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...

Dyrk1A (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A) is a serine/ threonine kinase essential for brain development and function, and its excessive activity is considered a pathogenic factor in Down syndrome. The development of potent, selective inhibitors of Dyrk1A would help to elucidate the molecular mechanisms of normal and diseased brains, and may provide a new lead compound for molecular-targeted drug discovery. Here, we report a novel Dyrk1A inhibitor, InDY, a benzothiazole derivative showing a potent ATPcompetitive inhibitory effect with IC 50 and K i values of 0.24 and 0.18 µm, respectively. X-ray crystallography of the Dyrk1A/InDY complex revealed the binding of InDY in the ATP pocket of the enzyme. InDY effectively reversed the aberrant tau-phosphorylation and rescued the repressed nFAT (nuclear factor of activated T cell) signalling induced by Dyrk1A overexpression. Importantly, proInDY, a prodrug of InDY, effectively recovered Xenopus embryos from head malformation induced by Dyrk1A overexpression, resulting in normally developed embryos and demonstrating the utility of proInDY in vivo.

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...

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by missplicing of exon 20, resulting from an intronic mutation in the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene encoding IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1). A newly established splicing reporter assay allowed us to visualize pathogenic splicing in cells and to screen small chemicals for the ability to correct the aberrant splicing of IKBKAP. Using this splicing reporter, we screened our chemical libraries and identified a compound, rectifier of aberrant splicing (RECTAS), that rectifies the aberrant IKBKAP splicing in cells from patients with FD. Here, we found that the levels of modified uridine at the wobble position in cytoplasmic tRNAs are reduced in cells from patients with FD and that treatment with RECTAS increases the expression of IKAP and recovers the tRNA modifications. These findings suggest that the missplicing of IKBKAP results in reduced tRNA modifications in patients with FD and that RECTAS is a promising therapeutic drug candidate for FD. IKAP is currently known as elongator protein 1 (ELP1), an integral component of the human Elongator complex, which was originally identified in Saccharomyces cerevisiae and shown to be well conserved among species (1). Although multiple functions of IKAP/ELP1 in JNK signaling, neuronal development during embryogenesis, exocytosis, and actin cytoskeleton regulation have been reported (reviewed in refs. 2, 3), yeast genetic analyses have shown that the Elongator complex is also required for the formation of the C5-substituent of 5-carbamoylmethyl (ncm 5 ), 5-methoxycarbonylmethyl (mcm 5 ), and its derivatives at the wobble uridine in tRNAs recognizing purine-ending codons (4, 5). Most recently, it was demonstrated that conditional IKAP/Elp1 KO in mouse testes results in male infertility by disrupting meiotic progression, along with the reduction of modified nucleosides [5-methoxycarbonylmethyl uridine (mcm 5 U), 5-carbamoylmethyl uridine (ncm 5 U), and 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U)] of total tRNAs in the testes (6). These modifications are highly likely to play critical roles in the maintenance of translational fidelity, suggesting that the defects in these modifications lead to the mistranslation of various proteins.Familial dysautonomia (FD; Riley-Day syndrome), an autosomal recessive neurodegenerative disease, is characterized by impaired development and progressive degeneration of the sensory and autonomic nerves. Patients who have FD exhibit various symptoms, including cardiovascular instability, recurrent pneumonia, vomiting/dysautonomic crisis, gastrointestinal dysfunction, decreased sensitivity to pain and temperature, and defective lacrimation. FD is a very common disorder in the Ashkenazi Jewish population, with a carrier frequency of 1 in 27. More than 99% of patients who have FD harbor a homozygous mutation in intron 20 (IVS20 + 6T > C: FD mutation)...

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Participation of multiple kinases in regulation of the binding of lamin B receptor (LBR) to chromatin was suggested previously (Takano, M., Takeuchi, M., Ito, H., Furukawa, K., Sugimoto, K., Omata, S., and Horigome, T. (2002) Eur. J. Biochem. 269, 943-953). To identify these kinases, regulation of the binding of the nucleoplasmic region (NK, amino acid residues 1-211) of LBR to sperm chromatin was studied using a cell cycle-dependent Xenopus egg extract in vitro. The binding was stimulated on specific phosphorylation of the NK fragment by an S-phase egg extract. Protein depletion with beads bearing SF2/ASF, which binds SR protein kinases, abolished this stimulation, suggesting that an SR protein kinase(s) is responsible for the activation of LBR. This was confirmed by direct phosphorylation and activation with recombinant SR protein-specific kinase 1. The binding of the NK fragment to chromatin pretreated with an S-phase extract was suppressed by incubation with an M-phase extract. Enzyme inhibitor experiments revealed that multiple kinases participate in the suppression. One of these kinases was shown to be cdc2 kinase using a specific inhibitor, roscovitine, and protein depletion with beads bearing p13, which specifically binds cdc2 kinase. Experiments involving a mutant NK fragment showed that the phosphorylation of serine 71 by cdc2 kinase is responsible for the suppression.The nuclear envelope separates the nucleoplasm from the cytoplasm and thereby organizes the nuclear architecture. The nuclear envelope consists of two lipid bilayers (inner and outer), nuclear pore complexes, and the nuclear lamina and undergoes repeated dynamic assembly and disassembly in every cell cycle in higher eukaryotes. Studies on the molecular mechanisms underlying nuclear envelope assembly and disassembly are important to understand the mechanisms underlying cell division and prenucleus formation. Studies on the organization of the nuclear architecture by the nuclear envelope during the cell cycle are also important to understand how mutants of nuclear envelope proteins, i.e. lamin A/C (1), emerin (2), and lamin B receptor (LBR) 1 (3), cause genetic diseases (4). The assembly and disassembly of the nuclear envelope in the cell cycle accompany association and dissociation of the inner nuclear membrane with and from chromatin, respectively. All major inner nuclear membrane proteins, such as the laminaassociated polypeptide 2 (LAP2) family, LBR, and emerin, are known to bind to chromatin in vitro (5, 6) and to accumulate on the surface of chromatin during the anaphase-telophase transition in vivo (7). Therefore, the binding of these proteins to chromatin is thought to support the binding of the inner nuclear membrane to chromatin. Indeed, LBR and LAP2 have been demonstrated to play functional roles in the interaction of the nuclear envelope and chromatin through in vitro experiments as follows. Depletion of LBR from nuclear envelope precursor vesicles suppresses nuclear assembly (8), neutralization of LBR by antibodies in a sea urc...

Prp4 is a protein kinase of Schizosaccharomyces pombe identified through its role in pre-mRNA splicing, and belongs to a kinase family including mammalian serine/arginine-rich protein-specific kinases and Clks, whose substrates are serine/arginine-rich proteins. We cloned human PRP4 (hPRP4) full-length cDNA and the antiserum raised against a partial peptide of hPRP4 recognized 170-kDa polypeptide in HeLa S3 cell extracts. Northern blot analysis revealed that hPRP4 mRNA was ubiquitously expressed in multiple tissues. The extended NH 2 -terminal region of hPRP4 contains an arginine/serine-rich domain and putative nuclear localization signals. hPRP4 phosphorylated and interacted with SF2/ASF, one of the essential splicing factors. Indirect immunofluorescence analysis revealed that endogenous hPRP4 was distributed in a nuclear speckled pattern and colocalized with SF2/ASF in HeLa S3 cells. Furthermore, hPRP4 interacted directly with Clk1 on its COOH terminus, and the arginine/serine-rich domain of hPRP4 was phosphorylated by Clk1 in vitro. Overexpression of Clk1 caused redistribution of hPRP4, from the speckled to the diffuse pattern in nucleoplasm, whereas inactive mutant of Clk1 caused no change of hPRP4 localization. These findings suggest that the NH 2 -terminal region of hPRP4 may play regulatory roles under an unidentified signal transduction pathway through Clk1.