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. 2018 Dec 14;37(24):e98658.
doi: 10.15252/embj.201798658. Epub 2018 Nov 22.

The IκB kinase complex is a regulator of mRNA stability

Nadine Mikuda  1 , Marina Kolesnichenko  1 , Patrick Beaudette  2 , Oliver Popp  2 , Bora Uyar  3 , Wei Sun  4 , Ahmet Bugra Tufan  1 , Björn Perder  1 , Altuna Akalin  3 , Wei Chen  4 , Philipp Mertins  2 , Gunnar Dittmar  2 , Michael Hinz  1 , Claus Scheidereit  5
Affiliations

The IκB kinase complex is a regulator of mRNA stability

Nadine Mikuda et al. EMBO J. .

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.

Keywords: IKK; EDC4; P bodies; RNA stability; post‐transcriptional regulation.

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Figures

Figure 1
Figure 1. EDC4 interacts with IKK in a stimulus‐sensitive manner

  1. Schematic diagram of the SILAC screen.

  2. 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.

  3. 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.

  4. 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.

  5. 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).

  6. 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.

  7. Co‐immunoprecipitation as in (F) with C‐terminally FLAG‐tagged IKKγ.

Source data are available online for this figure.
Figure 2
Figure 2. IKK phosphorylates EDC4

  1. 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.

  2. 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.

  3. 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.

  4. Diagram of EDC4 indicating IKKβ‐phosphorylated serines.

Source data are available online for this figure.
Figure 3
Figure 3. Phosphorylation of EDC4 by IKK promotes P body formation

  1. 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).

  2. 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).

  3. 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.

  4. 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.
Figure 4
Figure 4. EDC4 and IKK regulate stability of multiple transcripts

  1. 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.

  2. 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)

  3. 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).

  4. Analysis of mRNA expression in EDC4‐depleted cells relative to wt as in (A; Table EV4C).

  5. Analysis of mRNA stability in EDC4‐depleted cells relative to wt as in (B; Table EV4D).

  6. 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.

  7. Visualization of GO terms of mRNAs that are stabilized by IKK or EDC4, as in (F; Table EV4F).

Figure 5
Figure 5. Post‐transcriptional gene regulation by IKK
Proposed model for IKK in the regulation of mRNA stability versus transcription. IKK phosphorylates IκB proteins, promoting their degradation. Subsequently, NF‐κB translocates to the nucleus where it promotes transcription of dozens of target genes. Concurrently, IKK interacts with and phosphorylates EDC4, leading to an increase in P bodies. Recruited mRNAs are either degraded or stored in a translationally repressed state. This results in differential regulation of stability of hundreds of transcripts. In addition, IKK regulates various other RBPs contributing to the post‐transcriptional regulation of mRNA levels.
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