Deletion analysis of a unique 3' splice site indicates that alternating guanine and thymine residues represent an efficient splicing signal

Shelley, Carl Simon; Baralle, Francisco E.

doi:10.1093/nar/15.9.3787

Cited by 22 publications

(19 citation statements)

References 39 publications

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“…3B, lanes 2 and 3). This result supports previous studies showing that the removal of all the GU repeats in the apoA-II intron 2 caused ϳ90% of exon 3 to be skipped (11).…”

Section: Resultssupporting

confidence: 92%

“…In comparison with the splicing pattern of pApo-wt, the pCF/Apo18 construct showed 100% of exon 3 inclusion, thus supporting the hypothesis that the CFTR (GT) 11 (T) 5 polypyrimidine tract is stronger than that of the apoA-II (GT) 16 (Fig. 5B, lanes 1 and 2).…”

Section: Fig 3 In Vivo Effect Of Gu Deletion/replacementsupporting

confidence: 69%

“…A set of five CFTR/apoA-II hybrids was generated in which the exon 3 was progressively replaced by CFTR exon 9 and its 3Ј splice site replaced with the allelic configuration (GT) 11 (T) 5 . The constructs were named by indicating the number of apoA-II exon 3 nucleotides replaced by CFTR exon 9 (i.e.…”

Section: Fig 3 In Vivo Effect Of Gu Deletion/replacementmentioning

confidence: 99%

“…In fact, CFTR exon 9 undergoes alternative splicing, and its inclusion is inversely correlated with the length of the GU tract and directly proportional to the length of the poly(T) tract, (7,10) whereas apparently apoA-II exon 3 is constitutively spliced, and its inclusion is dependent on the presence of the GU tract (11). In other words, in the CFTR intron 8/exon 9 context, the stretch of pyrimidines alternated with purines alone is not equivalent to a functional continuous polypyrimidine tract (10), in contrast to what has been observed for the apoA-II gene (11). Altogether these observations prompted us to investigate the mechanisms underlying the constitutive splicing of apoA-II exon 3 and, in particular, to characterize the cis-acting elements and the trans-acting factors involved in apoA-II exon 3 definition.…”

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confidence: 99%

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An Exonic Splicing Enhancer Offsets the Atypical GU-rich 3′ Splice Site of Human Apolipoprotein A-II Exon 3

Arrisi-Mercado

Romano

Muro

et al. 2004

Journal of Biological Chemistry

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Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3 splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3 splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3 splice site.Pre-mRNA splicing is the process by which introns are removed and exons are joined together by a two-step trans-esterification reaction carried out by the spliceosome, a dynamic 60 S ribonucleoprotein particle (1). Formation of the spliceosome at particular splice junctions is triggered by recognition of the 5Ј splice site by the U1 small nuclear ribonucleoprotein and of the 3Ј splice site by U2AF followed by the U2 small nuclear ribonucleoprotein recognition of the branch point (2).The polypyrimidine tract is one of the important cis-acting sequences present in the 3Ј splice site of introns. The progressive deletion of the polypyrimidine tract abolish lariat formation, spliceosome assembly, and consequently the splicing process (3, 4), whereas increasing the length of the pyrimidine run can lead to improved efficiency of splicing in some systems (4).In vitro studies have demonstrated that the ability of polypyrimidine tracts to favor specific 3Ј splice site selection is not only determined by its length but also by its composition (4, 5). This feature coexists with a degree of flexibility in the specific sequence of a given tract.The human apolipoprotein A-II (apoA-II) 1 gene presents a peculiar arrangement of GT repeats within the polypyrimidine tract region at the intron 2/exon 3 junction that devia...

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“…3B, lanes 2 and 3). This result supports previous studies showing that the removal of all the GU repeats in the apoA-II intron 2 caused ϳ90% of exon 3 to be skipped (11).…”

Section: Resultssupporting

confidence: 92%

Section: Fig 3 In Vivo Effect Of Gu Deletion/replacementsupporting

confidence: 69%

Section: Fig 3 In Vivo Effect Of Gu Deletion/replacementmentioning

confidence: 99%

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confidence: 99%

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An Exonic Splicing Enhancer Offsets the Atypical GU-rich 3′ Splice Site of Human Apolipoprotein A-II Exon 3

Arrisi-Mercado

Romano

Muro

et al. 2004

Journal of Biological Chemistry

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“…In fact, in addition to the CFTR gene, the presence of simple (UG)mrepeated sequences has been described to influence the splicing process of at least two other genes: the apolipoprotein AII gene (10) and the human cardiac Na ϩ /Ca 2ϩ exchanger (11). In the Apo AII gene the UG tract was shown to be functionally equivalent to a polypyrimidine tract and required for efficient splicing of Apo AII exon 2 (10) while in the human cardiac Na ϩ /Ca 2ϩ exchanger (11) it acts as a strong intronic splicing enhancer situated in intron 2.…”

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confidence: 99%

Characterization and Functional Implications of the RNA Binding Properties of Nuclear Factor TDP-43, a Novel Splicing Regulator ofCFTR Exon 9

Buratti

Baralle

2001

Journal of Biological Chemistry

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626

606

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Variations in a polymorphic (TG)m sequence near exon 9 of the human CFTR gene have been associated with variable proportions of exon skipping and occurrence of disease. We have recently identified nuclear factor TDP-43 as a novel splicing regulator capable of binding to this element in the CFTR pre-mRNA and inhibiting recognition of the neighboring exon. In this study we report the dissection of the RNA binding properties of TDP-43 and their functional implications in relationship with the splicing process. Our results show that this protein contains two fully functional RNA recognition motif (RRM) domains with distinct RNA/DNA binding characteristics. Interestingly, TDP-43 can bind a minimum number of six UG (or TG) single-stranded dinucleotide stretches, and binding affinity increases with the number of repeats. In particular, the highly conserved Phe residues in the first RRM region play a key role in nucleic acid recognition.We have recently reported the identification of TDP-43 as a splicing regulator that specifically binds the (UG)m-repeated polymorphic region near the 3Ј-splice site of CFTR exon 9 and down-regulates its recognition by the splicing machinery (1). This region, acting in concert with the adjacent (u)n element, is one of the key cis-acting sequences which regulate the proportion of exon 9 skipping in the mature CFTR mRNA transcript (1-3). Considering that exon 9 skipping produces a non-functional CFTR protein (4, 5) the study of the RNA binding properties of TDP-43 is of considerable importance to gain further insight concerning the potential disease-causing consequences of its binding in vivo. Indeed, the clinical relevance of these studies is highlighted by the existence of a clear association between certain (TG)m(T)n alleles with distinct forms of Cystic Fibrosis (1, 6 -9).In addition, the study of (UG)m elements can provide further insight concerning the mRNA splicing process in general because (UG)m sequences have been described to act as splicing regulatory sequences in different genomic contexts. In fact, in addition to the CFTR gene, the presence of simple (UG)mrepeated sequences has been described to influence the splicing process of at least two other genes: the apolipoprotein AII gene (10) and the human cardiac Na ϩ /Ca 2ϩ exchanger (11). In the Apo AII gene the UG tract was shown to be functionally equivalent to a polypyrimidine tract and required for efficient splicing of Apo AII exon 2 (10) while in the human cardiac Na ϩ /Ca 2ϩ exchanger (11) it acts as a strong intronic splicing enhancer situated in intron 2. It should be noted that in contrast with these two genes, the CFTR (UG)m element was found to possess a strong inhibitory effect on CFTR exon 9 splicing, a property that may probably be linked to its peculiar evolutionary history. In fact, sequencing of the mouse CFTR exon 9 genomic region has shown that in the flanking introns, the (TG)m(T)n regulatory elements are absent and that the intron themselves are of very different length when compared with the human introns (...

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The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences

1992

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A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5' splice sites, 26 at 3' splice sites and 13 that result in the creation of novel splice sites. It is estimated that up to 15% of all point mutations causing human genetic disease result in an mRNA splicing defect. Of the 5' splice site mutations, 60% involved the invariant GT dinucleotide; mutations were found to be non-randomly distributed with an excess over expectation at positions +1 and +2, and apparent deficiencies at positions -1 and -2. Of the 3' splice site mutations, 87% involved the invariant AG dinucleotide; an excess of mutations over expectation was noted at position -2. This non-randomness of mutation reflects the evolutionary conservation apparent in splice site consensus sequences drawn up previously from primate genes, and is most probably attributable to detection bias resulting from the differing phenotypic severity of specific lesions. The spectrum of point mutations was also drastically skewed: purines were significantly over-represented as substituting nucleotides, perhaps because of steric hindrance (e.g. in U1 snRNA binding at 5' splice sites). Furthermore, splice sites affected by point mutations resulting in human genetic disease were markedly different from the splice site consensus sequences. When similarity was quantified by a 'consensus value', both extremely low and extremely high values were notably absent from the wild-type sequences of the mutated splice sites. Splice sites of intermediate similarity to the consensus sequence may thus be more prone to the deleterious effects of mutation. Regarding the phenotypic effects of mutations on mRNA splicing, exon skipping occurred more frequently than cryptic splice site usage. Evidence is presented that indicates that, at least for 5' splice site mutations, cryptic splice site usage is favoured under conditions where (1) a number of such sites are present in the immediate vicinity and (2) these sites exhibit sufficient homology to the splice site consensus sequence for them to be able to compete successfully with the mutated splice site. The novel concept of a "potential for cryptic splice site usage" value was introduced in order to quantify these characteristics, and to predict the relative proportion of exon skipping vs cryptic splice site utilization consequent to the introduction of a mutation at a normal splice site.

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Deletion analysis of a unique 3' splice site indicates that alternating guanine and thymine residues represent an efficient splicing signal

Cited by 22 publications

References 39 publications

An Exonic Splicing Enhancer Offsets the Atypical GU-rich 3′ Splice Site of Human Apolipoprotein A-II Exon 3

An Exonic Splicing Enhancer Offsets the Atypical GU-rich 3′ Splice Site of Human Apolipoprotein A-II Exon 3

Characterization and Functional Implications of the RNA Binding Properties of Nuclear Factor TDP-43, a Novel Splicing Regulator ofCFTR Exon 9

The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences

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