An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA

Ml, Zhang; Cl, Lorson; Androphy, Elliot J.; Zhou, Jianhua

doi:10.1038/sj.gt.3301550

Cited by 123 publications

(91 citation statements)

References 26 publications

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“…Previously, compounds have been identified which alter the incorporation of exon 7 in the SMN transcript from the SMN2 gene (32,33,35). In the present study, compounds such as forskolin and dibutyryl cAMP were shown to increase the expression of the SMN2 gene.…”

Section: Discussionmentioning

confidence: 53%

“…The presence of SMN2 in all SMA patients, the ability of more copies of SMN2 to modify the SMA phenotype, and the rescue of SMA mice by multiple copies of SMN2 make it an attractive therapeutic candidate. Molecules capable of inducing SMN2 expression (30,31) or altering the splicing of SMN2 such that more full-length SMN transcript is produced have been identified (32)(33)(34)(35)(36)(37). At the present time, there is limited information on the mode of action of these compounds as well as the protein complexes that interact with the SMN promoter.…”

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confidence: 99%

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Identification of a Novel Cyclic AMP-response Element (CRE-II) and the Role of CREB-1 in the cAMP-induced Expression of the Survival Motor Neuron (SMN) Gene

Majumder

Varadharaj

Ghoshal

et al. 2004

Journal of Biological Chemistry

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Spinal muscular atrophy, an autosomal recessive disorder, is caused by loss of the SMN1 (survival motor neuron) gene while retaining the SMN2 gene. SMN1 produces a majority of full-length SMN transcript, whereas SMN2 generates mostly an isoform lacking exon 7. Here, we demonstrate a novel cAMP-response element, CRE-II, in the SMN promoter that interacts with the cAMP-response element-binding (CREB) family of proteins. In vitro DNase I protection analysis and in vivo genomic footprinting of the SMN promoter using the brain and liver nuclei from SMN2 transgenic mice revealed footprinting at the CRE-II site. Site-directed mutation of the CRE-II element caused a marked reduction in the SMN promoter activity revealed by transient transfection assay. Activation of the cAMP pathway by dibutyryl cAMP (0.5 mM) alone or in combination with forskolin (20 M) caused a 2-5-fold increase in the SMN promoter activity but had no effect on the CRE-II mutated promoter. Electrophoretic mobility shift assay and a UV-induced DNA-protein crosslinking experiment confirmed that CREB1 binds specifically to the CRE-II site. Transient overexpression of CREB1 protein resulted in a 4-fold increase of the SMN promoter activity. Intraperitoneal injection of epinephrine in mice expressing two copies of the human SMN2 gene resulted in a 2-fold increase in full-length SMN transcript in the liver. Combined treatment with dibutyryl cAMP and forskolin significantly increased the level of both the full-length and exon 7-deleted SMN (exon⌬7SMN) transcript in primary hepatocytes from mice expressing two copies of human SMN2 gene. Similar treatments of type I spinal muscular atrophy mouse and human fibroblasts as well as HeLa cells resulted in an augmented level of SMN transcript. These findings suggest that the CRE-II site in SMN promoter positively regulates the expression of the SMN gene, and treatment with cAMP-elevating agents increases expression of both the full-length and exon⌬7SMN transcript.

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Section: Discussionmentioning

confidence: 53%

mentioning

confidence: 99%

Identification of a Novel Cyclic AMP-response Element (CRE-II) and the Role of CREB-1 in the cAMP-induced Expression of the Survival Motor Neuron (SMN) Gene

Majumder

Varadharaj

Ghoshal

et al. 2004

Journal of Biological Chemistry

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“…Both butyrate and valproic acid are known to act as histone deacetylase inhibitors enhancing transcription of some genes, among which are splicing factors of the SR family, whereas aclarubicin seems to increase the expression of transcription factors (38). Last, sodium vanadate has been reported to inhibit ATPase, alkaline, and tyrosine, as well as multiple other phosphatases, and its likely targets are SR proteins, snRNPs, and hnPNPs (39). This remark about the lack of specificity also stands for drugs that interfere with the kinase activity of DNA topo I.…”

Section: Discussionmentioning

confidence: 99%

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors

Soret

Bakkour

Maire

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

111

106

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The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have used in vitro splicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction. Importantly, compounds of this family specifically inhibit exonic splicing enhancer (ESE)-dependent splicing, because they interact directly and selectively with members of the serine-arginine-rich protein family. Treatment of cells expressing reporter constructs with ESE sequences demonstrated that selected indole derivatives mediate inhibition of ESE usage in vivo and prevent early splicing events required for HIV replication. This discovery opens the exciting possibility of a causal pharmacological treatment of aberrant splicing in human genetic disorders and development of new antiviral therapeutic approaches.splicing correction ͉ exonic splicing enhancer ͉ small chemicals ͉ pathologic splicing R emoval of introns from newly transcribed RNA polymerase II precursors (pre-mRNA) during splicing not only is an essential step for the expression of most genes in higher eukaryotic cells but also constitutes an important mechanism for generation of protein diversity and regulation of gene expression (1, 2). It is estimated that Ͼ70% of human genes are subjected to alternative splicing, and it is not surprising that many point mutations causing human diseases are associated with aberrant splicing (3, 4).Current models of constitutive and a fortiori alternative splicing suggest that splice site recognition is strongly modulated by the interaction of specific exonic and intronic pre-mRNA sequences with at least two classes of nonspliceosomal nuclear RNA-binding proteins: serine-arginine-rich (SR) proteins (5-7) and heterogeneous nuclear ribonucleoproteins (8-10). These proteins interact with spliceosomal components (5-7) and either activate or prevent the use of degenerate splice sites in their vicinity. Thus, binding of SR proteins to exonic splicing enhancers (ESE) through their RNA-recognition motif (RRM) promotes exon definition by recruiting constitutive factors via protein-protein interactions mediated by their arginine-serine-rich (RS) domain and prevents the action of nearby splicing silencers (4, 6, 11).Mutations causing human diseases may affect splice sites as well as regulatory sequences leading to the production of defective proteins (4, 11). Thus, targeting either the mutated sequences or the factors that bind them may prove to be a valuable strategy to correct aberrant splicing. Recently, antisense strategies targeting ESEdependent mechanisms have been used to induce skipping of exons containing nonsense mutations or, conversely, to restore exon inclusion by synthetic exon-specific effectors (bifunctional antisense peptide molecules or tailed antisense oligonucleotides) or spliceosome-mediat...

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“…14 As an indirect measurement of U7 snRNA expression, we analysed exon 7 inclusion in HeLa cells stably expressing an SMN2-luciferase reporter gene. 23 When these HS2 cells were transduced with pLV-T carrying the U7-ESE-B cassette, they showed a strong GFP fluorescence correlating with the expected increase in exon 7 inclusion of the SMN2 reporter gene, from a basal 13 ± 3 to 75 ± 7% (Figures 1d and e). These cells were subsequently transduced with a second lentiviral vector encoding the tTR-KRAB regulatory protein and a dsRed reporter gene and cultured either in the absence or in the presence of doxycycline.…”

Section: Development Of a Regulatable U7 Expression Systemmentioning

confidence: 78%

“…14,23 HeLa-705 cells stably express the human b-globin gene carrying the IVS2-705 mutation. 24 Conditions for cell culture, DNA transfections, lentiviral vector production in 293-T cells, titration on HeLa cells and transduction have been described.…”

Section: Cell Culturementioning

confidence: 99%

Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes

2008

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Many diseases affect pre-mRNA splicing, and alternative splicing is a major source of proteome diversity and an important mechanism for modulating gene expression. The ability to regulate a specific splicing event is therefore desirable; for example, to understand splicing-associated pathologies. We have developed methods based on modified U7 snRNAs, which allow us to induce efficient skipping or inclusion of selected exons. Here, we have adapted these U7 tools to a regulatable system that relies on a doxycyclinesensitive version of the Krü ppel-associated box (KRAB)/ KAP1 transcriptional silencing. Co-transduction of target cells with two lentiviral vectors, one carrying the KRAB protein and the other the regulatable U7 cassette, allows a tight regulation of the modified U7 snRNA. No leakage is observed in the repressed state, whereas full induction can be obtained with doxycycline in ng ml À1 concentrations. Only a few days are necessary for a full switch, and the induction/ repression can be repeated over several cycles without noticeable loss of control. Importantly, the U7 expression correlates with splicing correction, as shown for the skipping of an aberrant b-globin exon created by a thalassaemic mutation and the promotion of exon 7 inclusion in the human SMN2 gene, an important therapeutic target for spinal muscular atrophy.

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An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA

Abstract: Spinal muscular atrophy (SMA) is a degenerative motor neu

Cited by 123 publications

References 26 publications

Identification of a Novel Cyclic AMP-response Element (CRE-II) and the Role of CREB-1 in the cAMP-induced Expression of the Survival Motor Neuron (SMN) Gene

Identification of a Novel Cyclic AMP-response Element (CRE-II) and the Role of CREB-1 in the cAMP-induced Expression of the Survival Motor Neuron (SMN) Gene

Selective modification of alternative splicing by indole derivatives that target serine-arginine-rich protein splicing factors

Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes

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