Home About us Contact
|
Home About us Contact | ||
|
|
||
mRNA Splicing (mrna + splicing)
Selected AbstractsSystematic evaluation of the effect of common SNPs on pre-mRNA splicing,HUMAN MUTATION, Issue 4 2009Abdou ElSharawy Abstract The evolutionary and biomedical importance of differential mRNA splicing is well established. Numerous studies have assessed patterns of differential splicing in different genes and correlated these patterns to the genotypes for adjacent single-nucleotide polymorphisms (SNPs). Here, we have chosen a reverse approach and screened dbSNP for common SNPs at either canonical splice sites or exonic splice enhancers (ESEs) that would be classified as putatively splicing-relevant by bioinformatic tools. The 223 candidate SNPs retrieved from dbSNP were experimentally tested using a previously established panel of 92 matching DNAs and cDNAs. For each SNP, 16 cDNAs providing a balanced representation of the genotypes at the respective SNP were investigated by nested RT-PCR and subsequent sequencing. Putative allele-dependent splicing was verified by the cloning of PCR products. The positive predictive value of the bioinformatics tools turned out to be low, ranging from 0% for ESEfinder to 9% (in the case of acceptor-site SNPs) for a recently reported neural network. The results highlight the need for a better understanding of the sequence characteristics of functional splice-sites to improve our ability to predict in silico the splicing relevance of empirically observed DNA sequence variants. Hum Mutat 0, 1,9, 2009. © 2009 Wiley-Liss, Inc. [source] A specific GFP expression assay, penetrance estimate, and histological assessment for a putative splice site mutation in BRCA1,HUMAN MUTATION, Issue 1 2003M.C. Southey Abstract Genetic testing for cancer predisposing mutations in BRCA1 and BRCA2 has been of benefit to many individuals from breast and ovarian cancer-prone kindreds. However, a function has not been assigned to many of the domains that make up these complex proteins and hence, the significance of many sequence variants, including missense mutations, splice-site mutations, and in-frame deletions/insertions, remains unclear. We identified a putative splice site mutation (IVS6-2delA) in BRCA1 in a family attending a Familial Cancer Centre that had a significant history of both breast and ovarian cancer. This sequence variant was not novel but the exact effect on mRNA splicing and hence the biological impact of this sequence variation was unclear and therefore the finding was unable to be used in genetic counseling of the family. Via the construction of novel GFP-based expression fusion constructs, we demonstrated that this sequence variation prevented normal splicing of the BRCA1 transcript. By combining these data with an assessment of the histopathological features of the breast carcinomas in this family and mutation penetrance estimate we were able to conclude that this BRCA1 variant conveyed an increased risk of breast cancer. Hum Mutat 22:86,91, 2003. © 2003 Wiley-Liss, Inc. [source] Complementation of Physiological and Behavioral Defects by a Slowpoke Ca2+ -Activated K+ Channel TransgeneJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Robert Brenner Abstract: The Drosophila slowpoke gene encodes a large conductance calcium-activated potassium channel used in neurons, muscle, and some epithelial cells. Tissue-specific transcriptional promoters and alternative mRNA splicing generate a large array of transcripts. These distinct transcripts are thought to tailor the properties of the channel to the requirements of the cell. Presumably, a single splice variant cannot satisfy the specific needs of all cell types. To test this, we examined whether a single slowpoke splice variant was capable of complementing all slowpoke behavioral phenotypes. Null mutations in slowpoke cause animals to be semiflightless and to manifest an inducible "sticky-feet" phenotype. The well-characterized slowpoke transcriptional control region was used to direct the expression of a single slowpoke splice variant (cDNA H13) in transgenic flies. The endogenous gene in these flies had been inactivated by the slo4 mutation. Action-potential recordings and voltage-clamp recordings demonstrated the production of functional channels from the transgene. The transgene completely complemented the flight defect, but not the sticky-feet phenotype. We conclude that distinct slowpoke channel isoforms, produced by alternative splicing, are not interchangeable and are required for proper function of different cell types. [source] Altered mRNA splicing of dystrophin in type 1 myotonic dystrophyMUSCLE AND NERVE, Issue 2 2007Masayuki Nakamori MD Abstract Myotonic dystrophy type1 (DM1) is a multisystemic disorder caused by a CTG repeat expansion in the DMPK gene. Aberrant mRNA splicing of several genes has been reported to contribute to some of the symptoms, including myotonia and insulin resistance, but the cause of muscle wasting is unknown. Dystrophin is a cytoskeletal protein that is required for structural stability and signaling at the sarcolemma and has several spliced isoforms. We investigated the alternative splicing of dystrophin in skeletal and cardiac muscle of DM1 patients by using reverse transcriptase,polymerase chain reaction and found that dystrophin isoforms lacking exon 71 or 78, which is suggested to encode an important region for protein binding and hydrophobicity, were significantly increased. We suggest that the aberrantly spliced dystrophin is responsible for the muscle wasting in DM1. Muscle Nerve, 2007 [source] One novel and one recurrent mutation in the PROS1 gene cause type I protein S deficiency in patients with pulmonary embolism associated with deep vein thrombosisAMERICAN JOURNAL OF HEMATOLOGY, Issue 10 2006Kazuhiro Mizukami Abstract We investigated the molecular basis of type I protein S (PS) deficiency in two unrelated Japanese families, in which both probands developed pulmonary embolism associated with deep vein thrombosis. Nucleotide sequencing of amplified DNA revealed distinct point mutations in the PROS1 gene of the probands, which were designated protein S Sapporo 1 and protein S Sapporo 2. Additional mutations in the PROS1 gene were excluded by DNA sequencing of all exons and intron/exon boundaries. In the 25-year-old Japanese male patient who carried protein S Sapporo 1, we identified a heterozygous A-to-T change in the invariant ag dinucleotide of the acceptor splice site of intron f of the PROS1 gene. This mutation is a novel splice site mutation that impairs normal mRNA splicing, leading to exon 7 skipping, which was confirmed by platelet mRNA analysis. Translation of this mutant transcript would result in a truncated protein that lacks the entire epidermal growth factor-like domain 3 of the PS molecule. In a 31-year-old Japanese male and his younger brother who each carried protein S Sapporo 2, we detected a previously described heterozygous T-to-C transition at nucleotide position 1147 in exon 10 of the PROS1 gene, which predicts an amino acid substitution of tryptophan by arginine at residue 342 in the laminin G1 domain of the PS molecule. Both mutations would cause misfolding of the PS protein, resulting in the impairment of secretion, which is consistent with the type I PS deficiency phenotype. Am. J. Hematol., 2006. © 2006 Wiley-Liss, Inc. [source] RNA-mediated neurodegeneration in repeat expansion disordersANNALS OF NEUROLOGY, Issue 3 2010Peter K. Todd MD Most neurodegenerative disorders are thought to result primarily from the accumulation of misfolded proteins, which interfere with protein homeostasis in neurons. For a subset of diseases, however, noncoding regions of RNAs assume a primary toxic gain-of-function, leading to degeneration in many tissues, including the nervous system. Here we review a series of proposed mechanisms by which noncoding repeat expansions give rise to nervous system degeneration and dysfunction. These mechanisms include transcriptional alterations and the generation of antisense transcripts, sequestration of mRNA-associated protein complexes that lead to aberrant mRNA splicing and processing, and alterations in cellular processes, including activation of abnormal signaling cascades and failure of protein quality control pathways. We place these potential mechanisms in the context of known RNA-mediated disorders, including the myotonic dystrophies and fragile X tremor ataxia syndrome, and discuss recent results suggesting that mRNA toxicity may also play a role in some presumably protein-mediated neurodegenerative disorders. Lastly, we comment on recent progress in therapeutic development for these RNA-dominant diseases. ANN NEUROL 2010;67:291,300 [source] Growth and specification: fly Pax6 homologs eyegone and eyeless have distinct functionsBIOESSAYS, Issue 6 2004Aloma B. Rodrigues Development requires not only the correct specification of organs and cell types in the right places (pattern), but also the control of their size and shape (growth). Many signaling pathways control both pattern and growth and how these two are distinguished has been something of a mystery. In the fly eye, a Pax6 homolog (eyeless) controls eye specification together with several other genes. Now Dominguez et al.1 show that Notch signaling controls eye growth through a second Pax6 protein (Eyegone). In mice and humans the single Pax6 gene appears to encode both specification and growth controlling proteins through alternative mRNA splicing. BioEssays 26:600,603, 2004. © 2004 Wiley Periodicals, Inc. [source] The prevalence of, and molecular defects underlying, inherited protein S deficiency in the general populationBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2004Nicholas J. Beauchamp Summary The molecular basis of protein S (PS) deficiency was investigated in seven of eight donors identified with persistently low plasma PS levels from a survey of PS levels in 3788 Scottish blood donors. PROS1 gene analysis identified at least one defect in six donors. Five were heterozygous for the Heerlen polymorphism predicting a Ser460Pro substitution. Haplotype analysis revealed the possibility that this allele was inherited with the same haplotype in four of the five donors, suggesting a founder effect for the Heerlen allele in this population. One Heerlen allele carrier was also heterozygous for a 3 bp deletion 68,72 bp upstream of exon 2. Platelet PROS1 transcript analysis showed no reduction in mRNA expression from the affected allele in this donor. A T to G transversion 3 bp upstream of exon 12 was identified in one donor, which is predicted to reduce the efficiency of PS mRNA splicing. However, PROS1 transcript analysis showed no evidence of exon skipping or cryptic splicing. No PROS1 gene defect was detected in the remaining donor. This genetic information enabled us to refine our estimate of the prevalence of heritable PS deficiency in the Scottish population to between 0·16% and 0·21%, predominantly resulting from the presence of the Heerlen allele. [source] PROTEOMIC ANALYSIS IN CARDIOVASCULAR DISEASESCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2008C Cieniewski-Bernard SUMMARY 1Cardiovascular diseases are a major cause of morbidity and mortality in western countries. The molecular mechanisms responsible for heart dysfunction are still largely unknown, except in cases of genetic defects or alteration of genes and proteins. 2The publication of genome sequences from humans and other species has demonstrated the complexity of biology, including the finding that one gene does not encode for only one protein but for several, due to mRNA splicing and post-translational modifications. 3Proteomic analysis can provide an overall understanding of changes in the levels of protein expression. Differential proteomics is a powerful tool for improving our understanding of integrated biochemical responses. The main techniques used are two-dimensional electrophoresis (2D-gel) and Surface-Enhanced Laser Desorption/Ionization Time of Flight (SELDI-TOF) to separate proteins associated with mass spectrometry. Bioinformatic tools make it possible to compare protein profiles obtained from diverse biological samples. 4The combination of these approaches has proved to be particularly interesting for studying cardiovascular diseases and thereby improving our understanding of the mechanisms involved and identifying new biochemical factors and biomarkers involved in these diseases. [source] Silent exonic mutations in the low-density lipoprotein receptor gene that cause familial hypercholesterolemia by affecting mRNA splicingCLINICAL GENETICS, Issue 6 2008JC Defesche In a large group of patients with the clinical phenotype of familial hypercholesterolemia, such as elevated low-density lipoprotein (LDL) cholesterol and premature atherosclerosis, but without functional mutations in the genes coding for the LDL receptor and apolipoprotein B, we examined the effect of 128 seemingly neutral exonic and intronic DNA variants, discovered by routine sequencing of these genes. Two variants, G186G and R385R, were found to be associated with altered splicing. The nucleotide change leading to G186G resulted in the generation of new 3,-splice donor site in exon 4 and R385R was associated with a new 5,-splice acceptor site in exon 9 of the LDL receptor gene. Splicing of these alternate splice sites leads to an in-frame 75-base pair deletion in a stable mRNA of exon 4 in case of G186G and R385R resulted in a 31-base pair frame-shift deletion in exon 9 and non-sense-mediated mRNA decay. [source] | |||||||||||||||||||||||||