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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)...

The mammalian testis determining factor SRY and its related Sox factors are critical developmental regulators. They share significant similarity in their high mobility group (HMG) domain and display discrete patterns of tissue-specific expression. Here we show that SRY and the Sox protein SOX6 colocalize with splicing factors in the nucleus and are dynamically redistributed following the blockage of splicing in living cells. Anti-SOX6 antibodies supershift the spliceosomal complex from assembled splicing reactions and inhibit splicing in vitro of multiple pre-mRNA substrates. Most importantly, SOX6-depleted nuclear extracts have impaired splicing activity, which is efficiently restored by addition of the recombinant SOX6 HMG domain and also by recombinant SRY and the SOX9 HMG domain. These results reveal an unexpected biological function of the SRY, SOX6, and SOX9 gene products and provide a functional link to the biochemical mechanisms operating in mammalian sex determination and in other developmental processes regulated by Sox genes.T he high mobility group (HMG)-type DNA binding domain was originally described as a domain in the RNA polymerase I transcription factor hUBF homologous to two regions in the chromatin HMG protein 1 (1). Proteins containing HMG domains have been found in a large variety of species and are grouped into two families, based on evolutionary and functional conservation (reviewed in ref.2). One family comprises the chromosomal HMG domain group of proteins, which are provided with two or more HMG domains and bind DNA with low to moderate affinity and little sequence specificity. The other family includes sequence-specific transcription factors, which are provided with only one HMG domain and bind to DNA sequences in the promoter of target genes to elicit their transcriptional activation. HMG domain factors bind DNA in the minor groove, induce a relevant DNA bending on binding, and are capable of binding to special or distorted DNA structures, such as four-way junctions, cis-platinum adducts, and base bulges (reviewed in ref.3). Given these properties, HMG domain proteins are postulated to act as architectural elements, promoting formation of contacts between factors bound at distant sites on DNA or recruiting proteins that by themselves do not bind to DNA and facilitating the assembly of higher-order complexes involved in transcriptional control, gene recombination, and DNA repair.Among sequence-specific HMG-domain proteins, a distinct position is occupied by the Sox (SRY box) protein family. A common feature of these proteins is to share more than 50% similarity in their HMG domains to SRY, the testis-determining factor located on human and mouse Y chromosomes. Sox proteins have been identified in a number of vertebrates and invertebrates, including Drosophila melanogaster and Caenorhabditis elegans. Outside the HMG box, Sox proteins are poorly conserved, and many are expressed with a tissue-specific pattern. These factors play a crucial role in development, as also shown by their invol...

The DAX-1 (NR0B1) gene encodes an unusual member of the nuclear hormone receptor superfamily which acts as a transcriptional repressor. Mutations in the human DAX-1 gene cause X-linked adrenal hypoplasia congenita (AHC) associated with hypogonadotropic hypogonadism (HHG). We have studied the intracellular localization of the DAX-1 protein in human adrenal cortex and mouse Leydig tumor cells and found it to be both nuclear and cytoplasmic. A significant proportion of DAX-1 is associated with polyribosomes and is found complexed with polyadenylated RNA. DAX-1 directly binds to RNA, two domains within the protein being responsible for cooperative binding activity and specificity. Mutations in DAX-1 found in AHC-HHG patients significantly impair RNA binding. These findings reveal that DAX-1 plays multiple regulatory roles at the transcriptional and posttranscriptional levels.DAX-1 (NR0B1) (32) is an unusual member of the nuclear hormone receptor superfamily whose mutations cause the Xlinked form of adrenal hypoplasia congenita (AHC), which is constantly associated with hypogonadotropic hypogonadism (HHG). In addition, the DAX-1 gene locus is mapped inside the minimal region on the X chromosome whose duplication causes male-to-female sex reversal in persons with an intact SRY gene (dosage-sensitive sex reversal) (2,22,43,53).The human DAX-1 gene encodes a 470-amino-acid (aa) protein whose C terminus is similar to the ligand-binding domain (LBD) of nuclear hormone receptors, while its N terminus is composed of three repeats of 67 to 69 aa with no significant similarity to any other known protein (20, 53). All mutations found in AHC-HHG kindreds have in common the characteristic of altering the structure of the DAX-1 C-terminal domain. We and others have shown that DAX-1 is endowed with transcriptional repressor activity (16,20,54). This property is invariably abolished in mutated DAX-1 proteins from AHC-HHG patients (16,20). This finding suggests that the impairment of the DAX-1 transcriptional activity is directly linked to the pathogenesis of AHC-HHG.DAX-1 expression is restricted to steroidogenic tissues and to some critical sites in the reproductive axis (15,45). When introduced in steroidogenic Y-1 cells, DAX-1 blocks steroid biosynthesis by impairing the expression of the steroidogenic acute regulatory protein (StAR) and of the enzymes required to convert cholesterol into pregnenolone and progesterone (21, 54). We have shown that the block of StAR expression is dependent on the binding of DAX-1 to a DNA hairpin site in the StAR promoter, which allows the recruitment to the promoter of the powerful transcriptional repression activity present in the DAX-1 C terminus (54). Using a transgenic mouse model, dax-1 overexpression has also been shown to produce sex reversal in male animals harboring a weak sry allele (42). This phenotype is likely caused by inappropriate repression of testosterone biosynthesis in Leydig cells in the developing male gonad by the overexpressed dax-1 and by a direct repressive effect on the...

CHRNA1 gene, encoding the muscle nicotinic acetylcholine receptor alpha subunit, harbors an inframe exon P3A. Inclusion of exon P3A disables assembly of the acetylcholine receptor subunits. A single nucleotide mutation in exon P3A identified in congenital myasthenic syndrome causes exclusive inclusion of exon P3A. The mutation gains a de novo binding affinity for a splicing enhancing RNA-binding protein, hnRNP LL, and displaces binding of a splicing suppressing RNA-binding protein, hnRNP L. The hnRNP L binds to another splicing repressor PTB through the proline-rich region and promotes PTB binding to the polypyrimidine tract upstream of exon P3A, whereas hnRNP LL lacking the proline-rich region cannot bind to PTB. Interaction of hnRNP L with PTB inhibits association of U2AF65 and U1 snRNP with the upstream and downstream of P3A, respectively, which causes a defect in exon P3A definition. HnRNP L and hnRNP LL thus antagonistically modulate PTB-mediated splicing suppression of exon P3A.

Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.

Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease. Various attempts are underway to convert severe DMD to a milder phenotype by modulating the splicing of the dystrophin gene and restoring its expression. In our previous study, we reported TG003, an inhibitor of CDC2-like kinase 1 (CLK1), as a splice-modifying compound for exon-skipping therapy; however, its metabolically unstable feature hinders clinical application. Here, we show an orally available inhibitor of CLK1, named TG693, which promoted the skipping of the endogenous mutated exon 31 in DMD patient-derived cells and increased the production of the functional exon 31-skipped dystrophin protein. Oral administration of TG693 to mice inhibited the phosphorylation of serine/arginine-rich proteins, which are the substrates of CLK1, and modulated pre-mRNA splicing in the skeletal muscle. Thus, TG693 is a splicing modulator for the mutated exon 31 of the dystrophin gene in vivo, possibly possessing therapeutic potential for DMD patients.

Overexpression of high-mobility group A protein 1a (HMGA1a) causes aberrant exon 5 skipping of the Presenilin-2 (PS2) pre-mRNA, which is almost exclusively detected in patients with sporadic Alzheimer's disease. An electrophoretic mobility shift assay confirmed aberrant U1 small nuclear ribonucleoprotein particle (snRNP)-HMGA1a complex formation (via the U1-70K component), with RNA containing a specific HMGA1a-binding site and an adjacent 5 splice site. Psoralen cross-linking analysis demonstrated that the binding of HMGA1a adjacent to the 5 splice site induces unusually extended association of U1 snRNP to the 5 splice site. As a result, spliceosome assembly across either the intron or the exon is arrested at an early ATP-independent stage. We conclude that the HMGA1a-induced aberrant exon skipping is caused by impaired dissociation of U1 snRNP from the 5 splice site, leading to a defect in exon definition. The proposed molecular mechanism has profound implications for other known posttranscriptional modulation strategies in various organisms, all of which are triggered by aberrant U1 snRNP binding.Alternative splicing is an essential process applied to generate tremendous diversity in protein products from a limited number of genes (reviewed in references 1 and 39). Misregulation of this process is also a major source of splicing defects in disease-associated genes (reviewed in reference 23). Mutations in essential cis-acting elements and disorders of transacting factors can cause such splicing defects (reviewed in reference 22). The former is evident in many genetic diseases that are commonly caused by point mutations in splicing signals, e.g., conserved splice site sequences and splicing enhancer elements (reviewed in references 4, 14, and 22). However, the mechanisms via which trans-acting factors cause changes in the splicing pattern of disease-associated genes, often with severe clinical consequences, are poorly understood.The presenilin-2 (PS2) gene is one of the known Alzheimer's disease (AD)-associated genes (reviewed in reference 33). The specific aberrant splicing, skipping exon 5, generates the truncated deleterious protein PS2V, which accumulates as visible PS2V bodies at high frequency in the hippocampus of sporadic AD patients (29,30). We established an experimental system in which this particular exon 5 skipping event can be induced by hypoxia in cultured human neuroblastoma SK-N-SH cells (29,30). Using this cell line system, we purified and identified high-mobility group A protein 1a (HMGA1a) as a sequence-specific RNA-binding factor that is responsible for specific exon 5 skipping in the PS2 premRNA (18). We found that overexpressed HMGA1a bound to the specific target sequence, GCU(G)CUACAAG, adjacent to the 5Ј splice site of the PS2 pre-mRNA (18,19). This was a surprising discovery, since HMGA1a/b (formerly known as HMG-I/Y; http://www.informatics.jax.org/mgihome /nomen/hmg_family.shtml) proteins had been classified previously as nonhistone DNA-binding proteins (reviewed in reference 26). The ...