?(Fig.1,1, lanes 3 and 6). and that this is mediated at least in part by Hsp27. Splicing of mRNA precursors, like other steps in the gene expression pathway, is regulated by environmental stress (25). It was observed over 20 years ago that splicing is interrupted by heat shock in cells (29). The same phenomenon was subsequently reported in organisms from to humans (2, 28), suggesting that heat shock-induced repression of splicing is highly conserved. The biological significance of this phenomenon appears to be severalfold (16, 31). First, since most metazoan genes contain introns (21), splicing repression can contribute to the general SB 203580 hydrochloride inhibition of gene expression that occurs upon heat shock. Second, SB 203580 hydrochloride in many species, genes encoding heat shock proteins contain few or no introns while other protein-coding genes are intron rich (12). Therefore, heat shock-induced splicing repression results in inhibition of the expression of most genes, while expression of heat shock genes is generally not affected, thereby ensuring the preferential expression of heat shock proteins. Third, splicing Rabbit polyclonal to CREB1 repression following heat shock causes accumulation of unspliced RNAs, some of which are exported into the cytoplasm and translated into abnormal proteins (30). These aberrant products may be partially responsible for the detrimental effects of heat shock. Thermotolerance refers to the phenomenon that a mild heat treatment induces transient resistance to a second and more severe heat shock (5). Thermotolerance can be observed not only at the cell survival level but also at the molecular level. For example, following a mild heat shock, splicing can occur normally in response to a more severe heat shock that would otherwise inhibit it (2, 29). Similar to heat-induced splicing inhibition, splicing thermotolerance is also conserved from yeast to humans (31). Splicing thermotolerance requires protein synthesis, most likely of heat shock proteins (31). However, it remains unclear which specific heat shock protein(s) is involved and how it might function in establishing thermotolerance. In yeast, Hsp104 and Hsp70 contribute to reactivation of splicing after heat inactivation, but overexpression of these proteins does not lead to development of thermotolerance (27). Hsp27 has recently been reported to play a similar role in human cells (19). Thus, the mechanism of splicing thermotolerance remains unclear. Earlier work from our lab has identified SRSF10 (previously called SRp38; see reference 17) as a factor that mediates global splicing inhibition, both during M phase of the cell cycle and in response to heat shock (24, 26). SRSF10 belongs to the SR family of splicing regulators, all of which share a domain rich in arginine/serine dipeptide repeats called the RS domain as well as one or two RNP-type RNA binding domains (11, 18). The RS domains of these proteins are extensively phosphorylated. Most SR proteins function as essential but redundant general splicing activators protein synthesis of heat shock proteins. Finally we provide evidence that thermotolerance of SRSF10 is mediated at least in part by Hsp27, which interacts with SRSF10 and protects it from dephosphorylation by the heat shock-activated phosphatase PP1. MATERIALS AND METHODS Constructs and antibodies. The Hsp27 wild type and mutant mammalian expression constructs were kind gifts of Michael Welsh (University of Michigan). Hsp70 and Hsp90 expression constructs were provided by Ulrich Hartl (Max Planck Institute of Biochemistry). The PP1 expression construct was previously described (23). pSuper-based constructs (3) were used for RNA interference (RNAi) of HSF1, and the sequence CCAAGGAGGTGCTGCCCAA within the HSF1 open reading frame (ORF) was targeted. SRSF10 antibody was described previously (26). Hsp/Hsc70 monoclonal antibody was from StressGen (Michigan). Hsp27 and phospho-Hsp27 (Ser82) antibodies were from Cell Signaling (Massachusetts). HSF1 antibody was from Lab Vision (California). Cell culture and SB 203580 hydrochloride heat shock assays. HeLa.