Fig 1.
The MAPK signaling pathway regulates post-transcriptional stages of ribosome biogenesis.
(A) Serum-starved HEK293, eHAP1 or HeLa cells were stimulated (PMA) or not (Ctl) with PMA (100 ng/ml) for 30 min, then treated (PD) or not (-) with 10 μM of PD184352 for 3h. Total RNAs were extracted and the levels of precursor and mature rRNAs and 7SL1 RNA were monitored by Northern blotting (NB, see S1A Fig for a detailed representation of the human pre-rRNA processing pathway and position of the probes). Total proteins were extracted and analyzed by Western blotting (WB) using the indicated antibodies. (B) RAMP analyses of pre-rRNA levels obtained in (A) normalized to the 7SL1 signals after quantification using MultiGauge software (Fujifilm). Graphical representations show fold changes compared to the starved conditions (Ctl). Statistically significant differences are indicated by asterisks (***: P≤0.001, **: P≤0.01, *: P≤0.05, Two-way ANOVA, Bonferroni posttests). (C) Serum-starved (Ctl) HEK293 cells were treated (PD+PMA) or not (PMA) with MEK 1/2 inhibitor PD184352 prior to PMA stimulation. After a 1 h phosphate deprivation, neo-synthesized RNAs were labeled for 1 h with 32P-labeled orthophosphate. Cells were harvested 0, 60 and 180 min following addition of cold orthophosphate. Total RNAs were extracted, separated by electrophoresis and transferred to nylon membranes. The labeled (pre-)rRNAs indicated on the left and on the right were detected by autoradiography. (D) Graphical representation of radioactive signals obtained in (C) after quantification using MultiGauge software (Fujifilm).
Fig 2.
RIOK2 is phosphorylated at Ser483 upon activation of the MAPK pathway.
(A) HEK293 cells were transfected with a plasmid expressing HA-RIOK2 or an empty vector (Ctl). HA-RIOK2 was immunoprecipitated from serum-starved cells treated (+) or not (-) with PD184352 for 1h (10 μM) prior to PMA (100 ng/ml, 20 min) or EGF (25 μg/ml, 10 min) stimulation (+). Samples were analyzed by WB using anti-RXRXXpS/T or anti-HA antibodies. (B) RXRXXpS/T ((P)-RIOK2) and total RIOK2 signals obtained in (A) were quantified using ImageLab software and expressed as fold change relative to the PMA condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (C) HA-RIOK2 was immunoprecipitated from serum-starved (Ctl) HEK293 cells treated (PMA+PD) or not (PMA) with PD184352 (10 μM, 1h) prior to PMA stimulation (100 ng/ml, 20 min). Purified HA-RIOK2 was isolated following SDS-PAGE, in gel digested with trypsin and the resulting peptides were submitted to nano-LC-MS/MS analysis. Label-free quantitative analysis of phosphorylation of the different RIOK2 phospho-peptides was performed as specified in the Materials and Methods section. Data are representative of triple biological replicate experiments for each condition. (D) HEK293 cells expressing HA-tagged versions of WT or mutant versions of RIOK2 (T481A or S483A) were serum starved (-) prior to PMA (100 ng/ml, 20 min) stimulation (+). HA-RIOK2 was immunoprecipitated and samples were analyzed by WB as in (A). (E) RXRXXpS/T ((P)-RIOK2) and total RIOK2 signals obtained in (D) were quantified using ImageLab software and expressed as fold change relative to the WT + PMA condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (F) Serum-starved HEK293 cells were stimulated with different agonists of the MAPK pathway. Phosphorylation of endogenous RIOK2 at Ser483 ((P)-RIOK2) was monitored by WB using specific antibodies. (G) (P)-RIOK2 and total RIOK2 signals obtained in (F) were quantified using ImageLab software and expressed as fold change relative to the starved condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test).
Fig 3.
RIOK2 is a direct RSK substrate.
(A) Serum-starved (Ctl) HEK293 cells were treated or not (-) with PD184352 (PD), or LJH685 (10 μM) (LJH) for 1 h prior to PMA stimulation (100 ng/ml, 20 min). Phosphorylation of endogenous RIOK2 at Ser483 ((P)-RIOK2) was monitored by WB using specific antibodies. (B) (P)-RIOK2 and total RIOK2 signals obtained in (A) were quantified using ImageLab software and expressed as fold change relative to the PMA condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (C) HEK293 cells were transfected with vectors over-expressing HA-tagged RSK1, RSK2, RSK3 or RSK4, or the empty vector (Ctl). Following serum-starvation and PMA stimulation (100 ng/ml, 20 min), samples were analyzed by WB using the indicated antibodies. (D) (P)-RIOK2 and total RIOK2 signals obtained in (C) were quantified using ImageLab software and expressed as fold change relative to starved Ctl cells. Statistically significant differences between starved conditions relative to starved Ctl cells are indicated by hashes, and between PMA stimulated conditions relative to stimulated Ctl cells by asterisks (#/*: P≤0.05, ns: not statistically significant, One-tailed Mann Whitney test). (E) HEK293 cells expressing shRNA targeting an irrelevant sequence (Ctl) or both RSK1 and RSK2 (RSK1/2) were processed as in (C). (F) (P)-RIOK2 and total RIOK2 signals obtained in (E) were quantified using ImageLab software and expressed as fold change relative to the starved condition. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (G) Human activated RSK1 was incubated in the presence of γ[32P]-ATP with either GST alone, or a GST-RIOK2 peptide (D443-E552) containing either S483 (RIOK2WT) or the non-phosphorylatable version (RIOK2S483A). The resulting samples were analyzed by SDS-PAGE and revealed by autoradiography or Coomassie blue staining. Quantification of [32P] incorporation within each peptide is expressed as n-fold change compared to the absence of RSK1. (H) In vitro kinase assays performed as in (G) in the presence or not of RSK inhibitors BI-D1870 or LJH685 (10 mM).
Fig 4.
RIOK2 phosphorylation at Ser483 is required for efficient maturation of pre-40S particles.
(A) MTS assays were performed on Control (Ctl) and RIOK2S483A (1 to 3) eHAP1 cell lines at the indicated time points. ODs at 490 nm were measured using Spectramax. (B) Control (Ctl) and RIOK2S483A (1 to 3) eHAP1 cell lines were incubated with 1 μM puromycin for the indicated times. Levels of puromycin-labelled peptides were monitored by WB using anti-puromycin antibodies. WB signals were quantified using ImageLab software and expressed as arbitrary units (a.u.). (C), Total cellular RNAs were extracted from control (Ctl), RIOK2S483A (1 to 3) or RIOK2S483D eHAP1 cell lines. Accumulation levels of pre-rRNAs and mature rRNAs were analyzed by NB as in Fig 1. (D) RAMP analyses of pre-rRNA levels obtained in (C) normalized to the 26S signals after quantification using MultiGauge software (Fujifilm). Graphical representations show fold changes compared to the control condition (Ctl). Statistically significant differences are indicated by asterisks (***: P≤0.001, *: P≤0.05, Two-way ANOVA, Bonferroni posttests). (E) FISH experiments performed on RIOK2WT (Ctl) and RIOK2S483A eHAP1 cell lines. Pre-rRNAs were detected using a Cy5-labeled 5’-ITS1 probe. Cells were stained with DAPI to visualize nuclei, and images were captured in identical setting conditions. (F) Nucleolar, nuclear and cytoplasmic fluorescence signals were quantified using ImageJ software, as described in the “Materials and Methods” section and S7 Fig. Graph representations show fold change in RIOK2S483A relative to RIOK2WT cell line (n = 100 cells from different fields). Statistically significant differences are indicated by asterisks (***: P≤0.001, **: P≤0.01, *: P≤0.05, One-tailed Mann Whitney test).
Fig 5.
Loss of RIOK2 phosphorylation at Ser483 increases its association with cytoplasmic pre-40S particles.
(A) RIOK2 localization was analyzed by IF microscopy using anti-RIOK2 antibodies in RIOK2WT (Ctl) and RIOK2S483A eHAP1 cell lines. Nuclei were visualized by DAPI staining. (B) Quantification of fluorescence observed in (A) using ImageJ software, as described in S7 Fig, and expressed as fold change relative to Ctl (n = 100 cells from different fields). Statistically significant differences are indicated by asterisks (***: P<0.0001, One-tailed Mann Whitney test). (C) Nucleo-cytoplasmic fractionation of serum-growing RIOK2WT (Ctl) and RIOK2S483A eHAP1 cell lines. RIOK2 levels in the different fractions (total cell extract, cytoplasm, nucleus) were analyzed by WB. Fractionation quality was validated using antibodies detecting tubulin (cytoplasmic protein) and fibrillarin (nuclear protein). (D) WB signals obtained in the cytoplasmic and nuclear fractions from (C) were quantified. The graph represents cytoplasmic/nuclear intensity ratios. Statistically significant differences are indicated by asterisks (*: P≤0.05, One-tailed Mann Whitney test). (E) HEK293 cells were transfected with plasmids expressing HA-tagged RIOK2WT or RIOK2S483A, or with empty vector (-). HA-RIOK2 was immunoprecipitated and co-immunoprecipitated proteins and 18S-E pre-rRNA were analyzed by WB and NB, respectively. (F) Quantification of the WB and NB signals obtained in (E) expressed as fold change compared to immunoprecipitated HA-RIOK2WT. (G) HEK293 cells were co-transfected with plasmids expressing HA-NOB1 and either Flag-RIOK2WT or Flag-RIOK2S483A. HA-NOB1 was immunoprecipitated and the co-immunoprecipitated proteins were analyzed by WB.
Fig 6.
RIOK2 phosphorylation at Ser483 facilitates its release from pre-40S particles and re-import into the nucleus.
(A) HEK293 cells were co-transfected with plasmids expressing HA-NOB1 and Flag-RIOK2WT, Flag-RIOK2S483A or Flag-RIOK2S483D. Pre-40S particles were immunopurified via HA-NOB1 and subsequently incubated for 45 or 90 min with a buffer inducing RIOK2 release at 16°C. The presence of RIOK2, LTV1 and RPS7 proteins in supernatants (released proteins) and on beads (pre-40S-bound proteins) were analyzed by WB. Experiments with Flag-RIOK2S483A and Flag-RIOK2S483D were performed with different sets of Flag-RIOK2WT as controls. A representative WB experiment for Flag-RIOK2WT is shown. (B) Quantification of WB signals from (A) using ImageLab software and expressed as released over bound RIOK2 ratios. Statistically significant differences are indicated by asterisks (***: P≤0.001, **: P≤0.01, Two-way ANOVA test, Bonferroni posttests). (C) RIOK2WT and RIOK2S483A eHAP1 cells were treated with Leptomycin B (LMB, 20 nM) for the indicated times. Subcellular localization of RIOK2 was monitored by immunofluorescence microscopy using specific antibodies. Nuclei were visualized by DAPI staining. (D), Quantification of nuclear to cytoplasmic fluorescence ratios at the indicated time points obtained in (C) using ImageJ software (n = 100 cells from different fields), as described in S7 Fig. Statistically significant differences are indicated by asterisks (***: P≤0.001, *: P≤0.05, 2way ANOVA tests, Bonferroni posttests).
Fig 7.
RIOK2 phosphorylation at Ser483 by RSK facilitates late stages of pre-40S particle maturation.
RIOK2 is incorporated into pre-40S particles in the nucleus and participates to their export to the cytoplasm. Phosphorylation of RIOK2 by RSK at Ser483 facilitates its dissociation from pre-40S particles, which allows simultaneous or subsequent dissociation of other factors (ENP1, LTV1, DIM2, NOB1) to promote efficient maturation of the 18S-E pre-rRNA. This phosphorylation event is required for optimal protein synthesis and cell proliferation.