Volume 20, Issue 6, Pages 891-903 (December 2005) p38-Dependent Phosphorylation of the mRNA Decay-Promoting Factor KSRP Controls the Stability of Select Myogenic Transcripts  Paola Briata, Sonia Vanina Forcales, Marco Ponassi, Giorgio Corte, Ching-Yi Chen, Michael Karin, Pier Lorenzo Puri, Roberto Gherzi  Molecular Cell  Volume 20, Issue 6, Pages 891-903 (December 2005) DOI: 10.1016/j.molcel.2005.10.021 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 p38 Activation in C2C12 Myoblasts Induces Stabilization of Select Myogenic Transcripts (A) Expression of myogenic markers and control transcripts in C2C12 cells cultured in GM or DM analyzed by RT-PCR. The sequence of the primers used is shown in Table S2. (B and C) Semiquantitative RT-PCR analysis of both myogenic and nonmyogenic transcripts in C2C12 cells cultured either in GM (dashed lines) or DM (solid lines and yellow highlight) (B), and in C2C12 cells cultured in GM and either infected with pAdMKK6EE (MKK6EE, solid lines, blue highlight) or mock infected with the pAdCMV empty vector (GM, dashed lines) (C). Total RNA was isolated after addition of actinomycin D. The amount of each transcript was quantitated by densitometry and plotted with a linear regression program. The values shown are averages (±SEM) of three independent experiments performed in duplicates. (D and E) In vitro RNA degradation assays using S100s from C2C12 cells cultured as indicated. Internally 32P labeled, capped RNA substrates (see Table S1 for sequences) were incubated with S100s for the indicated times, and their decay was analyzed as described in the Experimental Procedures. (F) In vitro RNA degradation assays using S100s from C2C12 GM cells preincubated with either recombinant MKK6EE alone or recombinant MKK6EE plus recombinant p38β for 20 min at 30°C. Molecular Cell 2005 20, 891-903DOI: (10.1016/j.molcel.2005.10.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 KSRP Binds Myogenic Transcripts and Controls Their Decay (A) Interaction between 32P labeled AREmyogenin RNA and proteins present in S100s of C2C12 cells, cultured as indicated, was evaluated by UV-crosslinking analysis (left). The UV-crosslinking reactions were subject to immunoblot analysis with anti-KSRP (middle) and anti-α-tubulin (right) antibodies. (B) Anti-KSRP (left) or preimmune serum (PI) (middle) immunoprecipitation of UV-crosslinking reactions performed as above. The immunoprecipitated material was subject to immunoblot analysis with anti-KSRP antibody to control the amount of KSRP (right). (C) Immunoblot analysis of S100s from C2C12 GM cells that were depleted with either preimmune serum (PI) or anti-KSRP, using antibodies to either KSRP or α-tubulin (left). In vitro RNA degradation assays using control (I.D. PI, lanes 1–4) or KSRP-depleted (I.D. αKSRP, lanes 5–8) C2C12 GM S100s, and C2C12 MKK6EE S100s incubated with either BSA (lanes 9–12) or recombinant KSRP (30 nM, lanes 13–16) (right). (D and E) KSRP knock down by siRNA retards rapid decay of myogenin and p21 transcripts in C2C12 GM cells. (D) C2C12 GM cells were transfected with either control or KSRP siRNA. KSRP expression levels were monitored by immunoblotting. The asterisk marks the position of crossreacting FBP1. (E) Levels of myogenic transcripts were analyzed by semiquantitative RT-PCR. Total RNA was isolated at the indicated times after addition of actinomycin D. The amount of each transcript was determined as in Figure 1. The values shown are averages (±SEM) of three independent experiments performed in duplicates. Molecular Cell 2005 20, 891-903DOI: (10.1016/j.molcel.2005.10.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Role of KSRP in Regulation of ARE-Containing Myogenic Transcripts (A) Expression of myogenic markers monitored by RT-PCR using RNA from either mock- or FLAG-KSRP-stably transfected C2C12 cells cultured in GM or DM as indicated. The sequence of the primers used is shown in Table S2. (B) In vitro RNA degradation assays using S100s from either mock- or FLAG-KSRP-transfected C2C12 cells cultured as indicated. (C) Semiquantitative RT-PCR analysis of myogenic and control transcripts in either mock- (dashed line) or FLAG-KSRP- (solid line, purple highlight) stably transfected C2C12 DM cells. Total RNA was isolated after addition of actinomycin D, and the amounts of specific transcripts were quantitated as above. The values shown are averages (±SEM) of three independent experiments performed in duplicates. (D) C2C12 cells were transfected with either empty FLAG expression vector or FLAG-KSRP. After 48 hr of incubation in DM, cells were fixed (4% paraformaldehyde) and stained with anti-FLAG monoclonal antibody (green) and anti-MHC polyclonal antibody (red), and examined by indirect immunofluorescence. DAPI counter staining (blue) was performed to visualize nuclei. Error bars represent the SEM. (E) Either mock-transfected C2C12 or C2C12-KSRP cells were transiently transfected with either Myogenin-Luc or p21-Luc reporters and cultured for 2 days either in GM, DM, or DM + SB. Cells were then collected and luciferase activity measured. Error bars represent the SEM. (F) ChIP analysis of E box-containing regions of the myogenin promoter was performed on mock-transfected C2C12 and C2C12-KSRP cells cultured in GM or DM with the indicated antibodies. Molecular Cell 2005 20, 891-903DOI: (10.1016/j.molcel.2005.10.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 p38 Phosphorylates KSRP (A) C2C12 GM S100s were incubated with either a mixture of activated recombinant p38α and p38β in kinase buffer or buffer alone in the presence of γ[32P]ATP. Reactions were immunoprecipitated with anti-KSRP antibody. Labeled proteins were analyzed by SDS-PAGE and autoradiography. (B) Highly purified recombinant KSRP was incubated with γ[32P]ATP in the absence (−) or in the presence (+) of activated p38 (α + β) (left). Increasing amounts (20–500 nM) of recombinant KSRP were incubated with 10 ng p38β and γ[32P]ATP (right). (C) Coimmunoprecipitation of endogenous KSRP and HA-p38β in HA-p38β-transfected 293T cells. Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting with anti-HA antibody. (D) Coimmunoprecipitation of endogenous KSRP and p38 in C2C12 cells cultured either in GM or DM. Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting with anti-p38 antibody. The arrow marks the position of p38, whereas the asterisk marks Ig heavy chains. (E) In vivo metabolic labeling of C2C12-KSRP cells with [32P]orthophosphate. Lysates from cells cultured as indicated were immunoprecipitated with anti-FLAG antibody, separated by SDS-PAGE, and autoradiographed. (F) Schematic representation of KSRP and GST-fused deletion mutants. Asterisks indicate the position of putative MAPK phosphorylation sites (left). p38β kinase assays using the indicated recombinant protein substrates (right). (G) Sequence comparison of KSRP from six different species. The potential p38 phosphorylation site is highlighted. Accession numbers are provided in parentheses. (H) p38β kinase assays using wt, T692A-, or P693A-mutated KSRP569-711 as substrates. (I) p38β kinase assays using the indicated recombinant proteins as substrates in the absence or in the presence of 2.5 μM SB203580. Molecular Cell 2005 20, 891-903DOI: (10.1016/j.molcel.2005.10.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 p38-Mediated Phosphorylation Impairs KSRP-RNA Interaction and KSRP Destabilizing Function (A) The interaction between 32P labeled AREmyogenin RNA and recombinant purified KSRP (30–300 nM) subject to kinase reactions in the absence or in the presence of p38β (as indicated) was evaluated by UV crosslinking (top). The amount of KSRP was monitored by immunoblotting (bottom). (B) Kinase assays using active recombinant purified MK2 (10 ng) and either purified recombinant KSRP or His-TTP as substrates (left). The interaction between 32P labeled AREmyogenin RNA and purified recombinant KSRP (30–300 nM) subject to kinase reaction in the absence or in the presence of recombinant MK2 (as indicated) was analyzed by UV crosslinking (right). (C) In vitro RNA degradation assays using S100s from C2C12 DM cells preincubated with either BSA, KSRP, or p38β-phosphorylated KSRP (30 nM each), respectively. (D) In vitro RNA degradation assays using S100s from either mock-transfected (C2C12 DM) or FLAG-KSRP-stably transfected C2C12 cells (C2C12-KSRP) cultured in DM and either mock infected (DM) or infected with pAdMKK6EE (MKK6EE DM). (E) Expression of myogenic markers monitored by RT-PCR using RNA from C2C12-KSRP cultured in GM, DM plus mock-infection, or DM plus infection with pAdMKK6EE (as indicated). (F) Semiquantitative RT-PCR analysis of myogenic and control transcripts in C2C12-KSRP DM cells either mock infected (dashed line, C2C12-KSRP DM) or infected with pAdMKK6EE (solid line, pink highlight, C2C12-KSRP DM+MKK6EE). Total RNA was isolated after addition of actinomycin D, and the amount of specific transcripts were quantitated as above. The values shown are averages (±SEM) of three independent experiments performed in duplicates. (G) KSRP associates with PARN and the exosome in both C2C12 GM and C2C12 DM cells. Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting with either anti-PARN or anti-hRrp46 (a core exosome component) antibodies. Arrows point to the position of PARN (two isoforms) and hRrp46. Molecular Cell 2005 20, 891-903DOI: (10.1016/j.molcel.2005.10.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 The KSRP(T692A) Mutant Is a Dominant-Negative Inhibitor of KSRP RNA Destabilizing Function (A) The interaction between 32P labeled AREmyogenin RNA and either wt KSRP or KSRP(T692A) (25–400 nM each) was evaluated by UV crosslinking (top panels). Immunoblot analysis with anti-KSRP antibody of the UV-crosslinking reaction is shown in the bottom panels. (B) In vitro RNA degradation assays using S100s from C2C12 DM cells preincubated with either BSA, KSRP (30 nM), or KSRP(T692A) (30 nM), respectively. (C) In vitro RNA degradation assays using S100s from either C2C12-KSRP or C2C12-KSRP(T692A) stable cell lines cultured in GM or DM. (D) Semiquantitative RT-PCR analysis of myogenic and control transcripts in C2C12-KSRP(T692A) cells cultured either in GM (dashed line) or DM (solid line and green highlight). Total RNA was isolated at the indicated times after actinomycin D addition. The values shown are averages (±SEM) of three independent experiments performed in duplicates. (E) In vitro degradation assays of nonmyogenic RNAs performed by using S100s from either C2C12-KSRP or C2C12-KSRP(T692A) cells cultured in GM. (F) KSRP associates with PARN and the exosome in both C2C12-KSRP and C2C12-KSRP(T692A) cells. Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting with either anti-PARN or anti-hRrp46 (a core exosome component) antibodies. Arrows point to the position of PARN (two isoforms) and hRrp46. Molecular Cell 2005 20, 891-903DOI: (10.1016/j.molcel.2005.10.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 A Model for the Control of Myogenic Transcripts Stability during C2C12 Differentiation Molecular Cell 2005 20, 891-903DOI: (10.1016/j.molcel.2005.10.021) Copyright © 2005 Elsevier Inc. Terms and Conditions