Reprogramming Alternative Pre-messenger RNA Splicing through the Use of Protein-binding Antisense Oligonucleotides

Villemaire, Jonathan; Dion, Isabelle; Elela, Sherif Abou; Chabot, Benoît

doi:10.1074/jbc.m308897200

Cited by 64 publications

(62 citation statements)

References 45 publications

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“…For example, unavailability of a competing 3 0 ss likely forced the splicing machinery to select a structured AG. This interpretation is in agreement with previously reported studies in which the repression of a targeted splice site was significantly higher when an alternative splice site was available Villemaire et al 2003).…”

Section: Wwwrnajournalorg 1673supporting

confidence: 83%

An artificial riboswitch for controlling pre-mRNA splicing

KIM¹

2005

RNA

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Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3 0 splice site (3 0 ss) region of a model pre-mRNA (AdMLTheo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3 0 ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant.

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Section: Wwwrnajournalorg 1673supporting

confidence: 83%

An artificial riboswitch for controlling pre-mRNA splicing

KIM¹

2005

RNA

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“…Other, very elegant strategies to interfere with splicing events are based on the design of bifunctional oligoribonucleotides with a first domain complementary to an exonic enhancer or silencer element in the target pre-mRNA, and a second domain recruiting specific splicing factors near the splice site (35)(36)(37). Although effective, these technologies require the precise knowledge of cis-acting elements regulating splicing of the targeted exon.…”

Section: Discussionmentioning

confidence: 99%

Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies

Rodríguez-Martín

García-Blanco

Mansfield

et al. 2005

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

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Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3 end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9 -exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10 ؊ tau RNA into exon 10 ؉ tau RNA could be achieved with Ϸ34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.T au is a microtubule-associated protein predominantly expressed in neurons and specifically localized in axons that promotes microtubule polymerization and stabilization (1, 2). Mutations in the MAPT gene, encoding tau, on chromosome 17, cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) (3-5). FTDP-17 is characterized by intraneuronal aggregates of tau and, as such, belongs to the group of dementias that includes Alzheimer's disease and collectively referred to as tauopathies.The human MAPT gene is a large gene of Ϸ134 kb comprising 16 exons (6). Alternative splicing of exons 2, 3, and 10 in the tau pre-mRNA results in the expression of six isoforms in the brain. Exon 10 encodes the second of four imperfect microtubulebinding repeats in the C-terminal half of the tau protein.Exclusion or inclusion of exon 10 gives rise to tau isoforms with three (tau3R, exon 10 Ϫ ) or four (tau4R, exon 10 ϩ ) microtubulebinding repeats (7). Furthermore, inclusion or exclusion of exons 2 and 3 produces tau isoforms with zero, one, or two inserts near the N terminus. Only tau3R is expressed during embryogenesis whereas tau3R and tau4R are expressed in approximately equal amounts in adult human brain. Exon 10 splicing is regulated by multiple cis-acting elements comprising exonic and intronic silencers and enhancers (for reviews, see refs. 8 and 9). In particular, intron 10 contains a bipartite element comprising a silencer and a modulator downstream to the 5Ј splice...

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“…These compounds have been used to modify alternative splicing in the SMN2 (spinal muscular atrophy), Bcl-x, and C-myc genes (29)(30)(31)(32)(33)(34)(35)(36). Besides blocking cryptic splice sites, antisense oligonucleotides can block splice regulation sequences in the FGFR1 gene (37).…”

Section: Discussionmentioning

confidence: 99%

Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

Du¹,

Pollard²,

Gatti

2007

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

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We used antisense morpholino oligonucleotides (AMOs) to redirect and restore normal splicing of three prototypic splicing mutations in the ataxia-telangiectasia mutated (ATM) gene. Two of the mutations activated cryptic 5 or 3 splice sites within exonic regions; the third mutation activated a downstream 5 splice site leading to pseudoexon inclusion of a portion of intron 28. AMOs were targeted to aberrant splice sites created by the mutations; this effectively restored normal ATM splicing at the mRNA level and led to the translation of full-length, functional ATM protein for at least 84 h in the three cell lines examined, as demonstrated by immunoblotting, ionizing irradiation-induced autophosphorylation of ATM, and transactivation of ATM substrates. Ionizing irradiation-induced cytotoxicity was markedly abrogated after AMO exposure. The ex vivo data strongly suggest that the disease-causing molecular pathogenesis of such prototypic mutations is not the amino acid change of the protein but the mutated DNA code itself, which alters splicing. Such prototypic splicing mutations may be correctable in vivo by systemic administration of AMOs and may provide an approach to customized, mutation-based treatment for ataxia-telangiectasia and other genetic disorders.ataxia-telangiectasia mutated ͉ mutation-based treatment T his study addresses the restoration of normal splicing in three ataxia-telangiectasia mutated (ATM) splicing mutations by exposure of ataxia-telangiectasia (A-T) cells to antisense morpholino oligonucleotides (AMOs). Antisense oligonucleotides have been used to restore pre-mRNA splicing in other disease models (1-8); however, the therapeutic rationale for each varies considerably and, to our knowledge, none has addressed an intranuclear or DNA repair disorder. Furthermore, we believe that A-T offers several advantages for exploring the therapeutic potential of AMOs, such as (i) the availability of many surrogate markers for evaluating ATM function, ex vivo and in vivo, and (ii) a well characterized spectrum of ATM mutations supported by an extensive cell repository of lymphoblastoid cell lines (LCLs) derived from patients with those mutations (9-12). The LCLs allow pre-mRNA mechanisms to be dissected in great detail. The principles gleaned can then be extended to other tissue targets, such as neuronal cells.A-T is a progressive autosomal recessive neurodegenerative disorder resulting from mutations in ATM (13). This gene includes 66 exons and encodes a 13-kb mature transcript with an ORF of 9,168 nt. The ATM protein is a serine/threonine kinase with Ͼ30 phosphorylation targets (14, 15). It affects control of cell cycle checkpoints, repair of dsDNA breaks, responses to oxidative stress, and apoptosis and is a potent tumor suppressor (16)(17)(18)(19). ATM is constitutively expressed in all tissues, primarily in the nucleus. So far no therapy exists for this disorder.ATM protein is not detectable in most A-T patients. A subset of patients with notable protein levels (5-20%) has milder phenotypes (20). Pati...

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Reprogramming Alternative Pre-messenger RNA Splicing through the Use of Protein-binding Antisense Oligonucleotides

Cited by 64 publications

References 45 publications

An artificial riboswitch for controlling pre-mRNA splicing

An artificial riboswitch for controlling pre-mRNA splicing

Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies

Correction of prototypic ATM splicing mutations and aberrant ATM function with antisense morpholino oligonucleotides

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