on-retaining RNAs are anchored in the nucleus until the splicing reaction is completed because SF3b is required not only for splicing but also for nuclear retention of pre-mRNA. Clk1/4 plays a key role on rephosphorylation of SR proteins after stress Depletion of Clk1/4 pre-mRNAs and a consequent shortage of Clk1/4 mRNAs delayed the recovery of SRSF4 and SRSF10 phosphorylation reduced by heat shock. This is well consistent with the results of Clk1/4 knockdown and our previous study showing that Clk1 and Clk4 are unique kinases for hyperphosphorylation of SRSF4 among known SR protein kinases, such as Clks, SRPKs, DYRKs, and Prp4. Dephosphorylation of SR proteins, such as SRSF10, mediates heat shockinduced splicing arrest. Thus, Clk1/4-dependent rephosphorylation of SR proteins is considered to play a key role in resumption of splicing activity after stress removal. In comparison with SRSF4 and SRSF10, rephosphorylation of SRSF6 and SRSF5 just seems to partially depend on the Clk1/4 activity, suggesting that other SR kinases also serve in rephosphorylation of these SR proteins. Previous studies about SR protein kinases and their stressinduced regulation were mainly predicated on the idea that the stress-induced regulation of kinase activity is directed to phosphorylation changes of SR proteins and alteration of splice site selections under stress conditions. Here, we show that stress-induced Clk1/4 expression serves in recovery from stressed conditions, providing new insights into the role of SR protein kinases in stress response. For instance, viral infection, like heat shock, activates protein phosphatase PP1 and dephosphorylates SR proteins, resulting in interruption of viral RNA processing. This is assumed to be a protective response of host cells by inhibiting viral replication and propagation. Actually, overexpression of SRPK2 or SR PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19834673 protein facilitated viral production, and administration of TG003, a Clk1/4 inhibitor, also MedChemExpress 487-52-5 suppressed influenza virus proliferation. In contrast, for host cell reactivation at a period of latent infection, the elevation of SR protein kinase activity should be required. Here, we advocate a novel molecular mechanism named ReSCUE for the recovery of SR protein phosphorylation after stress removal. Our experimental data indicate that Clk1/4 proteins supplied through stress-induced splicing of the intronretaining pre-mRNAs are essential for rapid recovery of the phosphorylation state of SR proteins and contribute to the restart of splicing reactions after stress removal. Considering the unique induction system and enzymatic properties of ReSCUE model 
Ninomiya et al. 37 Clk1/4, they may serve as a guardian to maintain the phosphorylation state of SR proteins to expand the survival chance of cells after the stress. Materials and methods Cell culture, stress response experiment, and transfection Mammalian cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with antibiotics and 10% fetal calf serum at 37C in 5% CO2. For heat shock induction, the medium was changed to prewarmed medium and incubated at 43C in an incubator with 5% CO2. To perform time course analysis of heat shock recovery, after heat shock for 60 min, the medium was changed to a new one at 37C, and the cells were cultured at 37C. For a control experiment of heat shock in Fig. 7 C and Fig. S4 C, the medium was changed to a new one at 37C instead of a prewarmed one, cultured for 60 min at 37C, and changed again to a new medium at
