CD44 is a hyaluronan binding cell surface signal transducing receptor that influences motility, cell survival and proliferation as well as the formation of tumor microenvironment. CD44 contains two variable regions encoded by variable exons. Alternative splicing, which is often deregulated in cancer, can produce various isoforms of CD44 with properties that may have different tissue specific effects and therefore even diverse effects on cancer progression. This review summarizes and puts together all major regulators of alternative splicing of CD44 in cancer that have been documented so far and that have an experimentally proved effect on CD44 isoform switching. It is important to better understand the mechanisms of alternative splicing of CD44, where all the variability of CD44 originates, to be able to explain the isoform switching and occurrence of variant isoforms of CD44 (CD44v) in cancer.
- MeSH
- Alternative Splicing MeSH
- Hyaluronan Receptors genetics metabolism MeSH
- Epithelial-Mesenchymal Transition MeSH
- Humans MeSH
- Neoplastic Stem Cells metabolism MeSH
- Neoplasms metabolism pathology MeSH
- Protein Isoforms genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The DNA damage checkpoint kinase, CHK2, promotes growth arrest or apoptosis through phosphorylating targets such as Cdc25A, Cdc25C, BRCA1, and p53. Both germline and somatic loss-of-function CHEK2 mutations occur in human tumours, the former linked to the Li-Fraumeni syndrome, and the latter found in diverse types of sporadic malignancies. Here we examined the status of CHK2 by genetic and immunohistochemical analyses in 53 breast carcinomas previously characterized for TP53 status. We identified two CHEK2 mutants, 470T>C (Ile157Thr), and a novel mutation, 1368insA leading to a premature stop codon in exon 13. The truncated protein encoded by CHEK2 carrying the 1368insA was stable yet mislocalized to the cytoplasm in tumour sections and when ectopically expressed in cultured cells. Unexpectedly, we found CHEK2 to be subject to extensive alternative splicing, with some 90 splice variants detected in our tumour series. While all cancers expressed normal-length CHEK2 mRNA together with the spliced transcripts, we demonstrate and/or predict some of these splice variants to lack CHK2 function and/or localize aberrantly. We conclude that cytoplasmic sequestration may represent a novel mechanism to disable CHK2, and propose to further explore the significance of the complex splicing patterns of this tumour suppressor gene in oncogenesis.
- MeSH
- Alternative Splicing * MeSH
- Checkpoint Kinase 2 MeSH
- Immunohistochemistry MeSH
- Humans MeSH
- Molecular Sequence Data MeSH
- Mutation * MeSH
- DNA Mutational Analysis MeSH
- Breast Neoplasms * genetics metabolism MeSH
- Polymerase Chain Reaction MeSH
- Protein Serine-Threonine Kinases * genetics metabolism MeSH
- Base Sequence MeSH
- Check Tag
- Humans MeSH
- Female MeSH
Mutations in the C1 inhibitor (C1INH) encoding gene, SERPING1, are associated with hereditary angioedema (HAE) which manifests as recurrent submucosal and subcutaneous edema episodes. The major C1INH function is the complement system inhibition, preventing its spontaneous activation. The presented study is focused on SERPING1 exon 3, an alternative and extraordinarily long exon (499 bp). Endogenous expression analysis performed in the HepG2, human liver, and human peripheral blood cells revealed several exon 3 splicing variants alongside exon inclusion: a highly prevalent exon skipping variant and less frequent +38 and -15 variants with alternative 3' splice sites (ss) located 38 and 15 nucleotides downstream and upstream from the authentic 3' ss, respectively. An exon skipping variant introducing a premature stop codon, represented nearly one third of all splicing variants and surprisingly appeared not to be degraded by NMD. The alternative -15 3' ss was used to a small extent, although predicted to be extremely weak. Its use was shown to be independent of its strength and highly sensitive to any changes in the surrounding sequence. -15 3' ss seems to be co-regulated with the authentic 3' ss, whose use is dependent mainly on its strength and less on the presence of intronic regulatory motifs. Subtle SERPING1 exon 3 splicing regulation can contribute to overall C1INH plasma levels and HAE pathogenesis.
- MeSH
- Alternative Splicing genetics MeSH
- Cell Nucleus genetics MeSH
- Hep G2 Cells MeSH
- Exons genetics MeSH
- Complement C1 Inhibitor Protein genetics MeSH
- Humans MeSH
- RNA, Small Interfering metabolism MeSH
- RNA Splice Sites genetics MeSH
- Mutation genetics MeSH
- Nonsense Mediated mRNA Decay genetics MeSH
- Base Sequence MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND/AIM: Colorectal cancer is currently the third leading cause of cancer-related deaths and recently, alternative splicing has risen as its important regulator and potential treatment target. In the present study, we analyzed gene expression of the MBNL family of regulators of alternative splicing in various stages of colorectal cancer development, together with the MBNL-target splicing events in FOXP1 and EPB41L3 genes and tumor-related CD44 variants. MATERIALS AND METHODS: Samples of tumor tissue and non-malignant mucosa from 108 patients were collected. After RNA isolation and reverse transcription, the relative gene expression of a selected gene panel was tested by quantitative real-time PCR, followed by statistical analysis. RESULTS: MBNL expression was decreased in tumor tissue compared to non-tumor mucosa. In addition, lower expression was observed for the variants of FOXP1 and EPB41L3, while higher expression in tumor tissue was detected both for total CD44 and its cancer-related variants 3 and 6. Transcript levels of the MBNL genes were not found to be related to any of the studied clinicopathological characteristics. Multiple significant associations were identified in the target gene panel, including higher transcript levels of FOXP1 and CD44v3 in patients with distant metastases and connections between recurrence-free survival and altered levels of FOXP1 and CD44v3. CONCLUSION: Our results identified for the first-time deregulation of MBNL genes in colorectal cancer. Down-regulation of their transcripts in tumor tissue compared to matched non-tumor mucosa can lead to transition of alternative splicing patterns towards a less differentiated phenotype, which highlights the importance of alternative splicing regulation for tumor growth and propagation.
- MeSH
- Alternative Splicing MeSH
- Cell Differentiation physiology MeSH
- Adult MeSH
- Colorectal Neoplasms genetics metabolism pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- RNA-Binding Proteins biosynthesis genetics MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
There is increasing evidence to suggest that splicing decisions are largely made when the nascent RNA is still associated with chromatin. Here we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection. Using splicing-sensitive microarrays, we identified ∼700 genes whose splicing was altered after HDAC inhibition. We provided evidence that HDAC inhibition induced histone H4 acetylation and increased RNA Polymerase II (Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduced co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. We further showed that the depletion of HDAC1 had similar effect on fibronectin alternative splicing as global HDAC inhibition. Importantly, this effect was reversed upon expression of mouse HDAC1 but not a catalytically inactive mutant. These results provide a molecular insight into a complex modulation of splicing by HDACs and chromatin modifications.
- MeSH
- Alternative Splicing drug effects genetics physiology MeSH
- HeLa Cells MeSH
- Histone Deacetylase 1 genetics physiology MeSH
- Histone Deacetylases genetics metabolism physiology MeSH
- Histone Deacetylase Inhibitors pharmacology MeSH
- Nuclear Proteins genetics MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Microarray Analysis MeSH
- Mice MeSH
- RNA-Binding Proteins genetics MeSH
- Gene Expression Regulation drug effects MeSH
- Gene Expression Profiling MeSH
- Transfection MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Histone acetylation modulates alternative splicing of several hundred genes. Here, we tested the role of the histone acetyltransferase p300 in alternative splicing and showed that knockdown of p300 promotes inclusion of the fibronectin (FN1) alternative EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as p300 knockdown. Next we showed that p300 controls histone H4 acetylation along the FN1 gene. Consistently, p300 depletion and CRE deletion/mutation both reduced histone H4 acetylation on mini-gene reporters. Finally, we provide evidence that the effect of CRE inactivation on H4 acetylation and alternative splicing is counteracted by the inhibition of histone deacetylases. Together, these data suggest that histone acetylation could be one of the mechanisms how promoter and promoter binding proteins influence alternative splicing.
- MeSH
- Acetylation MeSH
- Alternative Splicing * MeSH
- Fibronectins genetics metabolism MeSH
- Gene Knockdown Techniques MeSH
- HeLa Cells MeSH
- Histones metabolism MeSH
- Integrases genetics MeSH
- Humans MeSH
- RNA, Messenger metabolism MeSH
- Promoter Regions, Genetic MeSH
- E1A-Associated p300 Protein genetics metabolism MeSH
- Genes, Reporter MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: The PLAUR gene encodes the urokinase-like plasminogen activator receptor (uPAR) and may undergo alternative splicing. Excluding cassette exons 3, 5 and 6 from the transcript results in truncated protein variants whose precise functions have not been elucidated yet. The PLAUR gene is one of several expressed in myeloid cells, where uPAR participates in different cellular processes, including the contact activation system and kallikrein-kinin system, which play an important role in hereditary angioedema (HAE) pathogenesis. A hypothesis about the PLAUR splicing pattern impact on HAE severity was tested. METHODS AND RESULTS: The RT-PCR quantified by capillary electrophoresis was used. Although no significant difference in alternative transcript frequency was observed between healthy volunteers and HAE patients, a significant increase in all cassette exon inclusion variants was revealed during monocyte-to-macrophage differentiation. CONCLUSIONS: PLAUR alternative splicing in monocytes and macrophages neither was different between HAE patients and healthy controls, nor reflected disease severity. However, the results showed an PLAUR splicing pattern was changing during monocyte-to-macrophage differentiation, but the significance of these changes is unknown and awaits future clarification.
- MeSH
- Alternative Splicing genetics MeSH
- Angioedemas, Hereditary * genetics pathology MeSH
- Leukocytes MeSH
- Humans MeSH
- Macrophages pathology MeSH
- Monocytes * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Despite the wide application of nanomaterials, toxicity studies of nanoparticles (NP) are often limited to in vitro cell models, and the biological impact of NP exposure in mammals has not been thoroughly investigated. Zinc oxide (ZnO) NPs are commonly used in various consumer products. To evaluate the effects of the inhalation of ZnO NP in mice, we studied splice junction expression in the lungs as a proxy to gene expression changes analysis. Female ICR mice were treated with 6.46 × 104 and 1.93 × 106 NP/cm3 for 3 days and 3 months, respectively. An analysis of differential expression and alternative splicing events in 298 targets (splice junctions) of 68 genes involved in the processes relevant to the biological effects of ZnO NP was conducted using next-generation sequencing. Three days of exposure resulted in the upregulation of IL-6 and downregulation of BID, GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junction expression; 3 months of exposure increased the expression of splice junctions in ALDH3A1, APAF1, BID, CASP3, DHCR7, GCLC, GCLM, GSR, GSS, EHHADH, FAS, HMOX-1, IFNγ, NF-kB1, NQO-1, PTGS1, PTGS2, RAD51, RIPK2, SRXN1, TRAF6, and TXNRD1. Alternative splicing of TRAF6 and TXNRD1 was induced after 3 days of exposure to 1.93 × 106 NP/cm3. In summary, we observed changes of splice junction expression in genes involved in oxidative stress, apoptosis, immune response, inflammation, and DNA repair, as well as the induction of alternative splicing in genes associated with oxidative stress and inflammation. Our data indicate the potential negative biological effects of ZnO NP inhalation.
- MeSH
- Alternative Splicing drug effects MeSH
- Administration, Inhalation MeSH
- Apoptosis drug effects MeSH
- Immunity, Cellular drug effects MeSH
- Cell Cycle drug effects MeSH
- Gene Expression drug effects MeSH
- Mice, Inbred ICR MeSH
- Mice MeSH
- Nanoparticles toxicity MeSH
- DNA Repair drug effects MeSH
- Zinc Oxide toxicity MeSH
- Oxidative Stress drug effects MeSH
- Lung metabolism pathology MeSH
- Inflammation MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- 5' Untranslated Regions genetics MeSH
- Alternative Splicing * MeSH
- Protein Biosynthesis MeSH
- RNA Splicing * genetics MeSH
- Publication type
- Comment MeSH
- Editorial MeSH
