Notably, RNaseI treatment had not been sufficient to fully digest RNAs cross-linked to Rbm7 even at high concentrations and resulted in a smear of various RNA-Rbm7 cross-linking products (Supplemental Fig. 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 p38MAPK/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. 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. To further validate this phosphorylation, mouse wild-type (WT) GFP-tagged Rbm7 was ectopically expressed in HeLa cells and enriched by a GFP-Pulldown (Rothbauer et al. 2008). Precipitated proteins were probed with either anti-p-PKD-substrate-motif (LXRXXpS/T) antibody or an anti-pS136-Rbm7 antibody, both of which gave rise to a phospho-specific signal when HeLa cells were stimulated with anisomycin (Fig. 1B). This indicated the involvement of p38MAPK in the phosphorylation event, as anisomycin is known to be a strong activator of this pathway (Hazzalin et al. 1996). Importantly, inhibition of p38 using BIRB796 (Kuma et al. 2005) led to a complete loss of the phospho-signal, further indicating a role of the p38MAPK/MK2 signaling module in the Rbm7 phosphorylation (Fig. 1B). This result was confirmed for human FLAG-tagged RBM7 in HeLa cells (Supplemental Fig. S1A). The putatively nonphosphorylatable mutant of murine Rbm7, Rbm7S136A, was generated and similar pulldown experiments were conducted and showed no phosphorylation of Rbm7S136A in response to anisomycin (Fig. 1B). Finally, the direct phosphorylation of Rbm7 by MK2 was analyzed by an in vitro phosphorylation assay (Fig. 1C). Recombinant MK2 and/or p38 were mixed with GFP-Rbm7 or GFP-Rbm7S136A and incubated at 30C in the presence of ATP. Subsequently, samples were resolved by SDS-PAGE and analyzed by Western blotting using the pS136-Rbm7-specific antibody. Only the p38-activated MK2 was able to phosphorylate WT Rbm7 in vitro and gave rise to a robust phosphorylation-specific signal, whereas Rbm7S136A phosphorylation was unaffected by the same treatment (Fig. 1C). Alterations in the appearance of nonphosphorylated GFP-fusion proteins were due to the fact that they migrated at the same position as recombinant MK2 (Fig. 1C, lower panel). The phosphorylation result was confirmed by a radioactive kinase assay using [-33P]ATP and showing that only activated MK2, and not p38 alone, was able to phosphorylate Rbm7 (Supplemental Fig. S1B). Notably, mutant Rbm7S136A was still phosphorylated by active MK2 to a certain degree (see Discussion). Taken together, our results indicate that RBM7S136 is a major MK2-phosphorylation site that is conserved in most vertebrates (Fig. 1A). Next, we measured the kinetics of anisomycin-induced phosphorylation of murine Rbm7 in HeLa cells. Rbm7 phosphorylation at S136 as well as activity of p38 and MK2 (as measured by the activating phosphorylations of pT180/pY182 and pT334, respectively) could be clearly detected after 1 h of anisomycin treatment and were sustained during the full 4 h observation time (Fig. 1D). Notably, only GFP-tagged Rbm7 protein was detected by the anti-pS136-Rbm7 antibody. Simultaneously, the subcellular localizations of the overexpressed GFP-Rbm7 and GFP-Rbm7S136A were monitored (Supplemental Fig. S2A). As reported for human RBM7 (Lubas et al. 2011; Hett and West 2014), these proteins were exclusively localized to the nuclear compartment and did not display notable changes in localization upon anisomycin-treatment. Established bona fide substrates of MK2, such as Hsp27 (Stokoe et al. 1992) and NOGO-B LIFR (Rousseau et al. 2005), displayed similar kinetics of phosphorylation (Fig. 1D). We also detected endogenous Acetylleucine NEXT complex proteins RBM7 and ZCCHC8 (Fig. 1D). Anisomycin stimulation resulted in a reduction of endogenous RBM7, which correlated well with the MK2-mediated phosphorylation of murine Rbm7 and could be used to monitor the in vivo phosphorylation of endogenous RBM7 (Fig. 1DCF). The disappearance of endogenous RBM7 signal was similar to the weaker signal of total MK2 upon anisomycin stimulation that is due Acetylleucine to weaker detection of the phospho-protein by the antibody (see MK2 panel in Fig. 1D and Tiedje et al. 2012). The effect on RBM7 detection was reversed by pretreatment with the p38MAPK-inhibitor BIRB796 (Fig. 1E) and could also indicate phosphorylation or protein stabilization. To exclude a role for stress-induced proteasomal degradation of RBM7, anisomycin stimulation was combined with proteasome inhibitor treatment. In the presence of the proteasome inhibitor MG-132, stress stimulation also altered the RBM7 signals (Supplemental Fig. S2B), arguing against the involvement of proteasomal degradation of RBM7 and substantiating the potential monitoring of endogenous RBM7 phosphorylation by immunoblotting signals. To confirm the in vivo phosphorylation of endogenous RBM7, lysates of HeLa cells were treated with calf intestine phosphatase (CIP) before analysis (Fig. 1F). Indeed, Acetylleucine the endogenous RBM7 signal was turned into a distinct intense band upon this.
Notably, RNaseI treatment had not been sufficient to fully digest RNAs cross-linked to Rbm7 even at high concentrations and resulted in a smear of various RNA-Rbm7 cross-linking products (Supplemental Fig