NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.     

Novel c.G630A TCIRG1 mutation causes aberrant splicing resulting in an unusually mild form of autosomal recessive osteopetrosis

Autosomal recessive osteopetrosis (ARO) is a severe genetic bone disease characterized by high bone density due to mutations that affect formation or function of osteoclasts. Mutations in the a3 subunit of the vacuolar-type H⁺-ATPase (encoded by T-cell immune regulator 1 [TCIRG1]) are responsible for ~50% of all ARO cases. We identified a novel TCIRG1 (c.G630A) mutation responsible for an unusually mild form of the disease. To characterize this mutation, osteoclasts were differentiated using peripheral blood monocytes from the patient (c.G630A/c.G630A), male sibling (+/+), unaffected female sibling (+/c.G630A), and unaffected parent (+/c.G630A). Osteoclast formation, bone-resorbing function, TCIRG1 protein, and mRNA expression levels were assessed. The c.G630A mutation did not affect osteoclast differentiation; however, bone-resorbing function was decreased. Both TCIRG1 protein and full-length TCIRG1 mRNA expression levels were also diminished in the affected patient's sample. The c.G630A mutation replaces the last nucleotide of exon 6 and may cause splicing defects. We analyzed the TCIRG1 splicing pattern between exons 4 to 8 and detected deletions of exons 5, 6, 7, and 5-6 (ΔE56). These deletions were only observed in c.G630A/c.G630A and +/c.G630A samples, but not in +/+ controls. Among these deletions, only ΔE56 maintained the reading frame and was predicted to generate an 85 kDa protein. Exons 5-6 encode an uncharacterized portion of the cytoplasmic N-terminal domain of a3, a domain not involved in proton translocation. To investigate the effect of ΔE56 on V-ATPase function, we transformed yeast with plasmids carrying full-length or truncated Vph1p, the yeast ortholog of a3. Both proteins were expressed; however, ΔE56-Vph1p transformed yeast failed to grow on Zn²⁺-containing plates, a growth assay dependent on V-ATPase-mediated vacuolar acidification. In conclusion, our results show that the ΔE56 truncated protein is not functional, suggesting that the mild ARO phenotype observed in the patient is likely due to the residual full-length protein expression.     

c.G2114A MYH9 mutation (DFNA17) causes non-syndromic autosomal dominant hearing loss in a Brazilian family

We studied a family presenting 10 individuals affected by autosomal dominant deafness in all frequencies and three individuals affected by high frequency hearing loss. Genomic scanning using the 50k Affymetrix microarray technology yielded a Lod Score of 2.1 in chromosome 14 and a Lod Score of 1.9 in chromosome 22. Mapping refinement using microsatellites placed the chromosome 14 candidate region between markers D14S288 and D14S276 (8.85 cM) and the chromosome 22 near marker D22S283. Exome sequencing identified two candidate variants to explain hearing loss in chromosome 14 [PTGDR – c.G894A:p.R298R and PTGER2 – c.T247G:p.C83G], and one in chromosome 22 [MYH9, c.G2114A:p.R705H]. Pedigree segregation analysis allowed exclusion of the PTGDR and PTGER2 variants as the cause of deafness. However, the MYH9 variant segregated with the phenotype in all affected members, except the three individuals with different phenotype. This gene has been previously described as mutated in autosomal dominant hereditary hearing loss and corresponds to DFNA17. The mutation identified in our study is the same described in the prior report. Thus, although linkage studies suggested a candidate gene in chromosome 14, we concluded that the mutation in chromosome 22 better explains the hearing loss phenotype in the Brazilian family.     

The control of lipid metabolism by mRNA splicing in Drosophila

The storage of lipids is an evolutionarily conserved process that is important for the survival of organisms during shifts in nutrient availability. Triglycerides are stored in lipid droplets, but the mechanisms of how lipids are stored in these structures are poorly understood. Previous in vitro RNAi screens have implicated several components of the spliceosome in controlling lipid droplet formation and storage, but the in vivo relevance of these phenotypes is unclear. In this study, we identify specific members of the splicing machinery that are necessary for normal triglyceride storage in the Drosophila fat body. Decreasing the expression of the splicing factors U1-70K, U2AF38, U2AF50 in the fat body resulted in decreased triglyceride levels. Interestingly, while decreasing the SR protein 9G8 in the larval fat body yielded a similar triglyceride phenotype, its knockdown in the adult fat body resulted in a substantial increase in lipid stores. This increase in fat storage is due in part to altered splicing of the gene for the β-oxidation enzyme CPT1, producing an isoform with less enzymatic activity. Together, these data indicate a role for mRNA splicing in regulating lipid storage in Drosophila and provide a link between the regulation of gene expression and lipid homeostasis.     

The G314S KCNQ1 mutation exerts a dominant-negative effect on expression of KCNQ1 channels in oocytes

Congenital long QT syndrome is a cardiac disorder characterized by prolongation of QT interval on the surface ECG associated with syncopal attacks and a high risk of sudden death. Mutations in the voltage-gated potassium channel subunit KCNQ1 induce the most common form of long QT syndrome (LQT1). We previously identified a hot spot mutation G314S located within the pore region of the KCNQ1 ion channel in a Chinese family with long QT syndrome. In the present study, we used oocyte expression of the KCNQ1 polypeptide to study the effects of the G314S mutation on channel properties. The results of electrophysiological studies indicate G314S, co-expressed with KCNE1 was unable to assemble to form active channel. G314S, co-expressed with WT KCNQ1 and KCNE1, suppressed I_ks currents in a dominant-negative manner, which is consistent with long QT syndrome in the members of the Chinese family carrying G314S KCNQ1 mutation.     

Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VASP

Filamin c is the predominantly expressed filamin isoform in striated muscles. It is localized in myofibrillar Z-discs, where it binds FATZ and myotilin, and in myotendinous junctions and intercalated discs. Here, we identify Xin, the protein encoded by the human gene 'cardiomyopathy associated 1' (CMYA1) as filamin c binding partner at these specialized structures where the ends of myofibrils are attached to the sarcolemma. Xin directly binds the EVH1 domain proteins Mena and VASP. In the adult heart, Xin and Mena/VASP colocalize with filamin c in intercalated discs. In cultured cardiomyocytes, the proteins also localize in the nonstriated part of myofibrils, where sarcomeres are assembled and an extensive reorganization of the actin cytoskeleton occurs. Unusual intraexonic splicing events result in the existence of three Xin isoforms that associate differentially with its ligands. The identification of the complex filamin c-Xin-Mena/VASP provides a first glance on the role of Xin in the molecular mechanisms involved in developmental and adaptive remodeling of the actin cytoskeleton during cardiac morphogenesis and sarcomere assembly.     

Association between the C.1161G>A and C.1779C>G Genetic Variants of XRCC1 Gene and Hepatocellular Carcinoma Risk in Chinese Population

The human X-ray repair complementing group 1 gene (XRCC1) is an important candidate gene influencing hepatocellular carcinoma (HCC) susceptibility. The objective of this study was to detect the association between c.1161G>A and c.1779C>G variants of XRCC1 gene and HCC risk. This study was conducted in Chinese population consisting of 623 HCC cases and 639 controls. These two genetic variants could be genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The association of XRCC1 gene variants with the risk of HCC was investigated under different genetic models. Our findings suggested that the genotypes/alleles from c.1161G>A and c.1779C>G genetic variants were statistically associated with HCC risk. As for the c.1161G>A, the AA genotype was statistically associated with the increased risk of HCC compared to GG wild genotype (OR = 2.36, 95% CI 1.63-3.40, P < 0.001). As for the c.1779C>G, the risk of HCC was significantly higher for GG genotype compared to CC wild genotype (OR = 2.17, 95% CI 1.51-3.12, P < 0.001). Furthermore, significant differences in the risk of HCC were also detected in other genetic models for these two variants. The allele-A of c.1161G>A and allele-G of c.1779C>G variants may contribute to the susceptibility of HCC (A versus G: OR = 1.48, 95% CI 1.26-1.75, P < 0.001 and G versus C: OR = 1.51, 95% CI 1.28-1.78, P < 0.001). Our data indicated that these two variants of XRCC1 gene were statistically associated with HCC risk in Chinese population.     

The association of the exon 4 variations of Tim-1 gene with allergic diseases in a Korean population

The family of T-cell immunoglobulin domain and mucin domain (TIM) proteins is identified to be expressed on T cells. A member of Tim family, TIM-1, is considered as a membrane protein that is associated with the development of Th2 biased immune responses and may be selectively expressed on Th2 cells. In the present study, we analyzed the association of allele and genotype frequencies between asthma or atopy patients and the controls without asthma and atopy using large sample size at 5383_5397del and 5509_5511delCAA variations of Tim-1 gene. Although the allele frequency of 5509_5511delCAA variation in asthma was not significantly different (P=0.085), the genotype of 5509_5511delCAA variation in asthma was significantly associated with the susceptibility to asthma (P=0.037). The genotype and allele frequencies of 5383_5397del variation in atopic dermatitis were significantly different from those in the non-asthmatic and non-atopic controls (P=0.005 and P=0.002, respectively). Our results strongly suggest that the 5383_5397del variation site of Tim-1 exon 4 might be associated with atopic dermatitis susceptibility.     

Role of six single nucleotide polymorphisms,risk factors in coronary disease,in OLR1 alternative splicing

The OLR1 gene encodes the oxidized low-density lipoprotein receptor (LOX-1), which is responsible for the cellular uptake of oxidized LDL (Ox-LDL), foam cell formation in atheroma plaques and atherosclerotic plaque rupture. Alternative splicing (AS) of OLR1 exon 5 generates two protein isoforms with antagonistic functions in Ox-LDL uptake. Previous work identified six single nucleotide polymorphisms (SNPs) in linkage disequilibrium that influence the inclusion levels of OLR1 exon 5 and correlate with the risk of cardiovascular disease. Here we use minigenes to recapitulate the effects of two allelic series (Low- and High-Risk) on OLR1 AS and identify one SNP in intron 4 (rs3736234) as the main contributor to the differences in exon 5 inclusion, while the other SNPs in the allelic series attenuate the drastic effects of this key SNP. Bioinformatic, proteomic, mutational and functional high-throughput analyses allowed us to define regulatory sequence motifs and identify SR protein family members (SRSF1, SRSF2) and HMGA1 as factors involved in the regulation of OLR1 AS. Our results suggest that antagonism between SRSF1 and SRSF2/HMGA1, and differential recognition of their regulatory motifs depending on the identity of the rs3736234 polymorphism, influence OLR1 exon 5 inclusion and the efficiency of Ox-LDL uptake, with potential implications for atherosclerosis and coronary disease.     

Characterization of C. elegans RING finger protein 1, a binding partner of ubiquitin-conjugating enzyme 1

In a yeast two-hybrid screen, RING finger protein 1 (RFP-1) and UBR1 were identified as potential binding partners of C. elegans UBC-1, a ubiquitin-conjugating enzyme with a high degree of identity to S. cerevisiae UBC2/RAD6. The interaction of RFP-1 and UBC-1 was confirmed by co-immunoprecipitation experiments. Yeast interaction trap experiments mapped the region of interaction to the basic N-terminal 313 residues of RFP-1. The acidic carboxy-terminal extension of UBC-1 was not required for the interaction with RFP-1. Western blot analysis and indirect immunohistochemical staining show that RFP-1 is present in embryos, larvae, and adults, where it is found in intestinal, nerve ring, pharyngeal, gonadal, and oocyte cell nuclei. Double-stranded RNA interference experiments against rfp-1 indicate that this gene is required for L1 development, vulval development, and for egg laying. By contrast, RNA interference against ubc-1 gave no obvious phenotype, suggesting that ubc-1 is nonessential or is functionally redundant.     

A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation,results in delayed degradation of maternal proteins and embryonic lethality

In vertebrates, oocytes undergo maturation, arrest in metaphase II, and can then be fertilized by sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. In Caenorhabditis elegans, where no delay between oocyte maturation and fertilization is apparent, oocyte maturation and fertilization must be tightly coordinated. It is not clear what coordinates the transition from an oocyte to an embryo in C. elegans, but regulated turnover of oocyte-specific proteins contributes to the process. We describe here a gain-of-function mutation (zu405) in a gene that is essential for oocyte maturation, oma-1. In wild type animals, OMA-1 protein is expressed at a high level exclusively in oocytes and newly fertilized embryos and is degraded rapidly after the first mitotic division. The zu405 mutation results in improper degradation of the OMA-1 protein in embryos. In oma-1(zu405) embryos, the C blastomere is transformed to the EMS blastomere fate, resulting in embryonic lethality. We show that degradation of several maternally supplied cell fate determinants, including SKN-1, PIE-1, MEX-3, and MEX-5, is delayed in oma-1(zu405) mutant embryos. In wild type embryos, SKN-1 functions in EMS for EMS blastomere fate specification. A decreased level of maternal SKN-1 protein in the C blastomere relative to EMS is believed to be responsible for this cell expressing the C, instead of the EMS, fate. Delayed degradation of maternal SKN-1 protein in oma-1(zu405) embryos and resultant elevated levels in C blastomere is likely responsible for the observed C-to-EMS blastomere fate transformation. These observations suggest that oma-1, in addition to its role in oocyte maturation, contributes to early embryonic development by regulating the temporal degradation of maternal proteins in early C. elegans embryos.     

A novel splicing mutation in COL1A1 gene caused type I osteogenesis imperfecta in a Chinese family

Osteogenesis imperfect (OI) is a heritable connective tissue disorder with bone fragility as a cardinal manifestation, accompanied by short stature, dentinogenesis imperfecta, hyperlaxity of ligaments and skin, blue sclerae and hearing loss. Dominant form of OI is caused by mutations in the type I procollagen genes, COL1A1/A2. Here we identified a novel splicing mutation c.3207+1G>A (GenBank ID: JQ236861) in the COL1A1 gene that caused type I OI in a Chinese family. RNA splicing analysis proved that this mutation created a new splicing site at c.3200, and then led to frameshift. This result further enriched the mutation spectrum of type I procollagen genes.     

Functional consequences of an in vivo mutation in exon 10 of the human GLUT1 gene

The functional consequences of an in vivo heterozygous insertion mutation in the human facilitated glucose transporter isoform 1 (GLUT1) gene were investigated. The resulting frameshift in exon 10 changed the primary structure of the C-terminus from 42 in native GLUT1 to 61 amino acid residues in the mutant. Kinetic studies on a patient's erythrocytes were substantiated by expressing the mutant cDNA in Xenopus laevis oocytes. K(m) and V(max) values were clearly decreased explaining pathogenicity. Targeting to the plasma membrane was comparable between mutant and wild-type GLUT1. Transport inhibition by cytochalasin B was more effective in the mutant than in the wild-type transporter. The substrate specificity of GLUT1 remained unchanged.     

The role of Schizosaccharomyces pombe DNA repair enzymes Apn1p and Uve1p in the base excision repair of apurinic/apyrimidinic sites

In Schizosaccharomyces pombe the repair of apurinic/apyrimidinic (AP) sites is mainly initiated by AP lyase activity of DNA glycosylase Nth1p. In contrast, the major AP endonuclease Apn2p functions by removing 3'-alpha,beta-unsaturated aldehyde ends induced by Nth1p, rather than by incising the AP sites. S. pombe possesses other minor AP endonuclease activities derived from Apn1p and Uve1p. In this study, we investigated the function of these two enzymes in base excision repair (BER) for methyl methanesulfonate (MMS) damage using the nth1 and apn2 mutants. Deletion of apn1 or uve1 from nth1Delta cells did not affect sensitivity to MMS. Exogenous expression of Apn1p failed to suppress the MMS sensitivity of nth1Delta cells. Although Apn1p and Uve1p incised the oligonucleotide containing an AP site analogue, these enzymes could not initiate repair of the AP sites in vivo. Despite this, expression of Apn1p partially restored the MMS sensitivity of apn2Delta cells, indicating that the enzyme functions as a 3'-phosphodiesterase to remove 3'-blocked ends. Localization of Apn1p in the nucleus and cytoplasm hints at an additional function of the enzyme other than nuclear DNA repair. Heterologous expression of Saccharomyces cerevisiae homologue of Apn1p completely restored the MMS resistance of the nth1Delta and apn2Delta cells. This result confirms a difference in the major pathway for processing the AP site between S. pombe and S. cerevisiae cells.     

The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line

To understand the molecular basis of sensory organ development and disease, we have cloned and characterized the zebrafish mutation dog-eared (dog) that is defective in formation of the inner ear and lateral line sensory systems. The dog locus encodes the eyes absent-1 (eya1) gene and single point mutations were found in three independent dog alleles, each prematurely truncating the expressed protein within the Eya domain. Moreover, morpholino-mediated knockdown of eya1 gene function phenocopies the dog-eared mutation. In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis but Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts. Increased levels of apoptosis occur in the migrating primordia of the posterior lateral line in dog embryos and as well as in regions of the developing otocyst that are mainly fated to give rise to sensory cells of the cristae. Importantly, mutation of the EYA1 or EYA4 gene causes hereditary syndromic deafness in humans. Determination of eya gene function during zebrafish organogenesis will facilitate understanding the molecular etiology of human vestibular and hearing disorders.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.