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1.
PLoS Genet ; 12(1): e1005827, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26824521

ABSTRACT

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an expansion of CUG repeats in the 3' UTR of the DMPK gene. The CUG repeats form aggregates of mutant mRNA, which cause misregulation and/or sequestration of RNA-binding proteins, causing aberrant alternative splicing in cells. Previously, we showed that the multi-functional RNA-binding protein Staufen1 (Stau1) was increased in skeletal muscle of DM1 mouse models and patients. We also showed that Stau1 rescues the alternative splicing profile of pre-mRNAs, e.g. the INSR and CLC1, known to be aberrantly spliced in DM1. In order to explore further the potential of Stau1 as a therapeutic target for DM1, we first investigated the mechanism by which Stau1 regulates pre-mRNA alternative splicing. We report here that Stau1 regulates the alternative splicing of exon 11 of the human INSR via binding to Alu elements located in intron 10. Additionally, using a high-throughput RT-PCR screen, we have identified numerous Stau1-regulated alternative splicing events in both WT and DM1 myoblasts. A number of these aberrant ASEs in DM1, including INSR exon 11, are rescued by overexpression of Stau1. However, we find other ASEs in DM1 cells, where overexpression of Stau1 shifts the splicing patterns away from WT conditions. Moreover, we uncovered that Stau1-regulated ASEs harbour Alu elements in intronic regions flanking the alternative exon more than non-Stau1 targets. Taken together, these data highlight the broad impact of Stau1 as a splicing regulator and suggest that Stau1 may act as a disease modifier in DM1.


Subject(s)
Alternative Splicing/genetics , Cytoskeletal Proteins/genetics , Myotonin-Protein Kinase/genetics , RNA-Binding Proteins/genetics , Trinucleotide Repeat Expansion/genetics , 3' Untranslated Regions , Alu Elements/genetics , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cytoskeletal Proteins/metabolism , Humans , Mice , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myoblasts/metabolism , Myoblasts/pathology , Myotonic Dystrophy , Myotonin-Protein Kinase/metabolism , Protein Binding , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism
2.
J Immunol ; 197(9): 3618-3627, 2016 11 01.
Article in English | MEDLINE | ID: mdl-27664281

ABSTRACT

Diverse signals received by CD8+ T cells are integrated to achieve the required magnitude of cell expansion and the appropriate balance of effector/memory CD8+ T cell generation. Notably, the strength and nature of TCR signaling influence the differentiation and functional capacity of effector and memory CD8+ T cells. Dok-1 and Dok-2, the two members of the Dok family expressed in T cells, negatively regulate TCR signaling in vitro. However, the role of Dok proteins in modulating T cell function in vivo has not yet studied. We studied the function of Dok-1 and Dok-2 proteins in the regulation of the CD8+ T cell response to vaccinia virus infection. Comparison of responses to vaccinia virus expressing OVA peptide SIINFEKL by wild-type and Dok-1/2-/- CD8+ OT-I cells showed that the absence of Dok-1 and Dok-2 slightly reduced the magnitude of virus-specific effector CD8+ T cell expansion. This was not due to reduced proliferation or enhanced apoptosis of effector CD8+ T cells. Dok-1/2-deficient effector CD8+ T cells showed increased cell surface TCR expression following virus infection in vivo and increased expression of granzyme B and TNF upon stimulation with peptide Ag ex vivo. Finally, Dok-1/2-deficient effector CD8+ T had a severe defect in survival that resulted in impaired generation of memory CD8+ T cells. These results reveal the critical involvement of Dok-1 and Dok-2 in a negative-feedback loop that prevents overactivation of CD8+ T cells and promotes memory formation.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , CD8-Positive T-Lymphocytes/immunology , DNA-Binding Proteins/metabolism , Immunologic Memory , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Vaccinia/immunology , Virus Diseases/immunology , Adaptor Proteins, Signal Transducing/genetics , Animals , CD8-Positive T-Lymphocytes/virology , Cell Differentiation , Cell Survival , Cells, Cultured , DNA-Binding Proteins/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphoproteins/genetics , RNA-Binding Proteins/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Signal Transduction
3.
BMC Mol Biol ; 18(1): 19, 2017 07 20.
Article in English | MEDLINE | ID: mdl-28728573

ABSTRACT

BACKGROUND: RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform. RESULTS: RBM10 knockdown (KD) provoked alterations in splicing events in 10-20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity. CONCLUSION: Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.


Subject(s)
RNA Precursors/genetics , RNA Splicing , RNA-Binding Proteins/metabolism , Alternative Splicing , Cell Line , Cluster Analysis , Computational Biology/methods , Exons , Fibroblasts , Gene Expression Profiling , Humans , Reproducibility of Results , Signal Transduction , Survival of Motor Neuron 2 Protein/genetics , ras Proteins/metabolism
4.
Nucleic Acids Res ; 43(3): 1869-82, 2015 Feb 18.
Article in English | MEDLINE | ID: mdl-25605796

ABSTRACT

Alternative splicing is the main source of proteome diversity. Here, we have investigated how alternative splicing affects the function of two human histone methyltransferases (HMTase): G9A and SUV39H2. We show that exon 10 in G9A and exon 3 in SUV39H2 are alternatively included in a variety of tissues and cell lines, as well as in a different species. The production of these variants is likely tightly regulated because both constitutive and alternative splicing factors control their splicing profiles. Based on this evidence, we have assessed the link between the inclusion of these exons and the activity of both enzymes. We document that these HMTase genes yield several protein isoforms, which are likely issued from alternative splicing regulation. We demonstrate that inclusion of SUV39H2 exon 3 is a determinant of the stability, the sub-nuclear localization, and the HMTase activity. Genome-wide expression analysis further revealed that alternative inclusion of SUV39H2 exon 3 differentially modulates the expression of target genes. Our data also suggest that a variant of G9A may display a function that is independent of H3K9 methylation. Our work emphasizes that expression and function of genes are not collinear; therefore alternative splicing must be taken into account in any functional study.


Subject(s)
Alternative Splicing , DNA Modification Methylases/genetics , Cell Line , DNA Modification Methylases/metabolism , Humans
5.
RNA ; 20(2): 189-201, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24335142

ABSTRACT

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.


Subject(s)
Alternative Splicing , Ovarian Neoplasms/metabolism , RNA, Messenger/genetics , Cell Line, Tumor , Epithelial Cells/metabolism , Female , Gene Expression , Humans , Laser Capture Microdissection , Organ Specificity , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Splice Sites , RNA Splicing Factors , RNA, Messenger/metabolism , RNA-Binding Proteins/physiology , Repressor Proteins/physiology , Stromal Cells/metabolism , Tumor Microenvironment
6.
BMC Cancer ; 15: 227, 2015 Apr 07.
Article in English | MEDLINE | ID: mdl-25884497

ABSTRACT

BACKGROUND: Modification of splicing by chemotherapeutic drugs has usually been evaluated on a limited number of pre-mRNAs selected for their recognized or potential importance in cell proliferation or apoptosis. However, the pathways linking splicing alterations to the efficiency of cancer therapy remain unclear. METHODS: Next-generation sequencing was used to analyse the transcriptome of breast carcinoma cells treated by cisplatin. Pharmacological inhibitors, RNA interference, cells deficient in specific signalling pathways, RT-PCR and FACS analysis were used to investigate how the anti-cancer drug cisplatin affected alternative splicing and the cell death pathway. RESULTS: We identified 717 splicing events affected by cisplatin, including 245 events involving cassette exons. Gene ontology analysis indicates that cell cycle, mRNA processing and pre-mRNA splicing were the main pathways affected. Importantly, the cisplatin-induced splicing alterations required class I PI3Ks P110ß but not components such as ATM, ATR and p53 that are involved in the DNA damage response. The siRNA-mediated depletion of the splicing regulator SRSF4, but not SRSF6, expression abrogated many of the splicing alterations as well as cell death induced by cisplatin. CONCLUSION: Many of the splicing alterations induced by cisplatin are caused by SRSF4 and they contribute to apoptosis in a process requires class I PI3K.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cisplatin/pharmacology , RNA Precursors/genetics , RNA Splicing/drug effects , RNA-Binding Proteins/metabolism , Cell Line, Tumor , Computational Biology , DNA Damage , Gene Expression Regulation, Neoplastic/drug effects , Humans , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , RNA Processing, Post-Transcriptional/drug effects , Serine-Arginine Splicing Factors , Signal Transduction/drug effects , Transcription Factors/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
7.
BMC Med Genet ; 15: 98, 2014 Aug 14.
Article in English | MEDLINE | ID: mdl-25115182

ABSTRACT

BACKGROUND: Mutations in the SQSTM1/p62 gene have been reported in Paget's disease of bone (PDB), but they are not sufficient to induce the pagetic osteoclast (OC) phenotype. We hypothesized that specific RNA isoforms of OC-related genes may contribute to the overactivity of pagetic OCs, along with other genetic predisposing factors. METHODS: Alternative splicing (AS) events were studied using a PCR-based screening strategy in OC cultures from 29 patients with PDB and 26 healthy donors (HD), all genotyped for the p62P392L mutation. Primer pairs targeting 5223 characterized AS events were used to analyze relative isoform ratios on pooled cDNA from samples of the four groups (PDB, PDBP392L, HD, HDP392L). Of the 1056 active AS events detected in the screening analysis, 192 were re-analyzed on non-amplified cDNA from each subject of the whole cohort. RESULTS: This analysis led to the identification of six AS events significantly associated with PDB, but none with p62P392L. The corresponding genes included LGALS8, RHOT1, CASC4, USP4, TBC1D25, and PIDD. In addition, RHOT1 and LGALS8 genes were upregulated in pagetic OCs, as were CASC4 and RHOT1 genes in the presence of p62P392L. Finally, we showed that the proteins encoded by LGALS8, RHOT1, USP4, TBC1D25, and PIDD were expressed in human OCs. CONCLUSION: This study allowed the identification of hitherto unknown players in OC biology, and our findings of a differential AS in pagetic OCs may generate new concepts in the pathogenesis of PDB.


Subject(s)
Alternative Splicing , Mutation , Osteitis Deformans/genetics , Osteoclasts/pathology , Adaptor Proteins, Signal Transducing/genetics , Cells, Cultured , Female , Gene Expression Profiling , Gene Expression Regulation , Genotype , Humans , Male , Osteitis Deformans/pathology , RNA/analysis , Sequestosome-1 Protein
8.
Hum Mol Genet ; 20(11): 2116-30, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-21378395

ABSTRACT

Proteins PRPF31, PRPF3 and PRPF8 (RP-PRPFs) are ubiquitously expressed components of the spliceosome, a macromolecular complex that processes nearly all pre-mRNAs. Although these spliceosomal proteins are conserved in eukaryotes and are essential for survival, heterozygous mutations in human RP-PRPF genes lead to retinitis pigmentosa, a hereditary disease restricted to the eye. Using cells from patients with 10 different mutations, we show that all clinically relevant RP-PRPF defects affect the stoichiometry of spliceosomal small nuclear RNAs (snRNAs), the protein composition of tri-small nuclear ribonucleoproteins and the kinetics of spliceosome assembly. These mutations cause inefficient splicing in vitro and affect constitutive splicing ex-vivo by impairing the removal of at least 9% of endogenously expressed introns. Alternative splicing choices are also affected when RP-PRPF defects are present. Furthermore, we show that the steady-state levels of snRNAs and processed pre-mRNAs are highest in the retina, indicating a particularly elevated splicing activity. Our results suggest a role for PRPFs defects in the etiology of PRPF-linked retinitis pigmentosa, which appears to be a truly systemic splicing disease. Although these mutations cause widespread and important splicing defects, they are likely tolerated by the majority of human tissues but are critical for retinal cell survival.


Subject(s)
RNA Precursors/genetics , RNA Splicing/genetics , RNA, Small Nuclear/genetics , Retinitis Pigmentosa/genetics , Spliceosomes/pathology , Alternative Splicing , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Eye Proteins/genetics , Eye Proteins/metabolism , Female , Genes, Dominant , Heterozygote , Humans , Introns , Male , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA-Binding Proteins , Retina/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear/genetics , Ribonucleoprotein, U4-U6 Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Spliceosomes/metabolism
9.
Eur J Immunol ; 42(9): 2491-504, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22736313

ABSTRACT

In T cells, two members of the Dok family, Dok-1 and Dok-2, are predominantly expressed. Recent evidence suggests that they play a negative role in T-cell signaling. In order to define whether Dok proteins regulate T-cell development, we have generated transgenic mice overexpressing Dok-1 in thymocytes and peripheral T cells. We show that overexpression of Dok-1 retards the transition from the CD4(-) CD8(-) to CD4(+) CD8(+) stage. Moreover, there is a specific expansion of PLZF-expressing Vγ1.1(+) Vδ6.3(+) T cells. This subset of γδ T cells acquires innate characteristics including rapid IL-4 production following stimulation and requiring SLAM-associated adaptor protein (SAP) for their development. Moreover, Dok-1 overexpression promotes the generation of an innate-like CD8(+) T-cell population that expresses Eomesodermin. Altogether, these findings identify a novel role for Dok-1 in the regulation of thymic differentiation and in particular, in the development of PLZF(+) γδ T cells.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Natural Killer T-Cells/metabolism , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/genetics , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Animals , DNA-Binding Proteins/metabolism , Female , Intracellular Signaling Peptides and Proteins/metabolism , Kruppel-Like Transcription Factors/biosynthesis , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphoproteins/metabolism , Promyelocytic Leukemia Zinc Finger Protein , RNA-Binding Proteins/metabolism , Signaling Lymphocytic Activation Molecule Associated Protein
10.
Mod Pathol ; 26(11): 1413-24, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23743930

ABSTRACT

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor's biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and <20% necrosis for genomic isolation. We validated our protocol in 40 patients who participated in a biopsy-driven study of therapeutic resistance in metastatic colorectal cancer. To ensure that our protocol was compatible with multiplex discovery platforms and that no component of the processing interfered with downstream enzymatic reactions, we performed array comparative genomic hybridization, methylation profiling, microRNA profiling, splicing variant analysis and gene expression profiling using genomic material isolated from liver biopsy cores. Our standard operating procedures for next-generation biobanking can be applied widely in multiple settings, including multicentered and international biopsy-driven trials.


Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Genetic Testing , Liver Neoplasms/genetics , Liver Neoplasms/secondary , Precision Medicine , Tissue Banks , Alternative Splicing , Biopsy, Large-Core Needle , Canada , Comparative Genomic Hybridization , DNA Methylation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genetic Predisposition to Disease , Genetic Testing/methods , High-Throughput Nucleotide Sequencing , Humans , MicroRNAs/analysis , Oligonucleotide Array Sequence Analysis , Patient Selection , Phenotype , Precision Medicine/methods , Predictive Value of Tests , Prognosis , Reproducibility of Results , Specimen Handling , Workflow
11.
BMC Cancer ; 11: 285, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21722370

ABSTRACT

BACKGROUND: Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. METHODS: Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. RESULTS: Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher levels of a splice variant of DR3 that has no trans-membrane domain and no death domain. CONCLUSION: Colon cancer cells acquire an increased capacity to survive via the activation of the PI3K/NFκB pathway following the stimulation of DR3 by E-selectin. Generation of a DR3 splice variant devoid of death domain can further contribute to protect against apoptosis.


Subject(s)
Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , E-Selectin/metabolism , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Tumor Necrosis Factor, Member 25/metabolism , Amino Acid Sequence , Apoptosis/physiology , Cell Adhesion , Cell Survival/physiology , Chromones/pharmacology , HT29 Cells , Human Umbilical Vein Endothelial Cells , Humans , Jurkat Cells , MAP Kinase Signaling System , Microscopy, Fluorescence , Molecular Sequence Data , Morpholines/pharmacology , Neoplasm Metastasis , Phosphorylation , Protein Isoforms , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Tumor Necrosis Factor, Member 25/chemistry , Receptors, Tumor Necrosis Factor, Member 25/genetics , src-Family Kinases/metabolism
12.
Nucleic Acids Res ; 36(10): 3320-31, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18440980

ABSTRACT

Alternative splicing of a single pre-mRNA transcript can produce protein isoforms that promote either cell growth or death. Here we show that Ro-31-8220 (Ro), an apoptotic agent that inhibits protein kinase C and activates the c-Jun N terminal kinase, decreased the proportion of the cell growth-promoting Bcl-xL splice variant. Targeted mutagenesis analyses narrowed down a critical sequence to a 16-nt G-tract element (Gt16). Transferring this element to a heterologous gene conferred Ro response on an otherwise constitutive exon. The Ro effect was reduced by okadaic acid, an inhibitor of protein phosphatases PP1 and PP2A, in a concentration-dependent manner. Search in the human genome followed by RT-PCR identified a group of genes that contain similar exonic G-tract elements and are responsive to Ro. Moreover, the Gt16 element also mediates the regulation of alternative splicing by other cell apoptosis-inducers particularly retinoic acid. Therefore, the G-tract element likely plays a role in the apoptotic agents-induced alternative splicing of a group of genes. The functions of these genes imply that this regulation will have impact on cell growth/death.


Subject(s)
Alternative Splicing , Apoptosis , RNA Precursors/chemistry , RNA, Messenger/chemistry , Regulatory Sequences, Ribonucleic Acid , bcl-X Protein/genetics , Alternative Splicing/drug effects , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Guanine/analysis , Humans , Indoles/pharmacology , Okadaic Acid/pharmacology , Phosphoric Monoester Hydrolases/antagonists & inhibitors , bcl-X Protein/metabolism
13.
BMC Biotechnol ; 6: 5, 2006 Jan 13.
Article in English | MEDLINE | ID: mdl-16412215

ABSTRACT

BACKGROUND: We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. RESULTS: We show that an oligonucleotide with a 5' tail containing the human beta-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. CONCLUSION: Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.


Subject(s)
Alternative Splicing/genetics , Gene Silencing , Genetic Engineering/methods , Oligonucleotides, Antisense/genetics , RNA Splice Sites/genetics , Gene Expression Regulation/genetics , Genetic Variation/genetics
14.
FEBS Lett ; 579(21): 4873-8, 2005 Aug 29.
Article in English | MEDLINE | ID: mdl-16111683

ABSTRACT

An increasing number of peptides translocate the plasma membrane of mammalian cells promising new avenues for drug delivery. However, only a few examples are known to penetrate the fungal cell wall. We compared the capacity of different fluorophore-labelled peptides to translocate into fission yeast and human cells and determined their intracellular distribution. Most of the 20 peptides tested were able to enter human cells, but only one, transportan 10 (TP10), efficiently penetrated fission yeast and was distributed uniformly inside the cells. The results show that the fungal cell wall may reduce, but does not block peptide uptake.


Subject(s)
Endocytosis/physiology , Peptides/metabolism , Recombinant Fusion Proteins/metabolism , Schizosaccharomyces/metabolism , Cell Wall/metabolism , Drug Carriers , Fluorescent Dyes/metabolism , Galanin , HeLa Cells , Humans , Schizosaccharomyces/cytology , Wasp Venoms
15.
Chem Biol ; 10(8): 769-78, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12954336

ABSTRACT

Thiostrepton and micrococcin inhibit protein synthesis by binding to the L11 binding domain (L11BD) of 23S ribosomal RNA. The two compounds are structurally related, yet they produce different effects on ribosomal RNA in footprinting experiments and on elongation factor-G (EF-G)-dependent GTP hydrolysis. Using NMR and an assay based on A1067 methylation by thiostrepton-resistance methyltransferase, we show that the related thiazoles, nosiheptide and siomycin, also bind to this region. The effect of all four antibiotics on EF-G-dependent GTP hydrolysis and EF-G-GDP-ribosome complex formation was studied. Our NMR and biochemical data demonstrate that thiostrepton, nosiheptide, and siomycin share a common profile, which differs from that of micrococcin. We have generated a three-dimensional (3D) model for the interaction of thiostrepton with L11BD RNA. The model rationalizes the differences between micrococcin and the thiostrepton-like antibiotics interacting with L11BD.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Ribosomes/metabolism , Thiazoles/metabolism , Anti-Bacterial Agents/chemical synthesis , Bacteriocins , Base Sequence , Binding Sites , Guanosine Triphosphate/metabolism , Hydrolysis , Magnetic Resonance Spectroscopy , Methylation , Methyltransferases/antagonists & inhibitors , Methyltransferases/metabolism , Methyltransferases/pharmacology , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Protein Binding , RNA, Ribosomal, 23S/drug effects , RNA, Ribosomal, 23S/metabolism , Thiazoles/chemistry , Thiostrepton/chemistry , Thiostrepton/metabolism
16.
Sci Rep ; 5: 14301, 2015 Sep 22.
Article in English | MEDLINE | ID: mdl-26391193

ABSTRACT

The SOCS1 (Suppressor Of Cytokine Signalling 1) protein is considered a tumour suppressor. Notably, the SOCS1 gene is frequently silenced in cancer by hypermethylation of its promoter. Besides blocking inflammation, SOCS1 tumour suppressor activity involves Met receptor inhibition and enhancement of p53 tumour suppressor activity. However, the role of SOCS1 in colorectal cancer (CRC) remains understudied and controversial. Here, we investigated SOCS1 relevance for CRC by querying gene expression datasets of human CRC specimens from The Cancer Genome Atlas (TCGA), and by SOCS1 gain/loss-of-function analyses in murine and human colon carcinoma cells. Our results show that SOCS1 mRNA levels in tumours were more often elevated than reduced with respect to matched adjacent normal tissue of CRC specimens (n = 41). The analysis of TCGA dataset of 431 CRC patients revealed no correlation between SOCS1 expression and overall survival. Overexpression of SOCS1 in CRC cells triggered cell growth enhancement, anchorage-independent growth and resistance to death stimuli, whereas knockdown of SOCS1 reduced these oncogenic features. Moreover, SOCS1 overexpression in mouse CT26 cells increased tumourigenesis in vivo. Biochemical analyses showed that SOCS1 pro-oncogenic activity correlated with the down-modulation of STAT1 expression. Collectively, these results suggest that SOCS1 may work as an oncogene in CRC.


Subject(s)
Cell Transformation, Neoplastic/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Suppressor of Cytokine Signaling Proteins/genetics , Aged , Aged, 80 and over , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic/metabolism , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/mortality , Disease Models, Animal , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Hepatocyte Growth Factor/metabolism , Humans , Interferon-gamma/metabolism , Male , Mice , Middle Aged , Neoplasm Grading , Neoplasm Metastasis , Neoplasm Staging , Prognosis , RNA, Messenger/genetics , STAT1 Transcription Factor/metabolism , Signal Transduction , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Up-Regulation
17.
PLoS One ; 9(9): e107324, 2014.
Article in English | MEDLINE | ID: mdl-25211016

ABSTRACT

With the goal of identifying splicing alterations in myotonic dystrophy 1 (DM1) tissues that may yield insights into targets or mechanisms, we have surveyed mis-splicing events in three systems using a RT-PCR screening and validation platform. First, a transgenic mouse model expressing CUG-repeats identified splicing alterations shared with other mouse models of DM1. Second, using cell cultures from human embryonic muscle, we noted that DM1-associated splicing alterations were significantly enriched in cytoskeleton (e.g. SORBS1, TACC2, TTN, ACTN1 and DMD) and channel (e.g. KCND3 and TRPM4) genes. Third, of the splicing alterations occurring in adult DM1 tissues, one produced a dominant negative variant of the splicing regulator RBFOX1. Notably, half of the splicing events controlled by MBNL1 were co-regulated by RBFOX1, and several events in this category were mis-spliced in DM1 tissues. Our results suggest that reduced RBFOX1 activity in DM1 tissues may amplify several of the splicing alterations caused by the deficiency in MBNL1.


Subject(s)
Muscle, Skeletal/metabolism , Myotonic Dystrophy/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/physiology , Adult , Alternative Splicing , Animals , Cell Line , Female , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Myoblasts/metabolism , Myotonic Dystrophy/genetics , RNA Splicing Factors , RNA-Binding Proteins/metabolism
18.
Nat Commun ; 5: 4760, 2014 Sep 11.
Article in English | MEDLINE | ID: mdl-25208576

ABSTRACT

Alternative splicing--the production of multiple messenger RNA isoforms from a single gene--is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2ß have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous--but not individual--depletion of Tra2α and Tra2ß induces substantial shifts in splicing of endogenous Tra2ß target exons, and that both constitutive and alternative target exons are under dual Tra2α-Tra2ß control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability.


Subject(s)
Alternative Splicing , Exons , Nerve Tissue Proteins/metabolism , Protein Kinases/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Cell Line, Tumor , Checkpoint Kinase 1 , Humans , MCF-7 Cells , Protein Kinases/metabolism , Serine-Arginine Splicing Factors
19.
Nat Commun ; 4: 2480, 2013.
Article in English | MEDLINE | ID: mdl-24048253

ABSTRACT

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has provided huge insight into the pathways, mechanisms and transcription factors that control differentiation. Here we use high-throughput RT-PCR technology to take a snapshot of splicing changes in the full spectrum of high- and low-expressed genes during induction of fibroblasts, from several donors, into iPSCs and their subsequent redifferentiation. We uncover a programme of concerted alternative splicing changes involved in late mesoderm differentiation and controlled by key splicing regulators MBNL1 and RBFOX2. These critical splicing adjustments arise early in vertebrate evolution and remain fixed in at least 10 genes (including PLOD2, CLSTN1, ATP2A1, PALM, ITGA6, KIF13A, FMNL3, PPIP5K1, MARK2 and FNIP1), implying that vertebrates require alternative splicing to fully implement the instructions of transcriptional control networks.


Subject(s)
Alternative Splicing , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Induced Pluripotent Stem Cells/metabolism , RNA-Binding Proteins/genetics , Repressor Proteins/genetics , Cell Differentiation , Cells, Cultured , Cellular Reprogramming/genetics , Fibroblasts/cytology , Gene Expression Profiling , Gene Regulatory Networks , Humans , Induced Pluripotent Stem Cells/cytology , Infant, Newborn , Mesoderm/cytology , Mesoderm/growth & development , Mesoderm/metabolism , Protein Binding , RNA Splicing Factors , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Signal Transduction
20.
Mol Cell Biol ; 33(2): 396-405, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23149937

ABSTRACT

Alternative splicing provides a critical and flexible layer of regulation intervening in many biological processes to regulate the diversity of proteins and impact cell phenotype. To identify alternative splicing differences that distinguish epithelial from mesenchymal tissues, we have investigated hundreds of cassette exons using a high-throughput reverse transcription-PCR (RT-PCR) platform. Extensive changes in splicing were noted between epithelial and mesenchymal tissues in both human colon and ovarian tissues, with many changes from mostly one splice variant to predominantly the other. Remarkably, many of the splicing differences that distinguish normal mesenchymal from normal epithelial tissues matched those that differentiate normal ovarian tissues from ovarian cancer. Furthermore, because splicing profiling could classify cancer cell lines according to their epithelial/mesenchymal characteristics, we used these cancer cell lines to identify regulators for these specific splicing signatures. By knocking down 78 potential splicing factors in five cell lines, we provide an extensive view of the complex regulatory landscape associated with the epithelial and mesenchymal states, thus revealing that RBFOX2 is an important driver of mesenchymal tissue-specific splicing.


Subject(s)
Alternative Splicing , Gene Expression Regulation , Mesenchymal Stem Cells/metabolism , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Cell Line, Tumor , Computational Biology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Exons , Fetus/cytology , Fetus/metabolism , Gene Expression Profiling , HeLa Cells , Humans , Mesenchymal Stem Cells/cytology , RNA Interference , RNA Splicing Factors , RNA-Binding Proteins/genetics , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction
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