The role of U2AF35 and U2AF65 in enhancer-dependent splicing

Graveley, Brenton R.; Hertel, Klemens J.; Maniatis, Tom

doi:10.1017/s1355838201010317

Cited by 133 publications

(125 citation statements)

References 41 publications

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“…In one model for their effects, they are bound by SR or other proteins that promote spliceosome formation, aiding the recognition of nearby splice sites and activating splicing (29)(30)(31)(32)(33). We have devised a strategy that takes advantage of an antisense oligonucleotide approach but, in contrast to the conventional use of such oligonucleotides as physical obstructions of a reaction at a target site or as mediators of RNase H degradation, we have used these oligonucleotides to Recruitment of SF2͞ASF to SMN2 exon 7 by the 5ЈGGA oligonucleotide.…”

Section: Discussionmentioning

confidence: 99%

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts

Skordis

Dunckley

Yue

et al. 2003

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

246

201

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The multiplicity of proteins compared with genes in mammals owes much to alternative splicing. Splicing signals are so subtle and complex that small perturbations may allow the production of new mRNA variants. However, the flexibility of splicing can also be a liability, and several genetic diseases result from single-base changes that cause exons to be skipped during splicing. Conventional oligonucleotide strategies can block reactions but cannot restore splicing. We describe here a method by which the use of a defective exon was restored. Spinal muscular atrophy (SMA) results from mutations of the Survival Motor Neuron (SMN) gene. Mutations of SMN1 cause SMA, whereas SMN2 acts as a modifying gene. The two genes undergo alternative splicing with SMN1, producing an abundance of full-length mRNA transcripts, whereas SMN2 predominantly produces exon 7-deleted transcripts. This discrepancy is because of a single nucleotide difference in SMN2 exon 7, which disrupts an exonic splicing enhancer containing an SF2͞ASF binding site. We have designed oligoribonucleotides that are complementary to exon 7 and contain exonic splicing enhancer motifs to provide trans-acting enhancers. These tailed oligoribonucleotides increased SMN2 exon 7 splicing in vitro and rescued the incorporation of SMN2 exon 7 in SMA patient fibroblasts. This treatment also resulted in the partial restoration of gems, intranuclear structures containing SMN protein that are severely reduced in patients with SMA. The use of tailed antisense oligonucleotides to recruit positively acting factors to stimulate a splicing reaction may have therapeutic applications for genetic disorders, such as SMA, in which splicing patterns are altered. P roximal spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the degeneration of the motor neurons in the anterior horn of the spinal cord, resulting in muscular atrophy and weakness. The overall incidence of SMA is 1 in 10,000 live births, with a carrier frequency of 1 in 50. Onset is primarily in childhood, and three different forms are recognized, type I SMA being the most severe form and type III SMA at the milder end of the scale. Children affected by SMA I never sit and usually die within the first year of life, whereas those with type III acquire the ability to walk and have a normal life expectancy. An intermediate category (SMA II) is also recognized, where affected individuals can sit unsupported but cannot walk (1). The gene implicated in SMA is the Survival of Motor Neuron (SMN) gene located on chromosome 5q13 (2). It consists of eight exons, the first seven of which encode a 294-aa protein (3). The human SMN gene exists as a mirror-image duplication with a telomeric (SMN1) and a centromeric (SMN2) copy. Mutations in SMN1 cause SMA (4), whereas the copy number of the residual SMN2 genes is believed to modify the severity of the phenotype, as suggested by the increased copy number in patients with a milder disease course (5). Deletions of both SMN1 and SMN2 have never been observed ...

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Section: Discussionmentioning

confidence: 99%

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts

Skordis

Dunckley

Yue

et al. 2003

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

246

201

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“…Two of the major players in establishing exon identity are the serine-and arginine-rich proteins (SR proteins) and the heterogeneous nuclear ribonucleoproteins (hnRNPs) (for review, see Wang and Burge 2008). SR proteins promote the initial stages of spliceosome assembly by binding to ESEs and recruiting basal splicing factors to adjacent splice sites or by antagonizing the effects of ESS elements (Kohtz et al 1994;Graveley et al 2001;Zhu et al 2001). In contrast, hnRNPs mediate the repressive effects of silencers and can alter recruitment of the core splicing machinery (Wang et al 2006;Yu et al 2008).…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Loss of exon identity is a common mechanism of human inherited disease

Sterne-Weiler¹,

Howard²,

Mort³

et al. 2011

Genome Res.

160

155

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It is widely accepted that at least 10% of all mutations causing human inherited disease disrupt splice-site consensus sequences. In contrast to splice-site mutations, the role of auxiliary cis-acting elements such as exonic splicing enhancers (ESE) and exonic splicing silencers (ESS) in human inherited disease is still poorly understood. Here we use a top-down approach to determine rates of loss or gain of known human exonic splicing regulatory (ESR) sequences associated with either disease-causing mutations or putatively neutral single nucleotide polymorphisms (SNPs). We observe significant enrichment toward loss of ESEs and gain of ESSs among inherited disease-causing variants relative to neutral polymorphisms, indicating that exon skipping may play a prominent role in aberrant gene regulation. Both computational and biochemical approaches underscore the relevance of exonic splicing enhancer loss and silencer gain in inherited disease. Additionally, we provide direct evidence that both SRp20 (SRSF3 ) and possibly PTB (PTBP1) are involved in the function of a splicing silencer that is created de novo by a total of 83 different inherited disease mutations in 67 different disease genes. Taken together, we find that~25% (7154/27,681) of known mis-sense and nonsense disease-causing mutations alter functional splicing signals within exons, suggesting a much more widespread role for aberrant mRNA processing in causing human inherited disease than has hitherto been appreciated.

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“…This family of proteins frequently enforces exon inclusion by binding to exonic splicing enhancers to stimulate the binding of generic splicing factors including U2AF, U2 snRNP, and U1 snRNP (Lavigueur et al 1993;Kohtz et al 1994;Wang et al 1995;Zuo and Maniatis 1996;Graveley et al 2001). However, SR proteins can also have negative impacts on splicing.…”

Section: Introductionmentioning

confidence: 99%

hnRNP A1 and hnRNP H can collaborate to modulate 5′ splice site selection

Fisette¹,

Toutant²,

Dugré-Brisson³

et al. 2009

RNA

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The mammalian proteins hnRNP A1 and hnRNP H control many splicing decisions in viral and cellular primary transcripts. To explain some of these activities, we have proposed that self-interactions between bound proteins create an RNA loop that represses internal splice sites while simultaneously activating the external sites that are brought in closer proximity. Here we show that a variety of hnRNP H binding sites can affect 59 splice site selection. The addition of two sets of hnRNP H sites in a model pre-mRNA modulates 59 splice site selection cooperatively, consistent with the looping model. Notably, binding sites for hnRNP A1 and H on the same pre-mRNA can similarly collaborate to modulate 59 splice site selection. The C-terminal portion of hnRNP H that contains the glycine-rich domains (GRD) is essential for splicing activity, and it can be functionally replaced by the GRD of hnRNP A1. Finally, we used the bioluminescence resonance energy transfer (BRET) technology to document the existence of homotypic and heterotypic interactions between hnRNP H and hnRNP A1 in live cells. Overall, our study suggests that interactions between different hnRNP proteins bound to distinct locations on a pre-mRNA can change its conformation to affect splicing decisions.

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The role of U2AF35 and U2AF65 in enhancer-dependent splicing

Cited by 133 publications

References 41 publications

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts

Loss of exon identity is a common mechanism of human inherited disease

hnRNP A1 and hnRNP H can collaborate to modulate 5′ splice site selection

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