The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes. The importance of alternative splicing is further illustrated by the increasing number of human diseases that have been attributed to mis-splicing events. Appropriate spatial and temporal generation of splicing variants demands that alternative splicing be subjected to extensive regulation, similar to transcriptional control. The Clk (Cdc2-like kinase) family has been implicated in splicing control and consists of at least four members. Through extensive screening of a chemical library, we found that a benzothiazole compound, TG003, had a potent inhibitory effect on the activity of Clk1/Sty. TG003 inhibited SF2/ASFdependent splicing of ␤-globin pre-mRNA in vitro by suppression of Clk-mediated phosphorylation. This drug also suppressed serine/arginine-rich protein phosphorylation, dissociation of nuclear speckles, and Clk1/ Sty-dependent alternative splicing in mammalian cells. Consistently, administration of TG003 rescued the embryonic defects induced by excessive Clk activity in Xenopus. Thus, TG003, a novel inhibitor of Clk family will be a valuable tool to dissect the regulatory mechanisms involving serine/arginine-rich protein phosphorylation signaling pathways in vivo, and may be applicable for the therapeutic manipulation of abnormal splicing.Recent whole genome sequence analyses revealed that a high degree of proteomic complexity is achieved with a limited number of genes. This surprising finding underscores the importance of alternative splicing, through which a single gene can generate multiple structurally and functionally distinct protein isoforms (1). Based on genome-wide analysis, 35-60% of human genes are thought to encode at least two alternatively spliced isoforms (2). The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing essential roles in many biological processes, such as embryonic development, cell growth, and apoptosis. Splicing mutations located in either intronic or exonic regions frequently cause hereditary diseases (reviewed in Refs. 3-5). More than 15% of mutations that cause genetic disease affect pre-mRNA splicing (6). Pre-mRNA splicing is also regulated in a tissue-specific or developmental stagespecific manner. Indeed, the selection of splice site can be altered by numerous extracellular stimuli, including growth factors, cytokines, hormones, depolarization, osmotic shock, and UVC irradiation through synthesis, phosphorylation, and a change in localization of serine/arginine-rich (SR) 1 proteins (7). SR proteins are a family of essential factors required for constitutive splicing of pre-mRNA (8) and play an important role in modulating alternative splicing (9). They are highly conserved in eukaryotes and are characterized by having one or two RNA-recognition motifs at the amino terminus a...

Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine-serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co-purified from HeLa cells with a 95-kDa protein, which was recognized by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser) 8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.Pre-mRNA splicing is an essential process required for the expression of most eukaryotic protein-coding genes. Splicing catalysis occurs in a spliceosome complex (1). Components of the spliceosome include the U1, U2, and U4/U6.U5 small nuclear ribonucleoprotein particles (snRNPs) 1 (2) and numerous non-snRNP protein factors (3). The latter include all members of the SR protein family, which play important roles during mammalian spliceosome assembly. All SR proteins have one or two N-terminal RNA-recognition motifs (RRMs) and a C-terminal domain rich in arginine-serine dipeptide repeats (RS domain). The RS domain is involved in protein-protein interactions with related domains of other splicing factors, and these interactions are thought to be important for splice site selection (4, 5).The SR proteins are phosphorylated at multiple serines located predominantly within the RS domain (6, 7). At least eight members of the SR family contain phosphopeptides that are recognized by the monoclonal antibody mAb104 (8). Analysis of tryptic phosphopeptides derived from SF2/ASF showed that the RS domain of this protein is phosphorylated at multiple sites both in vivo and in vitro (7). Although the physiological role of SR protein phosphorylation is unknown, recent studies suggested that phosphorylation of the RS domain of SF2/ASF enhances the interactions between this domain and the U1-70K polypeptide and that phosphorylation or dephosphorylation cycles may be required for splicing (9, 10).Several protein kinases can phosphorylate SR proteins, such as SF2/ASF, within its RS domain in vitro. A U1 sn...

Dendritic epidermal T cells (DETCs) found in mouse skin are NKG2D-positive γδ T cells involved in immune surveillance and wound repair. It is assumed that the interaction of an NKG2D receptor on DETCs and an MHC class I-like NKG2D ligand on keratinocytes activates DETCs, which then secrete cytokines promoting wound repair. However, direct evidence that DETC activation through NKG2D signaling promotes wound repair is not available. In the present study, we generated mAbs for an NKG2D ligand H60c previously suggested to be expressed specifically on skin keratinocytes. Local administration of H60c-specific mAb inhibited activation of DETCs and significantly delayed wound repair. Likewise, administration of NKG2D-specific mAb impaired wound repair to a similar extent. The delay in wound closure resulting from the blockade of the NKG2D pathway was comparable to that observed in γδ T cell-deficient mice. These results indicate that H60c/NKG2D interactions play a critical role in wound repair. Reassessment of binding affinities showed that H60c monomers bind to NKG2D with affinity (Kd = 26 ± 3.2 nM) comparable to those of other high-affinity NKG2D ligands. H60c is transcribed not only in skin but also in tissues such as tongue and female reproductive tract known to contain epithelium-resident γδ T cells expressing invariant TCRs, suggesting a more general role for H60c in the maintenance of epithelial integrity.

We have investigated physical distances and directions of transposition of the maize transposable element Ac in Arabidopsis thaliana. We prepared a transferred DNA (T-DNA) construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. Three transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. These transgenic plants were crossed with the Arabidopsis that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with endonuclease I-SceI, sizes of segment of DNA were determined by pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results showed that 50% of all transposition events had occurred within 1,700 kb on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac-Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites.

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Initial oxidation processes on Si(001) have been studied by means of surface differential reflectance (SDR). The time courses of the SDR spectra measured during thermal oxidation at 820 and 920 K allowed two different growth modes, Langmuir-type adsorption and two-dimensional island growth, to be distinguished. No photon energy dependence was observed in the time course of the SDR intensity at either temperature. On the other hand, different uptake curves were observed at different photon energies for oxidation at 300 K. The difference between the oxidation mechanisms at 300 K and at high temperatures was qualitatively apparent from SDR results, because significant photon energy dependence was observed only at 300 K. Possible assignments of the spectral components in the SDR spectra are discussed.

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