Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5′ end of exon 8, a 33 nt RNA from 3′ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.     

Missense mutations in exon 4 of the porcine LEPR gene encoding extracellular domain and their association with fatness traits

Three non-synonymous single nucleotide polymorphisms (T221C, T232A and C233T) were detected in exon 4 of the porcine leptin receptor (LEPR) gene. The T232A substitution could be identified as a (Tsp509I) restriction fragment length polymorphism. The frequency of genotype TT varied in six genetic groups from 0.62 (Duroc) to 0.99 (Polish Large White). Sequencing of exon 4, performed for 30 animals, revealed that only two intragenic haplotypes (TC and AT at nucleotide position 232-233) were present. The phenotypic effect of the Tsp509I polymorphism was tested for the Polish Landrace (n = 241) and a synthetic line 990 (n = 243). There was no statistical evidence for the direct effect of the LEPR polymorphisms on fatness traits. However, in Polish Landrace allele A at position 232 was associated with thicker backfat over shoulder.     

The iStem, a long-range RNA secondary structure element required for efficient exon inclusion in the Drosophila Dscam pre-mRNA

The Drosophila Dscam gene encodes 38,016 different proteins, due to alternative splicing of 95 of its 115 exons, that function in axon guidance and innate immunity. The alternative exons are organized into four clusters, and the exons within each cluster are spliced in a mutually exclusive manner. Here we describe an evolutionarily conserved RNA secondary structure we call the Inclusion Stem (iStem) that is required for efficient inclusion of all 12 variable exons in the exon 4 cluster. Although the iStem governs inclusion or exclusion of the entire exon 4 cluster, it does not play a significant role in determining which variable exon is selected. Thus, the iStem is a novel type of regulatory element that simultaneously controls the splicing of multiple alternative exons.     

A novel mutation in the neurofibromatosis type 1 (NF1) gene promotes skipping of two exons by preventing exon definition

Using a protein truncation assay, we have identified a new mutation in the neurofibromatosis type 1 (NF1) gene that causes a severe defect in NF1 pre-mRNA splicing. The mutation, which consists of a G to A transition at position +1 of the 5' splice site of exon 12a, is associated with the loss of both exons 11 and 12a in the NF1 mRNA. Through the use of in vivo and in vitro splicing assays, we show that the mutation inactivates the 5' splice site of exon 12a, and prevents the definition of exon 12a, a process that is normally required to stimulate the weak 3' splice site of exon 12a. Because the 5' splice site mutation weakens the interaction of splicing factors with the 3' splice site of exon 12a, we propose that exon 11/exon 12a splicing is also compromised, leading to the exclusion of both exons 11 and 12a. Our results provide in vivo support for the importance of the exon definition model during NF1 splicing, and suggest that the NF1 region containing exons 11 and 12a plays an important role in the activity of neurofibromin.     

Structural effects of clinically observed mutations in JAK2 exons 13-15: comparison with V617F and exon 12 mutations

Background  

The functional relevance of many of the recently detected JAK2 mutations, except V617F and exon 12 mutants, in patients with chronic myeloproliferative neoplasia (MPN) has been significantly overlooked. To explore atomic-level explanations of the possible mutational effects from those overlooked mutants, we performed a set of molecular dynamics simulations on clinically observed mutants, including newly discovered mutations (K539L, R564L, L579F, H587N, S591L, H606Q, V617I, V617F, C618R, L624P, whole exon 14-deletion) and control mutants (V617C, V617Y, K603Q/N667K).     

Screening for mutations in exon 4 of the LDL receptor gene in a German population with severe hypercholesterolemia

A group of 218 patients with severe hypercholesterolemia (LDL cholesterol >260 mg/dl) living in the Cologne area were screened for mutations in the 3 half of exon 4 of the low density lipoprotein (LDL) receptor gene by the single-strand conformation polymorphism (SSCP) method. The analysed fragment was 242 bp in length and comprised approximately 6% of the coding region. In 11 patients an abnormal SSCP pattern was observed. Two of the abnormal fragment patterns were identical. The results of the SSCP screening could be confirmed by direct DNA sequencing. Three of the ten different mutations were previously described (3 bp deletion: codon 197; Asp₂₀₀Gly; Glu₂₀₇stop). Of the newly identified mutations there were two deletions, two insertions, one combined insertion and deletion mutation and two single base pair substitutions [1 bp deletion: G in codon 197; 37 bp deletion: T in codon 196–208 or AT in 196–207 and GA in codon 208; 18 bp insertion: codon 201–206; 8 bp insertion: codon 155–156 and GA in codon 157; 6 bp insertion (codon 196–197) and 5 bp deletion (codon 199, C in codon 198 and G in codon 198 or 200); Asp₂₀₀Tyr; Asp₂₀₃Val]. The 8-bp insertion was detected in a second unrelated individual. The analysis of the functional consequences of the mutations indicates that all mutations were causative of the LDL cholesterol elevation.     

Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase.

Suppressors of the methyl methanesulfonate sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase turned out to contain semidominant mutations in Rad5l, a homolog of the bacterial RecA protein. The nature of these mutations was determined by direct sequencing. The 26 mutations characterized were single base substitutions leading to amino acid replacements at 18 different sites. The great majority of these sites (75%) are conserved in the family of RecA-like proteins, and 10 of them affect sites corresponding to amino acids in RecA that are probably directly involved in ATP reactions, binding, and/or hydrolysis. Six mutations are in domains thought to be involved in interaction between monomers; they may also affect ATP reactions. By themselves, all the alleles confer a rad5l null phenotype. When heterozygous, however, they are, to varying degrees, negative semidominant for radiation sensitivity; presumably the mutant proteins are coassembled with wild-type Rad51 and poison the resulting nucleofilaments or recombination complexes. This negative effect is partially suppressed by an SRS2 deletion, which supports the hypothesis that Srs2 reverses recombination structures that contain either mutated proteins or numerous DNA lesions.     

昆虫钠离子通道基因突变及其与杀虫剂抗性关系的研究进展

昆虫电压门控钠离子通道(voltage-gated sodium channel)存在于所有可兴奋细胞的细胞膜上,在动作电位的产生和传导上起重要作用,是有机氯和拟除虫菊酯杀虫剂的靶标位点。在农业和医学害虫控制过程中,由于有机氯和拟除虫菊酯杀虫剂的广泛使用,抗药性问题日益突出。其中,由于钠离子通道基因突变,降低了钠离子通道对有机氯和拟除虫菊酯类杀虫剂的亲和性,从而产生击倒抗性(knock-down resistance, kdr),已成为抗性产生的重要机制之一。本文综述了昆虫钠离子通道的跨膜拓扑结构、功能、进化及其基因的克隆;更重要的是总结了已报道的40多种昆虫40个钠离子通道基因非同义突变,以及钠离子通道基因选择性mRNA剪接和编辑,以及它们与杀虫剂抗性的关系;也评述了钠离子通道基因突变引起蛋白质结构的改变,从而对杀虫剂抗性的影响机制。这些研究对于进一步鉴定与杀虫剂抗性相关的突变及抗性机制,开发有机氯和拟除虫菊酯类杀虫剂抗性分子监测方法具有重要意义。     

Grey, a novel mutation in the murine Lyst gene, causes the beige phenotype by skipping of exon 25

The murine beige mutant phenotype and the human Chediak-Higashi syndrome are caused by mutations in the murine Lyst (lysosomal trafficking regulator) gene and the human CHS gene, respectively. In this report we have analyzed a novel murine mutant Lyst allele, called Lyst(bg-grey), that had been found in an ENU mutation screen and named grey because of the grey coat color of affected mice. The phenotype caused by the Lyst(bg-grey) mutation was inherited in a recessive fashion. Melanosomes of melanocytes associated with hair follicles and the choroid layer of the eye, as well as melanosomes in the neural tube-derived pigment epithelium of the retina, were larger and irregularly shaped in homozygous mutants compared with those of wild-type controls. Secretory vesicles in dermal mast cells of the mutant skin were enlarged as well. Test crosses with beige homozygous mutant mice (Lyst(bg)) showed that double heterozygotes (Lyst(bg)/Lyst(bg-grey)) were phenotypically indistinguishable from either homozygous parent, demonstrating that the ENU mutation was an allele of the murine Lyst gene. RT-PCR analyses revealed the skipping of exon 25 in Lyst(bg-grey) mutants, which is predicted to cause a missense D2399E mutation and the loss of the following 77 amino acids encoded by exon 25 but leave the C-terminal end of the protein intact. Analysis of the genomic Lyst locus around exon 25 showed that the splice donor at the end of exon 25 showed a T-to-C transition point mutation. Western blot analysis suggests that the Lyst(bg-grey) mutation causes instability of the LYST protein. Because the phenotype of Lyst(bg) and Lyst(bg-grey) mutants is indistinguishable, at least with respect to melanosomes and secretory granules in mast cells, the Lyst(bg-grey) mutation defines a critical region for the stability of the murine LYST protein.     

Rapid changes in the exon/intron structure of a mammalian thrombin inhibitor gene

The genomic organization of the heparin cofactor II (HCII) gene from rat and mouse was investigated and compared with their human counterpart. The genes share a common core structure consisting of five exons interrupted by four introns, but the mouse and rat gene reveal individual additional features. A unique differentially spliced exon is present in the 5'-untranslated region of the rat gene, which most probably has arisen de novo by point mutations in intronic sequences of the ancestor gene. In the mouse HCII gene, a novel intron/exon boundary has been created due to the presence of an additional DNA segment, which simultaneously provides a 3'-splice site and a polypyrimidine stretch leading to an alternatively used exon of increased size. Our data suggest that, in contrast to most other mammalian genes, the exon/intron pattern of the gene coding for HCII is in dynamic evolution.     

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.     

Molecular characterization of exons 6, 8 and 9 of ABCB4 gene in children with Progressive Familial Intrahepatic Cholestasis type 3

Aim of work: To estimate the frequency of mutations involving exons 6, 8 and 9 of Adenosine triphosphate-binding cassette, subfamily B, member 4 (ABCB4) gene among children with progressive intrahepatic cholestasis with high γ-GT activity (PFIC3).

Subjects and methods: Cross sectional study was conducted on 30 children with PFIC3. Genotyping was performed by sequencing analysis of exons 6, 8 and exon 9 of ABCB4 gene.

Results: Heterozygous synonymous polymorphic variant was detected in exon 6 (rs 1202283) and in exon 8 (rs 2109505). No mutations in studied exons were detected.

Conclusion: Exons 6, 8 and 9 mutations of ABCB4 gene are not common among Egyptian children with PFIC3.