprecipitation was performed with anti-FLAG sepharose from whole-cell lysates of irradiated (10 Gy) cells. Left panel: input, right panel: FLAG-IP. Asterisks denote specific bands. Note that the three EDC4 fragments reveal an aberrant migration relative to calculated molecular weight in SDS–PAGE (Braun et al, 2012). HEK293 cells expressing N-terminally His-tagged IKKγ deletion constructs (as shown in Appendix Fig S1H) or empty His-vector together with HA-tagged EDC4 WD40 domain (HA-EDC41–538). Immunoprecipitates from whole-cell lysates of irradiated cells with anti-HA sepharose were analysed by WB for His and HA. Left panel, input; right panel, HA-IP. Asterisks, specific bands. Co-immunoprecipitation as in (F) with C-terminally FLAG-tagged IKKγ. Source data are available online for this figure. Source Data for Figure 1 [embj201798658-sup-0010-SDataFig1.pdf] Download figure Download PowerPoint Co-immunoprecipitation of endogenous IKK and EDC4 confirmed the increased interaction in response to irradiation (Fig 1A and B, Appendix Fig S1A). To determine whether EDC4 is a substrate of the IKK complex, interaction of EDC4 with IKKβ was analysed. IKKβ is activated by DNA damage and also other canonical IKK stimuli, including TNFα. Reciprocal co-immunoprecipitation of endogenous IKKβ and EDC4 showed a basal interaction that was enhanced in response to IR and to TNFα (Fig 1C). To rule out the possibility that this interaction was mediated through RNA, reciprocal co-immunoprecipitations were repeated in the presence of RNase A and RNase T1 (Appendix Fig S1C). In situ analysis by proximity ligation assays (PLAs) revealed a direct and an IR- or TNFα-enhanced interaction between EDC4 and IKKβ (Fig 1D). These findings suggest that IKK-EDC4 interaction is not restricted to DNA damage signalling, but can be induced by different stimuli that activate IKK. We further expanded our analysis to other components of P bodies and to the IKK cascade and showed DNA damage-induced interaction of IKKγ with P body marker DDX6 (Appendix Fig S1D) and of EDC4 with TRAF6 (TNF receptor-associated factor 6), which is an essential component of the cytoplasmic IKK signalling cascade (Appendix Fig S1E; Hinz et al, 2010). We next asked which protein domains are required for the IKKγ-EDC4 interaction (Fig 1E–G, Appendix Fig S1F–H). The EDC4 protein contains an N-terminal WD40 domain and a C-terminal α-helical domain connected by a serine-rich linker region (Appendix Fig S1G). In vitro co-IP assays showed that the N-terminal WD40 domain is necessary and sufficient for interaction with full-length IKKγ (Appendix Fig S1F). Precipitation of sub-regions of EDC4 from irradiated cells confirmed that the WD40 domain of EDC4 interacts with IKKγ (Fig 1E). We further showed that the leucine zipper and zinc-finger motifs in IKKγ are required for binding to EDC4 (Fig 1F and G, Appendix Fig S1H). Collectively, these results demonstrate that cellular stress promotes interaction between EDC4 and the IKK complex. The IKK complex phosphorylates EDC4 on serines in the WD40 domain and in the serine-rich linker To identify IKK substrate sites in EDC4, we performed in vitro kinase assays with endogenous IKK and the purified recombinant sub-regions of EDC4. IR, TNFα and IL-1β all led to inducible phosphorylation of EDC4 in the WD40 domain and the serine-rich linker region, indicating that canonical IKKγ/IKKβ signalling mediates phosphorylation of EDC4 (Fig 2A). Figure 2. IKK phosphorylates EDC4 In vitro kinase assay (KA) using endogenous IKKβ from cells, unstimulated or stimulated with IR (20 Gy, 45 min; top panel) or IL-1β (10 ng/ml, 10 min; bottom panel) with purified GST-EDC4 domains as indicated. Lower panel: cold KA as above, Coomassie blue staining. Asterisks denote specific bands. IKK phosphosite identification in EDC4 by mass spectrometry (see Table EV2 for MS data). Endogenous IKK purified from unstimulated or TNFα-treated (10 ng/ml, 15 min) U2-OS cells by immunoprecipitation of IKKγ was used in a cold KA with recombinant EDC4 sub-regions, followed by MS analysis. Top, MS spectrum for phospho-serine 583. Bottom, MS spectrum for phospho-serine 855. In vitro KA of IKKβ (as in A) from TNFα-stimulated cells with purified recombinant Strep-EDC4 WD40 domain (EDC4 1–538) and point mutants for IKK phosphosites, S107A, S405A and S107/405A. Below: cold kinase assay. Diagram of EDC4 indicating IKKβ-phosphorylated serines. Source data are available online for this figure. Source Data for Figure 2 [embj201798658-sup-0011-SDataFig2.pdf] Download figure Download PowerPoint Subsequent MS analysis revealed serines 583 and 855 in the serine-rich linker region of EDC4 as IKK phosphosites (Fig 2B, Table EV2). Because of limited sequence coverage by MS, we searched the dbPTM database and found serines 107 and 405 as further potential IKK phosphorylation sites. Combined substitution of serines 107 and 405 with alanines, but not of each alone, resulted in complete loss of IKK-mediated phosphorylation (Fig 2C). These data confirmed that IKK phosphorylates EDC4 at two serines in the WD40 domain of EDC4 (Ser107 and Ser405) and at two serines in the serine-rich linker region of EDC4 (Ser583 and Ser855; Fig 2D). To confirm that phosphorylation of EDC4 by IKK occurs in cells, we additionally performed MS analysis on endogenous, immunoprecipitated EDC4 in IKKβ or EDC4 CRISPR knockout cell lines or control cells (Appendix Fig S2A–C, Table EV3). TNFα stimulation resulted in phosphorylation of EDC4 at Ser583 in an IKK-dependent manner. This site, along with serines 107 and 855, was likewise reported as phosphosite of EDC4 by the PhosphoSitePlus.org database (Appendix Fig S2D). In summary, we demonstrated that EDC4 is an IKK substrate in cells. Phosphorylation of EDC4 by IKK enhances formation of P bodies EDC4 is essential for the assembly of RBP complexes and RNA, whose microscopically detectable foci are referred to as P bodies (Yu et al, 2005). P bodies are detectable in eukaryotic cells under normal cellular conditions, but increase in size and number in response to exogenous and endogenous stress (Kedersha et al, 2005; Teixeira et al, 2005). Furthermore, P body size and number are proportional to and dependent on the pool of translationally silenced mRNAs (Parker & Sheth, 2007). Therefore, we analysed changes in P body formation following DNA damage. The number of P bodies was significantly increased after irradiation, as seen by immunofluorescence staining of cells with P body marker DDX6 in different human primary and cancer cell lines (Fig 3A and Appendix Fig S3A and B). Furthermore, both IKKβ and IKKγ co-localized to the P body foci and the number of foci containing EDC4 and IKKβ or IKKγ increased after stimulation (Appendix Fig S3C and D). Since IKK directly phosphorylates EDC4, we tested whether other known IKK stimuli would also affect P body formation. Indeed, TNFα, IL-1β or hydrogen peroxide-induced T-loop phosphorylation of IKKβ, as expected, and led to an increase in P body numbers (Appendix Fig S3E–J). Depletion of IKKβ or IKKγ attenuated P body induction in stimulated cells (Fig 3B and Appendix Fig S3K–N). Similarly, an IKK inhibitor abrogated P body induction in response to DNA damage (Appendix Fig S3O). Figure 3. Phosphorylation of EDC4 by IKK promotes P body formation Fluorescence microscopy using anti-DDX6 antibody (green) of untreated U2-OS cells, or 45 or 90 min post-irradiation (IR). Nuclei stained with DAPI (blue), scale bar 50 μm. Bottom panel: quantification of P-body foci from independent experiments (n = 3) by ImageJ software, 100 cells per experiment ± s.d. unpaired t-test, *P < 0.01. Equivalent results were obtained with staining for P body components EDC4, DCP1a and DCP2 (not shown). As described in panel (A), except in clonal U2-OS cells stably expressing pTRIPZ RFP-coupled shIKKβ. Cells were treated with doxycycline (IKKβsh) or left untreated (wt) and analysed 45 or 90 min after IR. Bottom panel: P body quantification as in (A). Fluorescence microscopy using EGFP-tagged EDC4 (green) and DAPI in clonal U2-OS cells stably expressing pTRIPZ RFP-coupled shEDC4 and reconstituted with either wild-type EGFP-tagged EDC4 or EGFP-tagged EDC4 phospho-deficient Ser107/405/583/855Ala mutant (SA). Cells were pre-treated with doxycycline to deplete endogenous EDC4 prior to IR. Immunoprecipitation of clonal U2-OS cells stably expressing pTRIPZ RFP-coupled shEDC4 to deplete endogenous EDC4 and reconstituted with either shRNA-resistant wild-type FLAG-tagged EDC4 or FLAG-tagged EDC4 phospho-deficient mutant (SA). Endogenous EDC4 was depleted with doxycycline treatment prior to IR and co-immunoprecipitation with anti-FLAG sepharose. IP lysates were analysed by Western blot with anti-FLAG, anti-DCP1a and anti-DCP2 antibodies. Left: input. Right: FLAG-IP. Source data are available online for this figure. Source Data for Figure 3 [embj201798658-sup-0012-SDataFig3.pdf] Download figure Download PowerPoint Depletion of EDC4 confirmed its essential role in P body formation (Appendix Fig S3P–S; Yu et al, 2005). CRISPR-mediated knockout of IKKβ or EDC4 confirmed the requirement of these two factors for the induction of P body formation in response to IR and TNFα (Appendix Fig S3S). Of note, extended absence of IKKβ in the CRISPR-mediated knockout cells led to a higher basal number of P bodies. But neither IR nor TNFα led to a further increase in foci number (Appendix Fig S3S, right panel), as also seen with IKKβ or IKKγ siRNA-mediated depletion or treatment with an IKK inhibitor. We reconstituted EDC4-depleted cells with EDC4 wild-type (wt) or with non-phosphorylatable EDC4 SA mutant to determine whether phosphorylation of EDC4 by IKK was necessary for P body induction. Reintroduction of wt EDC4 rescued formation of P bodies and their stimulus-dependent increase. In contrast, introduction of the EDC4 SA mutant led to a low basal level of P body formation and could not restore their amplification (Fig 3C). Moreover, the induced interaction of P body components DCP1a and DCP2 was only observed with the wt EDC4, but not with the EDC4 SA mutant (Fig 3D). Further factors required for mRNA stabilization and storage are likely assembled in the same manner. Taken together, these findings suggest that phosphorylation of EDC4 by IKK promotes assembly of P bodies in response to diverse stimuli. P bodies share many components with cytoplasmic stress granules (SGs; Kedersha et al, 2005). To determine whether SGs might be connected to the IKK-EDC4 response, we analysed whether SG formation would be affected by agents that activate IKK (Appendix Fig S4A). Only hydrogen peroxide treatment, as expected, but not TNFα, IL-1β or IR, led to significant SG formation at analysed time points. Thus, IKK signalling significantly affects the formation of P bodies but not of SGs (Appendix Fig S4B). IKK-EDC4 axis differentially regulates stability of hundreds of transcripts To identify transcripts post-transcriptionally regulated by IKK or EDC4 in unstimulated cells or in response to DNA damage, we performed actinomycin D chase experiments with wild-type, EDC4- or IKKβ-depleted cells followed by RNA-Seq (Fig 4). We used an early IR time point where we detected induction of P bodies (see above), but no significant transcriptional response via the IKK-NF-κB axis (Fig 4A, Tables EV4 and EV5 and Appendix Fig S5A and B). Transcript expression (due to changes in transcription, co-transcriptional regulation and post-transcriptional stabilization or degradation) was affected by DNA damage in both positive and negative manner (Appendix Fig S5A and B). Expression of genes responsible for cell proliferation and differentiation was negatively regulated (Appendix Fig S5C). A significant number of transcripts were, however, controlled solely post-transcriptionally (Appendix Fig S5D). The functional cluster "Transcription from RNA Polymerase II promoter" was attenuated both through altered expression and through destabilization (Appendix Fig S5C and D). Additionally, irradiation increased the stability of inflammatory and cytokine responses (Appendix Fig S5D–F). Figure 4. EDC4 and IKK regulate stability of multiple transcripts Schematic diagram of ActD chase experiments to determine stability of transcripts. pTRIPZ constructs encoding shIKKβ or shEDC4 (as above) were treated with dox to induce knockdown (KD, IKKβsh, EDC4sh) or left untreated (wt). ActD treatment, 120 min, IR at 60 min prior to harvest. RNA was analysed by qRT–PCR. Bar charts represent raw data. Line graphs represent normalized stability, were IR or unstimulated samples were set at 100% and ActD-treated samples represent residual expression. Graph showing mRNAs with increased (green) or decreased (red) expression in IKK-depleted cells (see A) relative to wt. mRNA expression was measured by RNA-Seq, and significance was calculated for two corresponding conditions and cut-off set for P-value < 0.1 and fold change > 2 (Table EV4A) Graph illustrating mRNAs with increased (green) or decreased (red) stability in IKK-depleted cells (see A) relative to wt. mRNA stability was determined by RNA-Seq (Table EV4B). Analysis of mRNA expression in EDC4-depleted cells relative to wt as in (A; Table EV4C). Analysis of mRNA stability in EDC4-depleted cells relative to wt as in (B; Table EV4D). GO terms of transcripts which are destabilized by IKK or EDC4 using REVIGO for removal of redundancy of GO terms (Supek et al, 2011). Scatterplot clustering of groups with functionally similar GO terms. Each circle represents a GO term; size indicative of gene target numbers; the x-axis and y-axis are semantic coordinates given by REVIGO; violet: EDC4-depleted, blue: IKK-depleted, orange: terms identical for both EDC4 and IKK-depleted samples; P-value < 0.05 (Table EV4E). Note that semantic co-positioning in the plot indicates functional similarity of GO terms. Visualization of GO terms of mRNAs that are stabilized by IKK or EDC4, as in (F; Table EV4F). Download figure Download PowerPoint A significant proportion of mRNAs whose expression changed in an IKKβ- or EDC4-dependent manner (Fig 4B and D) were regulated via stabilization of their transcripts (Fig 4C and E). Overall, IKKβ or EDC4 regulated the stability of a large number of transcripts, respectively (Fig 4A–E and Table EV4), a third of which showed regulation by both IKK and EDC4 (Table EV4). In addition to the IKK-EDC4 axis, both factors also independently regulated mRNA stability (Fig 4B–E, Table EV4). This likely involves other RBPs that interact with IKK (Appendix Fig S1A). Transcripts destabilized via the IKKβ-EDC4 axis were enriched for GO terms "response to wounding", "negative regulation of apoptotic process" and "cellular response to lipopolysaccharide" and included numerous NF-κB targets, which were regulated under these unstimulated conditions in an NF-κB-independent manner (Fig 4F, Tables EV4 and EV6). In unstimulated cells, stabilized targets showed enrichment for GO terms "cell adhesion" and "extracellular matrix organization" and "regulation of ion transmembrane transport" (Fig 4G and Table EV4). Transcripts which were destabilized via the IKK-EDC4 axis included IL-8, CSF2, CCL5, CEBPβ and KLF10, while selectively stabilized transcripts comprised CCL7, CCL11, FOXA1, IRF7 and p63 (Table EV4). IL-8 mRNA is one of the first identified transcripts whose stability is regulated by P bodies following IL-1β stimulation and whose transcription is regulated by NF-κB (Rzeczkowski et al, 2011). We therefore used IL-8 as a bona fide target of IKK and EDC4 for an in-depth analysis on the impact on mRNA stability. The stability of IL-8 mRNA increased after IR treatment compared to its baseline stability in wild-type cells (Appendix Fig S6A and B). We first compared exon–exon with exon–intron spanning IL-8 transcripts (Appendix Fig S6B) and did not detect any unprocessed exon–intron pre-mRNA for IL-8 in ActD-treated cells, confirming complete inhibition of transcription. Therefore, irradiation increased IL-8 mRNA expression through stabilization of pre-existing transcripts. Stabilization of IL-8 mRNA by DNA damage was confirmed in several cell types (Appendix Fig S6C–E) and using the other IKK-activating stimuli, TNFα, IL-1β or hy
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