Dysregulation of TDP‐43 intracellular localization and early onset ALS are associated with a TARDBP S375G variant

We investigated the Central Nervous System (CNS) and skeletal muscle tissue from A woman was clinically diagnosed with amyotrophic lateral sclerosis (ALS) at the age of 22. Neuropathologic evaluation showed upper and lower motor neuron loss, corticospinal tract degeneration and skeletal muscle denervation. Analysis of the patient's Deoxyribonucleic acid (DNA) revealed a AGT>GGT change resulting in an S375G substitution in the C‐terminal region of TDP‐43. This variant was previously reported as being benign. Considering the early onset and severity of the disease in this patient, we tested the effects of this genetic variant on TDP‐43 localization, pre‐mRNA splicing activity and toxicity, in parallel with the effects on known neighboring disease‐associated mutations. In cell lines, expressed in culture, S375G TDP‐43 appeared to be more significantly localized in the nucleus and to exert higher toxicity than wild‐type TDP‐43. Strikingly, a phosphomimic mutant at the same residue (S375E) showed a strong tendency to accumulate in the cytoplasm, especially under stress conditions, and molecular dynamics simulations suggest that phosphorylation of this residue can disrupt TDP‐43 intermolecular interactions. The results of the current study highlight the importance of phosphorylation and regulation of TDP‐43 nuclear‐cytoplasmic shuttling/redistribution, in relation to the pathogenetic mechanisms involved in different forms of ALS.     

Mutations in spliceosome genes and therapeutic opportunities in myeloid malignancies

Since the discovery of RNA splicing more than 40 years ago, our comprehension of the molecular events orchestrating constitutive and alternative splicing has greatly improved. Dysregulation of pre‐mRNA splicing has been observed in many human diseases including neurodegenerative diseases and cancer. The recent identification of frequent somatic mutations in core components of the spliceosome in myeloid malignancies and functional analysis using model systems has advanced our knowledge of how splicing alterations contribute to disease pathogenesis. In this review, we summarize our current understanding on the mechanisms of how mutant splicing factors impact splicing and the resulting functional and pathophysiological consequences. We also review recent advances to develop novel therapeutic approaches targeting splicing catalysis and splicing regulatory proteins, and discuss emerging technologies using oligonucleotide‐based therapies to modulate pathogenically spliced isoforms.     

Cloning, characterization and immunolocalization of human ameloblastin

Amelogenesis imperfecta is a broad classification of hereditary enamel defects, exhibiting both genetic and clinical diversity. Most amelogenesis imperfecta cases are autosomal dominant disorders, yet only the local hypoplastic form has been mapped to human chromosome 4q between D4S242 1 and the albumin gene. An enamel protein cDNA, termed ameloblastin (also known as amelin and sheathlin), has been isolated from rat, mouse and pig. Its human homolog has been mapped to chromosome 4q21 between markers D4S409 and D4S400, flanking the local hypoplastic amelogenesis imperfecta critical region. Therefore, ameloblastin is a strong candidate gene for this form of amelogenesis imperfecta. To facilitate genetic studies related to this dental disease, we isolated and characterized a human ameloblastin cDNA. A human third molar cDNA library was screened and two ameloblastin clones identified. Nucleotide sequencing of these cDNAs indicated alternative splicing of the putative open reading frame, use of different polyadenylation signals, and a high degree of similarity to reported rat, mouse and porcine cDNAs. Immunohistochemistry studies on embryonic human teeth using an antibody to recombinant ameloblastin indicated ameloblastin expression by ameloblasts with localization in the enamel matrix associated with the sheath structures.     

Viral RNA splicing and papillomavirus oncogenesis

Human papillomavirus (HPV) infection is an etiologic cause leading to development of cervical and other ano-genital cancers as well as a subset of oropharyngeal cancers.     

Functional splicing assay of DSPP mutations in hereditary dentin defects

Oral Diseases (2011) 17 , 690–695 Objective: Dentin sialophosphoprotein (DSPP) gene mutations have been identified in isolated hereditary dentin defects; however, the genotype–phenotype correlations are poorly understood. We performed in vitro splicing assays to test the hypothesis that DSPP mutations in splice junctions as well as proposed missense/nonsense mutations experimentally result in aberrant pre‐mRNA splicing. Materials and methods: The genomic fragment of the human DSPP gene was cloned into the pSPL3 splicing vector, and previously reported as well as informative de novo mutations were then introduced by PCR mutagenesis. The COS‐7 cells were transfected with each plasmid vector, and total RNA was isolated. RT‐PCR result was analyzed, and the band intensity of the product was calibrated using ImageJ. Results: The predictions by others of exon 3 skipping in specific DSPP mutations have been validated and a cryptic splicing donor site has been identified. However, the degree of mutational effect on pre‐mRNA splicing varied considerably depending on the changed nucleotide. Conclusions: The predictions of exon 3 skipping in specific DSPP mutations have been validated, and a cryptic splicing donor site has been identified. Our data may provide insight into the contribution of DSPP mutations in the pathogenesis and genotype–phenotype correlations of hereditary dentin defects.     

The role of mRNA splicing in prostate cancer

Alternative splicing （AS） is a crucial step in gene expression. It is subject to intricate regulation, and its deregulation in cancer can lead to a wide array of neoplastic phenotypes. A large body of evidence implicates splice isoforms in most if not all hallmarks of cancer, including growth, apoptosis, invasion and metastasis, angiogenesis, and metabolism. AS has important clinical implications since it can be manipulated therapeutically to treat cancer and represents a mechanism of resistance to therapy. In prostate cancer （PCa） AS also plays a prominent role and this review will summarize the current knowledge of alternatively spliced genes with important functional consequences. We will highlight accumulating evidence on AS of the components of the two critical pathways in PCa： androgen receptor （AR） and phosphoinositide 3-kinase （PI3K）. These observations together with data on dysregulation of splice factors in PCa suggest that AR and PI3K pathways may be interconnected with previously unappreciated splicing regulatory networks. In addition, we will discuss several lines of evidence implicating splicing regulation in the development of the castration resistance.