scholarly journals Aberrant Splicing of tau Pre-mRNA Caused by Intronic Mutations Associated with the Inherited Dementia Frontotemporal Dementia with Parkinsonism Linked to Chromosome 17

2000 ◽  
Vol 20 (11) ◽  
pp. 4036-4048 ◽  
Author(s):  
Zhihong Jiang ◽  
Jocelyn Cote ◽  
Jennifer M. Kwon ◽  
Alison M. Goate ◽  
Jane Y. Wu
Keyword(s):  
Alternative Splicing ◽  
Frontotemporal Dementia ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Mrna Splicing ◽  
Chromosome 17 ◽  
Mobility Shift ◽  
U1 Snrnp ◽  
Exon 11 ◽  
Exon 10

ABSTRACT Frontotemporal dementia accounts for a significant fraction of dementia cases. Frontotemporal dementia with parkinsonism linked to chromosome 17 is associated with either exonic or intronic mutations in the tau gene. This highlights the involvement of aberrant pre-mRNA splicing in the pathogenesis of neurodegenerative disorders. Little is known about the molecular mechanisms of the splicing defects underlying these diseases. To establish a model system for studying the role of pre-mRNA splicing in neurodegenerative diseases, we have constructed a tau minigene that reproduces tau alternative splicing in both cultured cells and in vitro biochemical assays. We demonstrate that mutations in a nonconserved intronic region of the human tau gene lead to increased splicing between exon 10 and exon 11. Systematic biochemical analyses indicate the importance of U1 snRNP and, to a lesser extent, U6 snRNP in differentially recognizing wild-type versus intron mutant tau pre-mRNAs. Gel mobility shift assays with purified U1 snRNP and oligonucleotide-directed RNase H cleavage experiments support the idea that the intronic mutations destabilize a stem-loop structure that sequesters the 5′ splice site downstream of exon 10 in tau pre-mRNA, leading to increases in U1 snRNP binding and in splicing between exon 10 and exon 11. Thus, mutations in nonconserved intronic regions that increase rather than decrease alternative splicing can be an important pathogenic mechanism for the development of human diseases.

scholarly journals RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 3020 ◽  
Author(s):  
Alan Ann Lerk Ong ◽  
Jiazi Tan ◽  
Malini Bhadra ◽  
Clément Dezanet ◽  
Kiran M. Patil ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Nucleic Acids ◽  
Splice Site ◽  
Peptide Nucleic Acids ◽  
Amino Sugar ◽  
Hairpin Structure ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Antisense Peptide ◽  
Exon 10

Alternative splicing of tau pre-mRNA is regulated by a 5′ splice site (5′ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5′ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3′ arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5′ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5′ arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5′ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

Evidence of the modulation of mRNA splicing fidelity in humans by oxidative stress and p53

Genome ◽  
2007 ◽  
Vol 50 (10) ◽  
pp. 946-953 ◽  
Author(s):  
Kim Disher ◽  
Adonis Skandalis
Keyword(s):  
Oxidative Stress ◽  
Alternative Splicing ◽  
Molecular Mechanisms ◽  
Splice Variants ◽  
Dna Lesions ◽  
Mrna Splicing ◽  
Aberrant Splice ◽  
Biological Phenomenon ◽  
Mrna Splice ◽  
Human Genes

The majority of human genes generate mRNA splice variants and while there is little doubt that alternative splicing is an important biological phenomenon, it has also become apparent that some splice variants are associated with disease. To elucidate the molecular mechanisms responsible for generating aberrant splice variants, we have investigated alternative splicing of the human genes HPRT and POLB following oxidative stress in different genetic backgrounds. Our study revealed that splicing fidelity is sensitive to oxidative stress. Following treatment of cells with H2O2, the overall frequency of aberrant, unproductive splice variants increased in both loci. At least in POLB, splicing fidelity is p53 dependent. In the absence of p53, the frequency of POLB splice variants is elevated but oxidative stress does not further increase the frequency of splice variants. Our data indicate that mis-splicing following oxidative stress represents a novel and significant genotoxic outcome and that it is not simply DNA lesions induced by oxidative stress that lead to mis-splicing but changes in the alternative splicing machinery itself.

scholarly journals SRp54 (SFRS11), a Regulator for tau Exon 10 Alternative Splicing Identified by an Expression Cloning Strategy

2006 ◽  
Vol 26 (18) ◽  
pp. 6739-6747 ◽  
Author(s):  
Jane Y. Wu ◽  
Amar Kar ◽  
David Kuo ◽  
Bing Yu ◽  
Necat Havlioglu
Keyword(s):  
Alternative Splicing ◽  
Fluorescent Protein ◽  
Neurodegenerative Disorder ◽  
Chromosome 17 ◽  
Expression Cloning ◽  
Splicing Regulators ◽  
Cloning Strategy ◽  
Green Fluorescent ◽  
And Function ◽  
Exon 10

ABSTRACT The tau gene encodes a microtubule-associated protein that is critical for neuronal survival and function. Splicing defects in the human tau gene lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), an autosomal dominant neurodegenerative disorder. Genetic mutations associated with FTDP-17 often affect tau exon 10 alternative splicing. To investigate mechanisms regulating tau exon 10 alternative splicing, we have developed a green fluorescent protein reporter for tau exon 10 skipping and an expression cloning strategy to identify splicing regulators. A role for SRp54 (also named SFRS11) as a tau exon 10 splicing repressor has been uncovered using this strategy. The overexpression of SRp54 suppresses tau exon 10 inclusion. RNA interference-mediated knock-down of SRp54 increases exon 10 inclusion. SRp54 interacts with a purine-rich element in exon 10 and antagonizes Tra2β, an SR-domain-containing protein that enhances exon 10 inclusion. Deletion of this exonic element eliminates the activity of SRp54 in suppressing exon 10 inclusion. Our data support a role of SRp54 in regulating tau exon 10 splicing. These experiments also establish a generally useful approach for identifying trans-acting regulators of alternative splicing by expression cloning.

scholarly journals Identifying biological pathways that underlie primordial short stature using network analysis

2014 ◽  
Vol 52 (3) ◽  
pp. 333-344 ◽  
Author(s):  
Dan Hanson ◽  
Adam Stevens ◽  
Philip G Murray ◽  
Graeme C M Black ◽  
Peter E Clayton
Keyword(s):  
Molecular Mechanisms ◽  
Growth Failure ◽  
Mrna Splicing ◽  
Hek293 Cells ◽  
Growth Disorders ◽  
Biological Functions ◽  
Altered Expression ◽  
Signalling Molecules ◽  
Cellular Pathways ◽  
Exon 11

Mutations in CUL7, OBSL1 and CCDC8, leading to disordered ubiquitination, cause one of the commonest primordial growth disorders, 3-M syndrome. This condition is associated with i) abnormal p53 function, ii) GH and/or IGF1 resistance, which may relate to failure to recycle signalling molecules, and iii) cellular IGF2 deficiency. However the exact molecular mechanisms that may link these abnormalities generating growth restriction remain undefined. In this study, we have used immunoprecipitation/mass spectrometry and transcriptomic studies to generate a 3-M ‘interactome’, to define key cellular pathways and biological functions associated with growth failure seen in 3-M. We identified 189 proteins which interacted with CUL7, OBSL1 and CCDC8, from which a network including 176 of these proteins was generated. To strengthen the association to 3-M syndrome, these proteins were compared with an inferred network generated from the genes that were differentially expressed in 3-M fibroblasts compared with controls. This resulted in a final 3-M network of 131 proteins, with the most significant biological pathway within the network being mRNA splicing/processing. We have shown using an exogenous insulin receptor (INSR) minigene system that alternative splicing of exon 11 is significantly changed in HEK293 cells with altered expression of CUL7, OBSL1 and CCDC8 and in 3-M fibroblasts. The net result is a reduction in the expression of the mitogenic INSR isoform in 3-M syndrome. From these preliminary data, we hypothesise that disordered ubiquitination could result in aberrant mRNA splicing in 3-M; however, further investigation is required to determine whether this contributes to growth failure.

Tau alternative splicing in familial and sporadic tauopathies

2012 ◽  
Vol 40 (4) ◽  
pp. 677-680 ◽  
Author(s):  
Michael Niblock ◽  
Jean-Marc Gallo
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Alternative Splicing ◽  
Chromosome 17 ◽  
Binding Motif ◽  
Neuronal Viability ◽  
Protein Tau ◽  
Rna Analysis ◽  
Tau Isoforms ◽  
Exon 10

Six tau isoforms differing in their affinity for microtubules are produced by alternative splicing from the MAPT (microtubule-associated protein tau) gene in adult human brain. Several MAPT mutations causing the familial tauopathy, FTDP-17 (frontotemporal dementia with parkinsonism linked to chromosome 17), affect alternative splicing of exon 10, encoding a microtubule-binding motif. Advanced RNA analysis methods have suggested that levels of exon 10-containing MAPT mRNA are elevated in Alzheimer's disease. Furthermore, the MAPT H1 haplotype, associated with Alzheimer's disease, promotes exon 10 inclusion in MAPT mRNA. Thus an accurate regulation of tau alternative splicing is critical for the maintenance of neuronal viability, and its alteration might be a contributing factor to Alzheimer's disease. Tau alternative splicing could represent a target for therapeutic intervention to delay the progression of pathology in familial as well as sporadic tauopathies.

A Mucin-Specific Protease Enables Molecular and Functional Analysis of Human Cancer-Associated Mucins

2018 ◽  
Author(s):  
Stacy A. Malaker ◽  
Kayvon Pedram ◽  
Michael J. Ferracane ◽  
Elliot C. Woods ◽  
Jessica Kramer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
High Resolution Mass Spectrometry ◽  
Human Cancer ◽  
Glycosylation Sites ◽  
Bacterial Protease ◽  
Specific Protease ◽  
To Come ◽  
And Function

<div> <div> <div> <p>Mucins are a class of highly O-glycosylated proteins that are ubiquitously expressed on cellular surfaces and are important for human health, especially in the context of carcinomas. However, the molecular mechanisms by which aberrant mucin structures lead to tumor progression and immune evasion have been slow to come to light, in part because methods for selective mucin degradation are lacking. Here we employ high resolution mass spectrometry, polymer synthesis, and computational peptide docking to demonstrate that a bacterial protease, called StcE, cleaves mucin domains by recognizing a discrete peptide-, glycan-, and secondary structure- based motif. We exploited StcE’s unique properties to map glycosylation sites and structures of purified and recombinant human mucins by mass spectrometry. As well, we found that StcE will digest cancer-associated mucins from cultured cells and from ovarian cancer patient-derived ascites fluid. Finally, using StcE we discovered that Siglec-7, a glyco-immune checkpoint receptor, specifically binds sialomucins as biological ligands, whereas the related Siglec-9 receptor does not. Mucin-specific proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of glycoprotein structure and function and for deorphanizing mucin-binding receptors. </p> </div> </div> </div>

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