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p38MAPK/MK2-mediated phosphorylation of RBM7 regulates the human nuclear exosome targeting complex - PubMed

p38MAPK/MK2-mediated phosphorylation of RBM7 regulates the human nuclear exosome targeting complex

Christopher Tiedje et al. RNA. 2015 Feb.

Abstract

The nuclear exosome targeting complex (NEXT) directs a major 3'-5' exonuclease, the RNA exosome, for degradation of nuclear noncoding (nc) RNAs. We identified the RNA-binding component of the NEXT complex, RBM7, as a substrate of p38(MAPK)/MK2-mediated phosphorylation at residue S136. As a result of this phosphorylation, RBM7 displays a strongly decreased RNA-binding capacity, while inhibition of p38(MAPK) or mutation of S136A in RBM7 increases its RNA association. Interestingly, promoter-upstream transcripts (PROMPTs), such as proRBM39, proEXT1, proDNAJB4, accumulated upon stress stimulation in a p38(MAPK)/MK2-dependent manner, a process inhibited by overexpression of RBM7(S136A). While there are no stress-dependent changes in RNA-polymerase II (RNAPII) occupation of PROMPT regions representing unchanged transcription, stability of PROMPTs is increased. Hence, we propose that phosphorylation of RBM7 by the p38(MAPK)/MK2 axis increases nuclear ncRNA stability by blocking their RBM7-binding and subsequent RNA exosome targeting to allow stress-dependent modulations of the noncoding transcriptome.

Keywords: RBM7; RNA binding; RNA stability; ncRNAs; p38MAPK/MK2 signaling; phosphorylation.

© 2015 Tiedje et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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Figures

FIGURE 1.
FIGURE 1.

RBM7 is phosphorylated by MK2 in vivo and in vitro. (A) Multiple sequence alignment of vertebrate RBM7 and its closest homolog RBM11. Displayed amino acid sequences span the human RBM7 S136 MK2 phosphorylation site (indicated by a star). The MK2-site is absent in RBM11. (B) Murine GFP-Rbm7 and GFP-Rbm7S136A were overexpressed in HeLa cells and treated as indicated. Upon precipitation with the help of a GFP-Nanotrap lysates and precipitates were developed with p(S/T)-PKD-substrate and pS136-Rbm7 antibodies to analyze Rbm7 phosphorylation and a GFP-antibody as loading control. Only wild-type Rbm7 gave rise to a specific phosphorylation on S136 upon anisomycin stimulation, whereas BIRB796-treatment and S136 to A mutation resulted in a loss of the phospho-signal. (C) Murine GFP-Rbm7 and GFP-Rbm7S136A were again overexpressed in HeLa cells and precipitated with a GFP-Nanotrap upon stimulation as indicated. The precipitates were then incubated with recombinant MK2 and p38 proteins for 30 min at 30°C to trigger in vitro phosphorylation. Afterward, samples were resolved by SDS-PAGE and either transferred to a nitrocellulose membrane and developed with the specific pS136-Rbm7- or GFP-antibody to monitor equal precipitation or stained by Coomassie. As demonstrated with the in vivo experiments (2B and 2C) only active MK2 (MK2 + p38) could phosphorylate Rbm7 in vitro. Precipitated GFP-Rbm7 and GFP-Rbm7S136A was detected at the same molecular weight as recombinant MK2. Its positions are indicated by an arrow. (D) Murine GFP-Rbm7 was expressed in HeLa cells and stimulated with anisomycin for 0, 1, 2, and 4 h to gain information on the phosphorylation kinetics of RBM7 and the p38MAPK-signaling pathway components and targets. The anti-pS136-Rbm7 antibody only detected the phosphorylated exogenous GFP-tagged Rbm7 protein. The positions of endogenous RBM7 and ZCCHC8 are indicated by arrows. Anisomycin stimulation altered the band intensities of RBM7 and ZCCHC8. (E) To analyze the potential phosphorylation of endogenous RBM7 and ZCCHC8 in more detail, extracts of HeLa cells treated as indicated were resolved on 8% and 12% SDS gels, respectively. Still, the endogenous RBM7-signal is weaker when cells are treated with anisomycin and the ZCCHC8 signal shifts slightly to a higher molecular weight. Both effects could point to a phosphorylation of the endogenous proteins. Unspecific bands coming up with antibodies detecting endogenous RBM7 and ZCCHC8 are marked with a star. (F) To verify endogenous RBM7 and ZCCHC8 phosphorylation, cell lysates that were treated as indicated and incubated with phosphatase (CIP, see Materials and Methods) were analyzed by Western blotting upon running 8% or 10% gels, respectively. (G) Endogenous RBM7 from HeLa cells that were treated as in F was immunoprecipitated using the anti-pS136-Rbm7 antibody (see Materials and Methods for details). Samples were analyzed for the presence of pS136-RBM7 using the same antibody and eEF2 for equal loading. The arrow indicates endogenous phosphorylated RBM7 and the stars indicate for the heavy and the light of the antibody used for IP.

FIGURE 2.
FIGURE 2.

Consequences of MK2-mediated phosphorylation of RBM7 on its protein–protein interactions and its RNA-binding capacity. (A) N-terminally GFP-tagged Rbm7 and Rbm7S136A were coexpressed with FLAG-tagged ZCCHC8 in HeLa cells. Their interaction was analyzed in nonstimulated cells, in cells stimulated with anisomycin for 2 h or in cells stimulated with anisomycin after BIRB796-mediated p38MAPK-inhibtion. GFP-fusion proteins were precipitated as mentioned in the Materials and Methods. Equal expression of GFP- and FLAG-tagged fusion proteins and GAPDH (loading control) in the lysate was monitored by Western blotting. In addition, the stimulation with anisomycin and the inhibition by BIRB796 was checked with the above-mentioned pS136-Rbm7-specific antibody. It became clear that the interaction of exogenous Rbm7 and ZCCHC8 independent of the activation status of the cells and the phosphorylation status of the proteins was not altered. (B) HeLa cells overexpressing GFP-Rbm7 or GFP-Rbm7S136A were cross-linked with 300 mJ/cm2 UV-C light prior to cell lysis to covalently connect Rbm7 to the RNAs to which it is bound. As indicated, different conditions were analyzed and the cross-linked RNAs were radioactively labeled, visualized by autoradiography and developed afterward with an anti-GFP-antibody by Western blotting. As a negative control GFP-Rbm7S136A expressing cells were not cross-linked but otherwise treated like the other samples. Consistently, the precipitate did not produce signals of copurifying RNAs. (C) Rbm7-bound RNA signals of the autoradiograph were normalized to anti-GFP Western blot signals derived from the same membrane (see Materials and Methods). The star indicates P < 0.01. The replicate for parts of this experiment is shown in Supplemental Figure S4B.

FIGURE 3.
FIGURE 3.

PROMPTs are accumulating by anisomycin- and UV-C light-induced p38MAPK-activation in HeLa cells. (A) Treatment of HeLa cells with 10 µg/mL anisomycin for 2 h or irradiation with 100 J/m2 UV-C light results in the strong activation of p38MAPK as shown by Western blot for phospho-T180 and phospho-Y182 in p38 and the activation of its downstream kinase MK2 as monitored by T334 phosphorylation. Inhibition of p38MAPK-activity by the inhibitor BIRB796 (BIRB) prevents p38- and MK2-phosphorylation. Total p38 and MK2 protein remained unchanged throughout the experiments. The translation elongation factor eEF2 served as a loading control and RBM7 and ZCCHC8 showed similar band patterns in response to stress like in previous experiments (see Figs. 1E–G, 4A,C). (B) Using the anisomycin stimulation experimental setup, expression of four well-characterized PROMPTs (proRBM39, proEXT1, proIFNAR1, and proDNAJB4) was analyzed at different time points (0, 60, 120, and 240 min anisomycin stimulation) by qRT-PCR. (C) As a control the anisomycin-induced downstream expression of RBM39 was analyzed. (D) Like the anisomycin-induced up-regulation of PROMPTs the UV-C light-mediated accumulation can be abolished by p38MAPK-inhibition using BIRB796. All results are shown as x-fold induction relative to the nonstimulated expression that was set to 1. All graphs are displayed with standard deviations. All experiments were performed independently a minimum of two times and showed similar effects. (E) Overexpression of GFP-RBM7S136A leads to diminished PROMPTs accumulation upon anisomycin stimulation indicating that stress targets the exosomal degradation of PROMPTs via RBM7. (F,G) HeLa cells were treated with either a Control siRNA or hRRP40- and RBM7-specific siRNAs and 48 h post-transfection treated as indicated. Levels of proRBM39 and proDNAJB4 were determined by qRT-PCR and accumulation was calculated relative to untreated cells for each siRNA knockdown. Knockdown efficiency of hRRP40 and RBM7 was assayed by Western blot (Supplemental Fig. S7B).

FIGURE 4.
FIGURE 4.

PROMPTs are accumulating by anisomycin and partially by LPS stimulations in monocytic THP-1 cells. (A) THP-1 cells were stimulated for 1 h with 10 µg/mL anisomycin in the presence or absence of the p38-inhibitor BIRB796 and the indicated proteins were assayed by Western blot. (B) THP-1 cells were stimulated for 2 h with 10 µg/mL anisomycin in the presence or absence of the p38-inhibitor BIRB796. Consequently, PROMPTs RNA levels were determined by qRT-PCR and shown as x-fold induction relative to the nonstimulated expression levels. (C) LPS stimulation (1 µg/mL) for 1 h of THP-1 cells resulted in a strong induction of TNFα mRNA in THP-1 cells (right panel). Induction of TNFα was sensitive to inhibition of p38MAPK activity. Blotting for endogenous proteins showed similar loss of RBM7 band intensity as in Figure 1E–G or A. (D) The same cDNAs as in C were analyzed for PROMPTs RNA levels. While proRBM39, proEXT1, and proDNJAB4 levels were not changed, proIFNAR1 levels were twofold up-regulated by LPS stimulation in a p38-dependent manner. All graphs are displayed with standard deviations. The star indicates P < 0.04 (as determined by two-sided Student's t-test) for the LPS-induced up-regulation of proIFNAR1.

FIGURE 5.
FIGURE 5.

PROMPTs accumulation is sensitive to inhibition of the p38MAPK-activted kinase MK2 in HeLa and HEK293T cells. (A) Prior to 2 h anisomycin-stimulation HeLa cells were preincubated for 90 min with MK2-inhibitor PHA781089 (15 µM). The phosphorylation of MK2's bona fide substrate Hsp27 was monitored through Western blot for pS82-Hsp27. Blots for total Hsp27 and GAPDH served as loading controls. (B) Under the same conditions as in A. PROMPTs RNA levels were determined and are shown as x-fold inductions. MK2-inhibition led to a strong down-regulation of PROMPTs levels under these conditions. (C) PROMPTs were detected in HEK293T cells. Although up-regulation of proIFNAR1 was not detectable in these cells, the anisomycin-induction of proRM39 and proEXT1 was highly sensitive to the p38MAPK-activated kinase MK2 as shown by qRT-PCR. (D) Three different stable HEK293T cell lines expressing two distinct MK2-specific shRNAs and a sh-control-RNA were generated. MK2-knockdown efficacy was controlled through blotting for MK2. GAPDH served as a loading control. MK2 levels were strongly reduced in the two MK2-specific knockdown cell lines when compared with the control cell line. (E–G) The anisomycin-induced levels of proRBM39 (E), proEXT1 (F), and proDNAJB4 (G) were assayed in the control and knockdown cell lines. Both knockdown cell lines showed reduced PROMPTs levels upon anisomycin stimulation compared with the control cell line. Notably, PROMPTs levels were further reduced by pretreatment of the cells with the p38-inhibitor BIRB796. All graphs are displayed with standard deviations. All experiments were performed independently a minimum of two times and showed similar effects.

FIGURE 6.
FIGURE 6.

PROMPTs are regulated at the level of stability and not by RNAPII-mediated changes in transcription regulation. (A) The ability of three different proRBM39 amplicons along the transcript (#1, #2, and #4) (Andersen et al. 2013) to assess the anisomycin-induced up-regulation of proRBM39 in comparison to the primer pair used in all other qRT-PCR experiments was proven. All primers were similarly effective in detecting the strong induction. We note that the ChIP primer showed higher background amplification than the common qRT-PCR primers. (B) RNAPII ChIP analyses were performed after treating HeLa cells for 30 and for 60 min with anisomycin (10 µg/mL). The DNA-fragments cross-linked to RNAPII and to the control IgG were analyzed by qPCR. Only minor differences in RNAPII occupancy of the proRBM39 transcript region were detected. Notably, the occupancy along the transcript region decreases strongly toward upstream. (C) The anisomycin-induced up-regulation shows a pattern similar to PROMPTs, but the RNAPII occupancy pattern differs strikingly from PROMPTs. (D) Regardless of the duration of anisomycin stimulation the occupancy along the transcript is increased under stimulatory conditions. (E) The stability of PROMPTs was assayed in nonstimulated HeLa cells or in cells after 2 h of anisomycin stimulation by inhibiting RNA-Polymerase II transcription by the addition of 10 µg/mL actinomycin D. Every 5 min the PROMPTs RNA levels were determined by qRT-PCR and are shown as the remaining RNA amount (% of time point 0, start of actinomycin D addition). proRBM39, proIFNAR1, and proDNAJB4 transcripts were stabilized upon stimulation (increase of their half-life from 5–10 min to 20–35 min). The half-life of proEXT1 during anisomycin stimulation was ∼20 min and in the same range of the other PROMPTs that were analyzed (Supplemental Fig. S10A). Under nonstimulated conditions only the first time point before adding actinomycin D was accessible for qRT-PCR measurement as levels after actinomycin treatment were equal to background detection. (F) BIRB796-mediated p38MAPK-inhibtion reverses the gain in RNA stability of PROMPTs. The stability of proRBM39, proIFNAR1, and proDNJAB4 from derived HeLa cells treated as indicated was determined. BIRB796-treament led to an intermediate half-life of PROMPTs RNAs. All graphs are displayed with standard deviations. All experiments were performed independently a minimum of two times and showed similar effects.

FIGURE 7.
FIGURE 7.

Proposed model for the regulation of PROMPTs RNAs by the p38MAPK/MK2 signaling module upon stress. Upon stress, induced by anisomycin- or UV-C light-stimulation, p38MAPK becomes activated through phosphorylation. In turn, activated p38 is able to phosphorylate MK2, its downstream kinase. MK2 phosphorylates RBM7 at S136. It is possible that p38 or other stress-activated kinases are able to phosphorylate ZCCHC8 in parallel. Phosphorylated RBM7, as part of the trimeric NEXT complex (composed of RBM7, ZCCHC8, and hMTR4) is less effective in binding and in properly guiding PROMPTs to nuclear exosomal degradation. This is supported by a massive gain in PROMPTs RNA transcript stability and easy detection in qRT-PCR analyses because of their accumulation. Notably, p38MAPK/MK2-activation seems to be necessary but not sufficient for complete activation of this mechanism as LPS-, Il-1α stimulation and high osmolarity only lead to moderate and selective PROMPTs accumulation (Fig. 4D; Supplemental Fig. S8A,B).

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