U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites

Uchikawa, Hideki; Fujii, Kiyotaka; Kohno, Yoichi; Katsumata, Noriyuki; Nagao, Keiko; Yamada, Masao; Miyashita, Toshiyuki

doi:10.1007/s10038-007-0192-8

Cited by 17 publications

(8 citation statements)

References 30 publications

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“…To clarify if the enhancing effect is specific for the U1 snRNA particle, we inserted the tail of two active ExSpeU1s, fix10 and cf11, in the U7 snRNA. U7 is not involved in splicing and a modified version has been extensively used to express antisense RNA sequences (29–35). The resulting U7 fix10 and U7 cf11 were cotransfected with −8G, −2T mutants and +3G and Y577Y, respectively.…”

Section: Resultsmentioning

confidence: 99%

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

Alanis¹,

Pinotti²,

Mas³

et al. 2012

Human Molecular Genetics

145

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A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition.

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Section: Resultsmentioning

confidence: 99%

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

Alanis¹,

Pinotti²,

Mas³

et al. 2012

Human Molecular Genetics

145

View full text Add to dashboard Cite

show abstract

“…If there are no other cryptic splice sites that are stronger than the mutated authentic splice site, the ASO block may reactivate the mutated authentic splice site. This technique as has been used successfully for 5′ss mutations in BRCA1 and PTCH1 26. These types of splice site mutations have also been rescued using small molecules that can promote splicing to the inactivated splice site.…”

Section: Targeting Splicing In Disease: the Fixesmentioning

confidence: 99%

Targeting RNA splicing for disease therapy

2013

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Splicing of pre-messenger RNA into mature messenger RNA is an essential step for expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease-associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans-splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice-modulating approaches, and emerging therapeutics.

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“…Finally, our findings open the way to development of novel therapeutic strategies aimed at rescuing splicing inhibition in patient cells. In general, pseudoexon inclusion in pathological situations can be targeted through use of antisense oligonucleotides or modified U7 snRNA molecules against the cryptic 5′ and 3′ splice sites, as recently described for PCCA , PCCB , PTCH1 and BRCA1 [25,26]. Although these strategies may represent viable therapeutic approaches for repression of the NF1 pseudoexon, the results presented in this work have expanded the list of potential options.…”

Section: Discussionmentioning

confidence: 93%

Polypyrimidine tract binding protein regulates alternative splicing of an aberrant pseudoexon in NF1

et al. 2008

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Pseudoexons are intronic sequences that are approximately the same length as exons (200 bp) with apparently viable donor and acceptor splice sites but which are not normally spliced in the mature mRNA transcript. Despite the known abundance of exon splicing silencer regulatory elements within introns [1], it is not possible to formulate general rules for pseudoexon repression without remembering that splicing is very much dependent upon local context [2,3]. Normal exons need to encode the information for both protein synthesis and RNA splicing, so one might expect differences in pseudoexons with regard to several factors, including the distribution of splice consensus sequences, splicing enhancers, splicing silencers and secondary structures. In line with these considerations, bioinformatics studies have observed enrichment of exon splicing silencer elements within pseudoexons relative to exonic splicing enhancer elements [4,5]. Furthermore, it has been suggested that it is defective splice sites rather than the lack of enhancers that account for non-splicing of pseudoexon sequences, although even perfect consensus sequences are not always adequate for correct splicing [6]. To complicate matters further, it has been recently suggested that some pseudoexons are authentic exons whose inclusion leads to efficient nonsense-mediated decay, such as the pseudoexon located downstream of mutually exclusive exons 2 and 3 of the rat a-tropomyosin gene, which acts both as an alternative exon that leads to nonsense-mediated decay and as a zero-length exon [7].Nonetheless, in a clinical setting, pathological pseudoexon sequences have been identified by simple activation of cryptic splice sites which reinforce their strength, de novo creation, the inactivation ⁄ activation In disease-associated genes, understanding the functional significance of deep intronic nucleotide variants represents a difficult challenge. We previously reported that an NF1 intron 30 exonization event is triggered from a single correct nomenclature is 'c.293 27,[294][295]. In this paper, we investigate which characteristics play a role in regulating inclusion of the aberrant pseudoexon. Our investigation shows that pseudoexon inclusion levels are strongly downregulated by polypyrimidine tract binding protein and its homologue neuronal polypyrimidine tract binding protein. In particular, we provide evidence that the functional effect of polypyrimidine tract binding protein is proportional to its concentration, and map the cisacting elements that are principally responsible for this negative regulation. These results highlight the importance of evaluating local sequence context for diagnostic purposes, and the utility of developing therapies to turn off activated pseudoexons.Abbreviations NF1, neurofibromatosis type 1; nPTB, neuronal polypyrimidine tract binding protein; PTB, polypyrimidine tract binding protein.

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U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites

Cited by 17 publications

References 30 publications

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

Targeting RNA splicing for disease therapy

Polypyrimidine tract binding protein regulates alternative splicing of an aberrant pseudoexon in NF1

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