Publications by authors named "Roscoe Klinck"

29 Publications

  • Page 1 of 1

Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA.

BMC Mol Biol 2017 07 20;18(1):19. Epub 2017 Jul 20.

Département de Microbiologie et d'infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12867-017-0096-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520337PMC
July 2017

Dok-1 and Dok-2 Regulate the Formation of Memory CD8+ T Cells.

J Immunol 2016 11 23;197(9):3618-3627. Epub 2016 Sep 23.

Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Quebec H7V 1B7, Canada;

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1600385DOI Listing
November 2016

Staufen1 Regulates Multiple Alternative Splicing Events either Positively or Negatively in DM1 Indicating Its Role as a Disease Modifier.

PLoS Genet 2016 Jan 29;12(1):e1005827. Epub 2016 Jan 29.

Department of Cellular and Molecular Medicine, University of Ottawa; Centre for Neuromuscular Disease, Ottawa, Ontario, Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1005827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733145PMC
January 2016

Tumour-promoting role of SOCS1 in colorectal cancer cells.

Sci Rep 2015 Sep 22;5:14301. Epub 2015 Sep 22.

Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, J1E 4K8, Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep14301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585755PMC
September 2015

Role of the splicing factor SRSF4 in cisplatin-induced modifications of pre-mRNA splicing and apoptosis.

BMC Cancer 2015 Apr 7;15:227. Epub 2015 Apr 7.

Laboratory of Connective Tissues Biology, GIGA-Cancer, University of Liège, avenue de l'Hôpital 1, 4000, Liège, Belgium.

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 P110β 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12885-015-1259-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399393PMC
April 2015

Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions.

Nucleic Acids Res 2015 Feb 20;43(3):1869-82. Epub 2015 Jan 20.

Institut Pasteur, Département de Biologie du Développement et Cellules Souches, CNRS URA2578, Unité de Régulation Epigénétique, 25 rue du Docteur Roux, Paris, 75015, France

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330376PMC
February 2015

RBFOX1 cooperates with MBNL1 to control splicing in muscle, including events altered in myotonic dystrophy type 1.

PLoS One 2014 11;9(9):e107324. Epub 2014 Sep 11.

Department of Microbiology and Infectiology, Faculty of Medicine and Heath Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada; Laboratory of Functional Genomics, Faculty of Medicine and Heath Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107324PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161394PMC
October 2015

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons.

Nat Commun 2014 Sep 11;5:4760. Epub 2014 Sep 11.

Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle NE1 3BZ, UK.

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 Tra2β 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 Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α-Tra2β 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms5760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175592PMC
September 2014

Alternative splicing in osteoclasts and Paget's disease of bone.

BMC Med Genet 2014 Aug 14;15:98. Epub 2014 Aug 14.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12881-014-0098-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143580PMC
August 2014

Tumor microenvironment-associated modifications of alternative splicing.

RNA 2014 Feb 11;20(2):189-201. Epub 2013 Dec 11.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.042168.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895271PMC
February 2014

MBNL1 and RBFOX2 cooperate to establish a splicing programme involved in pluripotent stem cell differentiation.

Nat Commun 2013 ;4:2480

1] CNRS, Institut de Génétique Moléculaire de Montpellier, Montpellier F-34293, France [2] Institute of Genetic Medicine, Newcastle University, Newcastle-upon-Tyne NE1 3BZ, UK [3] Université Montpellier 2, F-34293 Montpellier cedex 5, France [4] Université Montpellier 1, F-34094 Montpellier cedex 5, France [5].

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms3480DOI Listing
April 2014

Next-generation biobanking of metastases to enable multidimensional molecular profiling in personalized medicine.

Mod Pathol 2013 Nov 7;26(11):1413-24. Epub 2013 Jun 7.

Quebec Clinical Research Organization in Cancer, Montreal, QC, Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/modpathol.2013.81DOI Listing
November 2013

RBFOX2 is an important regulator of mesenchymal tissue-specific splicing in both normal and cancer tissues.

Mol Cell Biol 2013 Jan 12;33(2):396-405. Epub 2012 Nov 12.

Laboratoire de génomique fonctionnelle de l'Université de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.01174-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554129PMC
January 2013

Dok-1 overexpression promotes development of γδ natural killer T cells.

Eur J Immunol 2012 Sep 16;42(9):2491-504. Epub 2012 Jul 16.

Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.201242421DOI Listing
September 2012

The Max b-HLH-LZ can transduce into cells and inhibit c-Myc transcriptional activities.

PLoS One 2012 22;7(2):e32172. Epub 2012 Feb 22.

Département de Pharmacologie, Faculté de Médicine et des Sciences de la Santé, Université de Sherbrooke, Québec, Canada.

The inhibition of the functions of c-Myc (endogenous and oncogenic) was recently shown to provide a spectacular therapeutic index in cancer mouse models, with complete tumor regression and minimal side-effects in normal tissues. This was achieved by the systemic and conditional expression of omomyc, the cDNA of a designed mutant of the b-HLH-LZ of c-Myc named Omomyc. The overall mode of action of Omomyc consists in the sequestration of Max and the concomitant competition of the Omomyc/Max complex with the endogenous c-Myc/Max heterodimer. This leads to the inhibition of the transactivation of Myc target genes involved in proliferation and metabolism. While this body of work has provided extraordinary insights to guide the future development of new cancer therapies that target c-Myc, Omomyc itself is not a therapeutic agent. In this context, we sought to exploit the use of a b-HLH-LZ to inhibit c-Myc in a cancer cell line in a more direct fashion. We demonstrate that the b-HLH-LZ domain of Max (Max*) behaves as a bona fide protein transduction domain (PTD) that can efficiently transduce across cellular membrane via through endocytosis and translocate to the nucleus. In addition, we show that the treatment of HeLa cells with Max* leads to a reduction of metabolism and proliferation rate. Accordingly, we observe a decrease of the population of HeLa cells in S phase, an accumulation in G1/G0 and the induction of apoptosis. In agreement with these phenotypic changes, we show by q-RT-PCR that the treatment of HeLa cells with Max* leads to the activation of the transcription c-Myc repressed genes as well as the repression of the expression of c-Myc activated genes. In addition to the novel discovery that the Max b-HLH-LZ is a PTD, our findings open up new avenues and strategies for the direct inhibition of c-Myc with b-HLH-LZ analogs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032172PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3284561PMC
August 2012

Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators.

Mol Cell Biol 2012 Mar 27;32(5):954-67. Epub 2011 Dec 27.

Département de Microbiologie et d’Infectiologie, Université de Sherbrooke, Sherbrooke, Québec, Canada.

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.06130-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295189PMC
March 2012

Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3.

BMC Cancer 2011 Jul 1;11:285. Epub 2011 Jul 1.

Le Centre de recherche en cancérologie de l'Université Laval et Centre de recherche du CHUQ, l'Hôtel-Dieu de Québec, 9 rue McMahon, Québec G1R 2J6 Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2407-11-285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177907PMC
July 2011

Alternative splicing of SYK regulates mitosis and cell survival.

Nat Struct Mol Biol 2011 Jun 8;18(6):673-9. Epub 2011 May 8.

Laboratoire de Génomique Fonctionnelle, Université de Sherbrooke, Sherbrooke, Québec, Canada.

Most human genes produce multiple mRNA isoforms through alternative splicing. However, the biological relevance of most splice variants remains unclear. In this study, we evaluated the functional impact of alternative splicing in cancer cells. We modulated the splicing pattern of 41 cancer-associated splicing events and scored the effects on cell growth, viability and apoptosis, identifying three isoforms essential for cell survival. Specifically, changing the splicing pattern of the spleen tyrosine kinase gene (SYK) impaired cell-cycle progression and anchorage-independent growth. Notably, exposure of cancer cells to epithelial growth factor modulated the SYK splicing pattern to promote the pro-survival isoform that is associated with cancer tissues in vivo. The data suggest that splicing of selected genes is specifically modified during tumor development to allow the expression of isoforms that promote cancer cell survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.2040DOI Listing
June 2011

PRPF mutations are associated with generalized defects in spliceosome formation and pre-mRNA splicing in patients with retinitis pigmentosa.

Hum Mol Genet 2011 Jun 5;20(11):2116-30. Epub 2011 Mar 5.

Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddr094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3090192PMC
June 2011

Control of alternative splicing through siRNA-mediated transcriptional gene silencing.

Nat Struct Mol Biol 2009 Jul 21;16(7):717-24. Epub 2009 Jun 21.

Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina.

When targeting promoter regions, small interfering RNAs (siRNAs) trigger a previously proposed pathway known as transcriptional gene silencing by promoting heterochromatin formation. Here we show that siRNAs targeting intronic or exonic sequences close to an alternative exon regulate the splicing of that exon. The effect occurred in hepatoma and HeLa cells with siRNA antisense strands designed to enter the silencing pathway, suggesting hybridization with nascent pre-mRNA. Unexpectedly, in HeLa cells the sense strands were also effective, suggesting that an endogenous antisense transcript, detectable in HeLa but not in hepatoma cells, acts as a target. The effect depends on Argonaute-1 and is counterbalanced by factors favoring chromatin opening or transcriptional elongation. The increase in heterochromatin marks (dimethylation at Lys9 and trimethylation at Lys27 of histone H3) at the target site, the need for the heterochromatin-associated protein HP1alpha and the reduction in RNA polymerase II processivity suggest a mechanism involving the kinetic coupling of transcription and alternative splicing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.1620DOI Listing
July 2009

Cancer-associated regulation of alternative splicing.

Nat Struct Mol Biol 2009 Jun 17;16(6):670-6. Epub 2009 May 17.

Laboratoire de génomique fonctionnelle de l'Université de Sherbrooke, Sherbrooke, Québec, Canada.

Alternative splicing of pre-mRNA increases the diversity of protein functions. Here we show that about half of all active alternative splicing events in ovarian and breast tissues are changed in tumors, and many seem to be regulated by a single factor; sequence analysis revealed binding sites for the RNA binding protein FOX2 downstream of one-third of the exons skipped in cancer. High-resolution analysis of FOX2 binding sites defined the precise positions relative to alternative exons at which the protein may function as either a silencer or an enhancer. Most of the identified targets were shifted in the same direction by FOX2 depletion in cell lines as they were in breast and ovarian cancer tissues. Notably, we found expression of FOX2 itself is downregulated in ovarian cancer and its splicing is altered in breast cancer samples. These results suggest that the decreased expression of FOX2 in cancer tissues modulates splicing and controls proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.1608DOI Listing
June 2009

Identification of alternative splicing markers for breast cancer.

Cancer Res 2008 Nov;68(22):9525-31

Laboratoire de génomique fonctionnelle de l'Université de Sherbrooke, Québec, Canada.

Breast cancer is the most common cause of cancer death among women under age 50 years, so it is imperative to identify molecular markers to improve diagnosis and prognosis of this disease. Here, we present a new approach for the identification of breast cancer markers that does not measure gene expression but instead uses the ratio of alternatively spliced mRNAs as its indicator. Using a high-throughput reverse transcription-PCR-based system for splicing annotation, we monitored the alternative splicing profiles of 600 cancer-associated genes in a panel of 21 normal and 26 cancerous breast tissues. We validated 41 alternative splicing events that significantly differed in breast tumors relative to normal breast tissues. Most cancer-specific changes in splicing that disrupt known protein domains support an increase in cell proliferation or survival consistent with a functional role for alternative splicing in cancer. In a blind screen, a classifier based on the 12 best cancer-associated splicing events correctly identified cancer tissues with 96% accuracy. Moreover, a subset of these alternative splicing events could order tissues according to histopathologic grade, and 5 markers were validated in a further blind set of 19 grade 1 and 19 grade 3 tumor samples. These results provide a simple alternative for the classification of normal and cancerous breast tumor tissues and underscore the putative role of alternative splicing in the biology of cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-08-1769DOI Listing
November 2008

Multiple and specific mRNA processing targets for the major human hnRNP proteins.

Mol Cell Biol 2008 Oct 21;28(19):6033-43. Epub 2008 Jul 21.

Département de Microbiologie et d'Infectiologie, Faculté de Médecine et des Sciences de la Santé, 3001, 12th Avenue Nord, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.

Alternative splicing is a key mechanism regulating gene expression, and it is often used to produce antagonistic activities particularly in apoptotic genes. Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins form a family of RNA-binding proteins that coat nascent pre-mRNAs. Many but not all major hnRNP proteins have been shown to participate in splicing control. The range and specificity of hnRNP protein action remain poorly documented, even for those affecting splice site selection. We used RNA interference and a reverse transcription-PCR screening platform to examine the implications of 14 of the major hnRNP proteins in the splicing of 56 alternative splicing events in apoptotic genes. Out of this total of 784 alternative splicing reactions tested in three human cell lines, 31 responded similarly to a knockdown in at least two different cell lines. On the other hand, the impact of other hnRNP knockdowns was cell line specific. The broadest effects were obtained with hnRNP K and C, two proteins whose role in alternative splicing had not previously been firmly established. Different hnRNP proteins affected distinct sets of targets with little overlap even between closely related hnRNP proteins. Overall, our study highlights the potential contribution of all of these major hnRNP proteins in alternative splicing control and shows that the targets for individual hnRNP proteins can vary in different cellular contexts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00726-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2547008PMC
October 2008

A G-tract element in apoptotic agents-induced alternative splicing.

Nucleic Acids Res 2008 Jun 24;36(10):3320-31. Epub 2008 Apr 24.

Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College. Kunming, China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkn207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2425498PMC
June 2008

Multiple alternative splicing markers for ovarian cancer.

Cancer Res 2008 Feb;68(3):657-63

Laboratoire de génomique fonctionnelle de l'Université de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada.

Intense efforts are currently being directed toward profiling gene expression in the hope of developing better cancer markers and identifying potential drug targets. Here, we present a sensitive new approach for the identification of cancer signatures based on direct high-throughput reverse transcription-PCR validation of alternative splicing events. This layered and integrated system for splicing annotation (LISA) fills a gap between high-throughput microarray studies and high-sensitivity individual gene investigations, and was created to monitor the splicing of 600 cancer-associated genes in 25 normal and 21 serous ovarian cancer tissues. Out of >4,700 alternative splicing events screened, the LISA identified 48 events that were significantly associated with serous ovarian tumor tissues. In a further screen directed at 39 ovarian tissues containing cancer pathologies of various origins, our ovarian cancer splicing signature successfully distinguished all normal tissues from cancer. High-volume identification of cancer-associated splice forms by the LISA paves the way for the use of alternative splicing profiling to diagnose subtypes of cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-07-2580DOI Listing
February 2008

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals.

BMC Biotechnol 2006 Jan 13;6. Epub 2006 Jan 13.

Département de Microbiologie et d'Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1472-6750-6-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1379639PMC
January 2006

Free uptake of cell-penetrating peptides by fission yeast.

FEBS Lett 2005 Aug;579(21):4873-8

Centre de Génomique Fonctionnelle de Sherbrooke, Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbooke, Sherbooke, Que., Canada.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2005.07.064DOI Listing
August 2005

Structural basis for contrasting activities of ribosome binding thiazole antibiotics.

Chem Biol 2003 Aug;10(8):769-78

RiboTargets, Ltd., Granta Park, Abington, CB1 6GB, Cambridge, United Kingdom.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s1074-5521(03)00173-xDOI Listing
August 2003

A conserved RNA structure within the HCV IRES eIF3-binding site.

Nat Struct Biol 2002 May;9(5):375-80

RiboTargets Ltd., Granta Park, Abington, Cambridge, CB1 6GB, UK.

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is recognized specifically by the small ribosomal subunit and eukaryotic initiation factor 3 (eIF3) before viral translation initiation. Using extensive mutagenesis and structure probing analysis, we show that the eIF3-binding domain of the HCV IRES contains an internal loop structure (loop IIIb) and an adjacent mismatched helix that are important for IRES-dependent initiation of translation. NMR studies reveal a unique three-dimensional structure for this internal loop that is conserved between viral isolates of varying primary sequence in this region. These data indicate that internal loop IIIb may be an attractive target for structure-based design of new antiviral agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsb785DOI Listing
May 2002