Spinal muscular atrophy results from the loss of functional survival motor neuron (SMN1) alleles. Two nearly identical copies of SMN exist and differ only by a single non-polymorphic C to T transition in exon 7. This transition leads to alteration of exon 7 splicing; that is, SMN1 produces a full-length transcript, whereas SMN2 expresses a low level of full-length transcript and predominantly an isoform lacking exon 7. The truncated transcript of SMN encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. In this paper, we identified a cis-acting element (element 1), which is composed of 45 bp in intron 6 responsible for the regulation of SMN exon 7 splicing. Mutations in element 1 or treatment with antisense oligonucleotides directed toward element 1 caused an increase in exon 7 inclusion. An ϳ33-kDa protein was demonstrated to associate with a pre-mRNA sequence containing both element 1 and the C to T transition in SMN exon 7 but not with the sequence containing mutated element 1, suggesting that the binding of the ϳ33-kDa protein plays crucial roles in the skipping of SMN exon 7 containing the C to T transition. Spinal muscular atrophy (SMA)1 is a common autosomal recessive disorder with progressive paralysis caused by the degeneration of motor neurons in the spinal cord (1). The survival of the motor neurons (SMN) gene has been identified as the disease gene of SMA and is present on chromosome 5 at 5q13 (2, 3). Humans contain two nearly identical copies of the SMN gene, SMN1 and SMN2. These genes encode an identical protein, a 294-amino acid RNA-binding protein. Only homozygous deletions or mutations of SMN1 result in the SMA phenotype, and the levels of SMN expression driven by SMN2 in motor neurons inversely correlate with the severity of the disease (4 -15).SMN1 mRNA expresses a full-length transcript, whereas SMN2 produces a low level of full-length transcript and predominantly an isoform lacking exon 7 (SMN⌬7) (2, 16, 17). The SMN⌬7 is less stable (18), and it was reported that SMN⌬7 cannot oligomerize or self-associate as efficiently as the protein produced from the full-length SMN transcript (2,19,20). Therefore, a deficiency in the full-length SMN protein correlates with the disease. The critical difference between SMN1 and SMN2 is a silent nucleotide transition in SMN exon 7. SMN1 contains a C located six nucleotides inside exon 7, whereas SMN2 contains a T at this position. This transition is considered to inhibit one of the splicing regulatory elements within exon 7, which are called exonic splicing enhancers (ESE) (21). A recent report demonstrated the presence of an ESE within exon 7 and that human Tra2-␤1, a member of the serine-arginine-related proteins of splicing factors, binds to the elements and stimulates an ESE (22). However, the critical C to T transition is not contained within the element. Furthermore, the transition does not change the binding activity of Tra2-␤1 to the ESE. Thus, it is still unclear why the C to T trans...

Spinal muscular atrophy is caused by the homozygous loss of survival motor neuron 1 (SMN1). SMN2, a nearly identical copy gene, differs from SMN1 only by a single nonpolymorphic C to T transition in exon 7, which leads to alteration of exon 7 splicing; SMN2 leads to exon 7 skipping and expression of a nonfunctional gene product and fails to compensate for the loss of SMN1. The exclusion of SMN exon 7 is critical for the onset of this disease. Regulation of SMN exon 7 splicing was determined by analyzing the roles of the cis-acting element in intron 7 (element 2), which we previously identified as a splicing enhancer element of SMN exon 7 containing the C to T transition. The minimum sequence essential for activation of the splicing was determined to be 24 nucleotides, and RNA structural analyses showed a stemloop structure. Deletion of this element or disruption of the stem-loop structure resulted in a decrease in exon 7 inclusion. A gel shift assay using element 2 revealed formation of RNA-protein complexes, suggesting that the binding of the trans-acting proteins to element 2 plays a crucial role in the splicing of SMN exon 7 containing the C to T transition. Spinal muscular atrophy (SMA)1 is a common autosomal recessive disorder characterized by the loss of motor neurons in the spinal cord, which presents as proximal, symmetrical limb, and trunk muscle weakness that ultimately leads to death (1). The survival of the motor neuron (SMN) gene has been identified as the disease-causing gene of SMA and is present on chromosome 5 at 5q13 (2, 3). Humans contain two nearly identical copies of the SMN gene, SMN1 and SMN2. These genes encode an identical protein, a 294-amino acid RNA-binding protein. Only homozygous deletions or mutations of SMN1 result in the SMA phenotype (4 -15).SMN1 mRNA expresses a full-length transcript, whereas SMN2 produces low levels of the full-length transcript and high levels of an isoform lacking exon 7 (SMN⌬7) (2,16,17). The SMN⌬7 protein is presumed to be less stable (18) and has a reduced ability to oligomerize, explaining why SMN2 cannot prevent SMA (2,19,20). The critical difference between SMN1 and SMN2 is a silent nucleotide transition in SMN exon 7. SMN1 contains a C located six nucleotides inside exon 7, whereas SMN2 contains a T at this position. This transition is believed to inhibit one of the splicing regulatory elements, called exonic splicing enhancers (ESE), within exon 7 (21). A previous report demonstrated the presence of an ESE within exon 7 and that human Tra2-␤1, a member of the serinearginine-related proteins of splicing factors, binds to the elements and stimulates an ESE (22). Recently, it was discovered that a single nucleotide change occurs within a heptamer motif of the ESE, which in SMN1 is recognized directly by SF2/ASF (23). The abrogation of the SF2/ASF-dependent ESE is considered to be the basis for the inefficient inclusion of exon 7 in SMN2. However, it is unclear whether Tra2-␤1 and SF2/ASF functionally cooperate to promote the inclusion of the exon and w...

IntroductionHsp90 is an abundant, evolutionarily conserved molecular chaperone whose function depends on its ability to bind and hydrolyze ATP. Through an ATPase cycle, Hsp90 facilitates proper folding of "client" proteins, thereby regulating their stability, protein interactions, intracellular trafficking, and functions. 1,2 To fulfill these functions, Hsp90 interacts with its cofactors and cochaperones including Hsp70, immunophilins, and p23, to form the Hsp90-based chaperone complex. 1,2 Natural compounds such as geldanamycin and radicicol bind the ATP-binding pocket of Hsp90 and disrupt its chaperone function. 3,4 Hsp90 is required for function and stability of diverse signal transduction proteins including oncogenic proteins such as ErbB2 and Raf-1. [2][3][4][5][6][7] Hence, the chaperone is an attractive target for cancer therapeutics. Indeed, Hsp90 inhibitors show antitumor activities in preclinical models, and geldanamycin analogues such as 17-allylamino-17-demethoxygeldanamycin (17-AAG) are currently undergoing clinical trials. 3,4 Importantly, Hsp90 inhibitors sensitize tumor cells to various genotoxic agents used for standard cancer therapeutics, including DNA crosslinkers, 8,9 ionizing radiation, 10 and replication inhibitors. 11,12 In line with these observations, it is suggested that Hsp90 regulates cell cycle checkpoints and DNA repair, 11-13 but the underlying mechanisms are poorly understood.Fanconi anemia (FA) is a genetically heterogeneous inherited disorder characterized by progressive bone marrow failure, cancer susceptibility, and cellular hypersensitivity to DNA cross-linkers such as mitomycin C (MMC). [14][15][16] Multiple FA proteins cooperate in a common biochemical pathway, termed the FA pathway, which is involved in cellular response to DNA damage. At least 8 FA proteins, specifically, FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL/PHF9, and FANCM, form a nuclear multiprotein complex (FA core complex), which is required for FANCD2 monoubiquitination in response to DNA damage. 17-32 DNA crosslinkers and replication inhibitors such as hydroxyurea (HU) are potent inducers of FANCD2 monoubiquitination. [14][15][16] The monoubiquitinated form of FANCD2 is targeted to the chromatin and participates in maintenance of genomic stability interacting with BRCA1 and BRCA2/FANCD1, at least in part, through homologydirected repair. 25,[33][34][35] FANCJ/BRIP1, previously identified as a BRCA1-interacting helicase, may function downstream of FANCD2 activation or independently of the FA pathway. [36][37][38] Previous studies suggested that nuclear levels of FANCA have profound effects on FA core complex formation. [17][18][19][20]24,25,27 Nuclear levels of FANCA are determined by protein synthesis, degradation, and nucleocytoplasmic shuttling mediated by a bipartite nuclear localization signal (NLS) and 3 leucine-rich nuclear export signals. 18,39,40 However, little is known about the regulatory mechanisms for intracellular turnover and trafficking of FANCA.In an attempt to elucidate the molecular m...

H19 is a tumor-suppressor gene, and changes in the methylation of the H19-differential methylation region (H19-DMR) are related to human health. However, little is known about the factors that regulate the methylation levels of H19-DMR. Several recent studies have shown that maternal environmental factors during pregnancy, such as smoking, drinking, chemical exposure, and nutrient intake, can alter the methylation levels of several genes in fetal tissues. In this study, we examined the effects of maternal factors on changes in the methylation levels of H19-DMR in the human umbilical cord (UC), an extra-embryonic tissue. Participants from the Chiba study of Mother and Children's Health (C-MACH) were enrolled in this study. Genomic DNA was extracted from UC samples, and the methylation level of H19-DMR was evaluated by methylation-sensitive high resolution melting analysis. Individual maternal and paternal factors and clinical information for newborns at birth were examined using questionnaires prepared in the C-MACH study, a brief-type self-administered diet history questionnaire (BDHQ) during early pregnancy (gestational age of 12 weeks), and medical records. Univariate and multivariate logistic regression analyses indicated that reduced H19-DMR methylation (<50% methylation) in UC tissues was positively related to decreased head circumference in newborns [odds ratio (OR) =2.82; 95% confidence intervals (CI): 1.21-6.87; p=0.0183 and OR =2.51; 95% CI: 1.02-6.46; p=0.0499, respectively]. Moreover, multiple comparison test showed that H19-DMR methylation in UC tissues was significantly reduced in the low calorie group (intake of less than 1,000kcal/day; methylation level: 40.98%; 95% CI: 33.86-48.11) compared with that in the middle (1,000-1,999kcal/day; methylation level: 51.28%; 95% CI: 48.28-54.27) and high (≥2,000kcal/day; methylation level: 52.16%; 95% CI: 44.81-59.51) calorie groups (p=0.0054 and 0.047, respectively). In the subpopulations with low to moderate calorie intake (<2,000kcal/day), reduced H19-DMR methylation in UC tissues was significantly related to serum homocysteine concentration (OR =0.520; 95% CI: 0.285-0.875; p=0.019), maternal age (OR =1.22; 95% CI: 1.01-1.52; p=0.049), and serum folate levels (OR =0.917; 95% CI: 0.838-0.990; p=0.040). These data indicated that H19-DMR methylation levels in human UC tissues could be modulated by maternal factors during early pregnancy and may affect fetal and newborn growth.