Determinants of Exon 7 Splicing in the Spinal Muscular Atrophy Genes, SMN1 and SMN2

Cartegni, Luca; Hastings, Michelle L.; Calarco, John A.; Stanchina, Elisa de; Krainer, Adrian R.

doi:10.1086/498853

Cited by 273 publications

(256 citation statements)

References 62 publications

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“…Although our data are consistent with the view that this is the sole factor responsible for SMN2 exon 7 exclusion (ref. 29; unpublished data), it remains possible that an alternative mechanism, such as loss of ESE function (27,28), also contributes to SMN2 exon 7 exclusion. However, we do not address this possibility here, but rather describe additional experiments designed to elucidate how the SMN2-specific ESS functions.…”

Section: Resultsmentioning

confidence: 99%

“…This base change, which is one of only four in Ͼ1.25 kb encompassing exon 7 and much of the flanking introns (23), does not affect either the exon 7 5Ј splice site or an ESE in the middle of the exon dependent on the splicing regulator Tra2 (25,26). Krainer and colleagues have presented evidence that the ϩ6 base change disrupts an ESE in SMN1 that depends on a specific serine/arginine-rich protein, ASF/SF2, which they suggest results in SMN2 exon 7 exclusion (27,28). In contrast, Kashima and Manley (29) provided data indicating that the C 3 T change creates an hnRNP A1-dependent ESS that recruits hnRNP A1 to SMN2 but not SMN1 premRNA, thereby repressing SMN2 exon 7 splicing.…”

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confidence: 99%

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An intronic element contributes to splicing repression in spinal muscular atrophy

Kashima

Rao

Manley

2007

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

115

109

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The neurodegenerative disease spinal muscular atrophy is caused by mutation of the survival motor neuron 1 (SMN1) gene. SMN2 is a nearly identical copy of SMN1 that is unable to prevent disease, because most SMN2 transcripts lack exon 7 and thus produce a nonfunctional protein. A key cause of inefficient SMN2 exon 7 splicing is a single nucleotide difference between SMN1 and SMN2 within exon 7. We previously provided evidence that this base change suppresses exon 7 splicing by creating an inhibitory element, a heterogeneous nuclear ribonucleoprotein (hnRNP) A1-dependent exonic splicing silencer. We now find that another rare nucleotide difference between SMN1 and SMN2, in intron 7, potentially creates a second SMN2-specific hnRNP A1 binding site. Remarkably, this single base change does indeed create a highaffinity hnRNP A1 binding site, and base substitutions that disrupt it restore exon 7 inclusion in vivo and prevent hnRNP A1 binding in vitro. We propose that interactions between hnRNP A1 molecules bound to the exonic and intronic sites cooperate to exclude exon 7 and discuss the significance of this exclusion with respect to SMN expression and splicing control more generally.alternative splicing ͉ exonic splicing silencer ͉ hnRNP A1 ͉ intronic splicing silencer A lternative splicing of mRNA precursors is an important mechanism that regulates gene expression and generates increased protein diversity from a limited number of genes. Indeed, expression of 70% or more of human genes is now estimated to involve alternative splicing (1, 2). In higher eukaryotes, alternative splicing plays an essential role in diverse cellular functions, contributing to such basic processes as cell growth, differentiation, and cell death (3, 4). Several types of cis-acting RNA elements and trans-acting protein factors help to regulate alternative splicing and do so by diverse mechanisms. In general, however, non-spliceosomal proteins that participate in regulating alternative splicing associate with regulatory elements in exons and/or introns. Serine/arginine-rich proteins (5) are typically involved in positive regulation of splicing, stimulating splicing by interacting with exonic splicing enhancer elements (ESEs) or intronic splicing enhancer elements. In contrast, negative regulation is promoted most frequently by heterogeneous nuclear ribonucleoproteins (hnRNPs) (6), which function by binding sequences known as exonic splicing silencers (ESSs) or intronic splicing silencers.Several hnRNP proteins have been identified as key splicing repressors. Among these, the abundant hnRNP A1 protein has been extensively characterized. The first hnRNP A1-dependent ESS was identified in studies of HIV tat exon 2 repression (7-9). This ESS was found to bind hnRNP A1, and mutations disrupting the ESS prevented hnRNP A1 binding and allowed enhanced exon 2 splicing. Several mechanisms have been proposed to explain hnRNP A1-mediated splicing repression. In one, hnRNP A1 binds to an ESS and through direct protein-protein interactions, recruits more ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

An intronic element contributes to splicing repression in spinal muscular atrophy

Kashima

Rao

Manley

2007

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

115

109

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“…However, the mutations in SMN2 pre-mRNA cause predominantly skipping of exon 7, which produces SMNΔ7, a truncated and less stable protein with reduced self-oligomerization activity. Alternative exon 7 splicing of SMN pre-mRNA was modulated by orchestrated RNAprotein and protein-protein interactions, secondary structures of RNA, and RNA sequences (25)(26)(27). Among the mutations on SMN2 pre-mRNA, the most functionally understood one is the C-to-U point mutation on exon 7, which plays an important role in alternative splicing of exon 7 (25-27).…”

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confidence: 99%

“…Alternative exon 7 splicing of SMN pre-mRNA was modulated by orchestrated RNAprotein and protein-protein interactions, secondary structures of RNA, and RNA sequences (25)(26)(27). Among the mutations on SMN2 pre-mRNA, the most functionally understood one is the C-to-U point mutation on exon 7, which plays an important role in alternative splicing of exon 7 (25)(26)(27). In vitro analysis using HeLa nuclear extract and S100 extract demonstrates that SRSF1 promotes exon 7 inclusion through contacting the enhancer sequence on exon 7 of SMN1 pre-mRNA, and that C-to-U mutation on SMN2 pre-mRNA disrupts SRSF1 binding and then consequently disrupts the enhancer function of SRSF1 (28).…”

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confidence: 99%

Splicing inhibition of U2AF ⁶⁵ leads to alternative exon skipping

Cho

Moon

Loh

et al. 2015

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

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U2 snRNP auxiliary factor 65 kDa (U2AF 65 ) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF 65 stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5′ splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF 65 . U2AF 65 overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF 65 inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF 65 effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF 65 even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF 65 possesses a splicing Inhibitory function that leads to alternative exon skipping.U2AF 65 | pre-mRNA splicing | splicing inhibition | exon exclusion | SMN P re-mRNA splicing is a process in which noncoding intron sequences are removed and exon sequences are then ligated together (1, 2). Pre-mRNA splicing is carried out by spliceosome, a large RNA-protein complex that contains five small nuclear ribonucleoproteins (U snRNPs) and more than 100 additional proteins (3). Pre-mRNA splicing occurs in the consensus sequences at the 5′ splice-site, 3′ splice-site, and branch point that are necessary for splicing. The sequence between 3′ AG dinucleotide and branch point is the polypyrimidine (Py) tract that directs spliceosome assembly on the 3′ splice-site. Alternative splicing provides an important regulatory mechanism in higher eukaryotes for multiple proteins produced from a single gene (4, 5).The U2 snRNP auxiliary factor 65 kDa (U2AF 65 ) exists as a heterodimer with U2AF 35 (6). U2AF 65 contains three C-terminal RNA recognition motifs (RRMs) and an N-terminal arginine/ serine-rich (RS) domain (7,8). Using U2AF 65 depletion/adding back technology with in vitro HeLa nuclear extract, it was demonstrated that U2AF 65 is an essential splicing factor (9). Whereas U2AF 65 binds to Py tract to promote prespliceosome assembly and branchpoint/U2 snRNA base pairing, U2AF 35 plays a role in the 3′ splice-site (10, 11). As U2AF 65 prefers high C/U-rich sequences in the Py tract, a stronger interaction between U2AF 65 and Py tract promotes prespliceosome assembly (12). U2AF 65 is also essential in vertebrate development (13,14). Its expression level is related to myotonic dystrophy, cystic fibrosis, and cancers (15, 16).Proximal spinal muscular atrophy (SMA) is an autosomal recessive genetic disease (17) and a leading cause of infant mortality. The motor neurons in the anterior horn of spinal cord are severely damaged in patients with type 1 SMA, usually leading to death before age 2 y as a result of a lack of respiratory support (18,19). In patients with SMA, the SMN1 gene is deleted or mutated, whereas the SMN2 gene, a duplicate of the SMN1 gene, is included (20). SMN2 genomic DNA...

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“…Ce variant altère non seulement une séquence ESE sur laquelle se fixe la protéine SR SF2/ASF (serine/arginine-rich splicing factor 2), mais entraîne également la création d'une séquence ESS sur laquelle se fixe la protéine hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1). Cela induit le saut de l'exon 7 dans certains transcrits : la protéine résultante est non-fonctionnelle et ne permet pas de compenser la perte de fonction de SMN1 [29].…”

Section: Exemples De Mutations Agissant En Cis Au Niveau D'une Régionunclassified

Altérations de l’épissage et maladies rares

Grange

2016

Med Sci (Paris)

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> La majorité des gènes codant des protéines chez l'homme sont soumis à l'épissage alternatif, le principal mécanisme permettant d'augmenter la diversité du transcriptome et du protéome. La régulation de l'épissage dépend de complexes ribonucléoprotéiques qui composent le splicéosome, ainsi que de protéines régulatrices qui reconnaissent des séquences au niveau des ARN pré-messagers. De plus en plus de mutations au niveau de ces séquences, mais également au niveau de composants du splicéosome ou de facteurs d'épissage, sont décrites pour être responsables du développement de maladies rares. Depuis ces dernières années, des approches de thérapie ciblée sont développées pour restaurer au moins partiellement des événements d'épissage altérés dans le cadre de ces maladies. < La Figure 1 présente les grandes étapes de la régulation de l'expression des gènes. Un gène peut être à l'origine de la synthèse de plusieurs transcrits distincts traduits en protéines différentes, qui peuvent avoir des fonctions ou des localisations cellulaires différentes. Certains transcrits ont également un rôle régulateur et sont dégradés par la voie du NMD (nonsense-mediated mRNA decay) [2]. Ces transcrits sont reconnus par le NMD parce qu'ils présentent un codon stop pré-maturé (ou PTC pour premature stop codon). L'épissage alternatif n'est pas une exception mais plutôt une règle puisque ce mécanisme concerne la très large majorité des gènes humains codants. Si on considère les gènes avec plusieurs exons, 90 % des gènes humains codants sont soumis à épissage alternatif [3,4]. Il y a en moyenne environ quatre transcrits différents par gène [3,4] et pour un tiers des gènes, l'épissage alternatif est à l'origine de la synthèse d'au moins cinq ARN messagers différents [3,4]. L'estimation de cette diversité des ARN messagers se fonde sur les connaissances actuelles, et notamment sur les bases de données de transcrits qui restent limitées : en fonction des types cellulaires et tissulaires, des stades de développement, des pathologies et des traitements appliqués (par exemple, siARN [small interfering RNA], composés chimiques, rayonnements ultra-violet, stress cytotoxique, etc.), à peine plus de la moitié seulement des transcrits seraient mis en évi-

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Determinants of Exon 7 Splicing in the Spinal Muscular Atrophy Genes, SMN1 and SMN2

Cited by 273 publications

References 62 publications

An intronic element contributes to splicing repression in spinal muscular atrophy

An intronic element contributes to splicing repression in spinal muscular atrophy

Splicing inhibition of U2AF ⁶⁵ leads to alternative exon skipping

Altérations de l’épissage et maladies rares

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Determinants of Exon 7 Splicing in the Spinal Muscular Atrophy Genes, SMN1 and SMN2

Cited by 273 publications

References 62 publications

An intronic element contributes to splicing repression in spinal muscular atrophy

An intronic element contributes to splicing repression in spinal muscular atrophy

Splicing inhibition of U2AF 65 leads to alternative exon skipping

Altérations de l’épissage et maladies rares

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Splicing inhibition of U2AF ⁶⁵ leads to alternative exon skipping