Identification of an Intronic Single-Point Mutation in<i>RP2</i>as the Cause of Semidominant X-linked Retinitis Pigmentosa

Pomares, Esther; Riera, Marina; Castro-Navarro, J.; Andrés-Gutiérrez, Ángeles; Gonzàlez‐Duarte, Roser; Marfany, Gemma

doi:10.1167/iovs.08-3208

Cited by 21 publications

(21 citation statements)

References 24 publications

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“…S4A). As reported previously (30), nearly all of the detected RP2 mRNAs composing the WT Py tract included the central exon (exon 4) (Fig. S4B).…”

Section: Resultssupporting

confidence: 84%

“…Although numerous examples of diseasecausing mutations in Py tracts have been identified, few studies are available on the consequences of these mutations for the splice site affinities of U2AF 65 . To investigate the involvement of U2AF 65 inhibition, we examined the effects on U2AF 65 association of mutations in two representative Py tracts (Table 1): (i) a U→A transversion in the Py tract of the neurofibromin 1 gene [NF1(U3 > A)], which creates a new 3′ splice site junction (consensus AG) and leads to familial neurofibromatosis type I (29), and (ii) a U→A transversion in the Py tract of the retinitis pigmentosa 2 (RP2) gene [RP2(U4 > A)] that also introduces an AG dinucleotide and induces exon skipping and, consequently, X-linked retinitis pigmentosa (30). We titrated the Py tract recognition domain of U2AF 65 comprising RRM1 and RRM2 and bordering residues into either the WT or mutated fluoresceinlabeled Py tract RNAs, and then fit the apparent equilibrium dissociation constant (K D ) values to anisotropy changes as described previously (31) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We next tested whether the synthetic U2AF 65 -D231V variant could correct defective splicing of the retinitis pigmentosacausing RP2 mutation [RP2(U4 > A)] using a splicing reporter minigene in a human cell line (30) (Fig. 2 and Fig.…”

Section: Resultsmentioning

confidence: 99%

“…As such, cotransfection with either the WT or D231V-modified U2AF 65 increased exon inclusion only slightly (by 4% or 7% of the total spliced transcript, respectively). Conversely, nearly all of the RP2 (U4 > A) transcript harboring the retinitis pigmentosa-causing mutation skipped exon 4, instead joining exons 3 and 5 (30) (Fig. 2B and Fig.…”

Section: Resultsmentioning

confidence: 99%

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Structure-guided U2AF ⁶⁵ variant improves recognition and splicing of a defective pre-mRNA

Agrawal

McLaughlin

Jenkins

et al. 2014

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

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Purine interruptions of polypyrimidine (Py) tract splice site signals contribute to human genetic diseases. The essential splicing factor U2AF 65 normally recognizes a Py tract consensus sequence preceding the major class of 3′ splice sites. We found that neurofibromatosisor retinitis pigmentosa-causing mutations in the 5′ regions of Py tracts severely reduce U2AF 65 affinity. Conversely, we identified a preferred binding site of U2AF 65 for purine substitutions in the 3′ regions of Py tracts. Based on a comparison of new U2AF 65 structures bound to either A-or G-containing Py tracts with previously identified pyrimidine-containing structures, we expected to find that a D231V amino acid change in U2AF 65 would specify U over other nucleotides. We found that the crystal structure of the U2AF 65 -D231V variant confirms favorable packing between the engineered valine and a target uracil base. The D231V amino acid change restores U2AF 65 affinity for two mutated splice sites that cause human genetic diseases and successfully promotes splicing of a defective retinitis pigmentosa-causing transcript. We conclude that reduced U2AF 65 binding is a molecular consequence of disease-relevant mutations, and that a structure-guided U2AF 65 variant is capable of manipulating gene expression in eukaryotic cells.pre-mRNA splicing | protein-RNA complex | protein engineering | crystal structure | RRM A pproximately 15% of the documented disease-causing point mutations disrupt consensus splice site elements in premRNAs, including a polypyrimidine (Py) tract between a branch point sequence (BPS) and an AG dinucleotide at the junction of the 3′ splice site (1) (Fig. 1A). For example, disease-causing mutations in Py tracts have been documented in ∼3,000 genes in the Human Gene Mutation Database (2), and an estimated 20% of these mutations affect regulatory splice site signals (3, 4). One of the earliest reports of a splice site mutation as a major cause of inherited human disease was for β-thalassemia (reviewed in ref. 5), for which splice site mutations in the human β-globin gene (HBB) are found in ∼14% of patients, causing symptoms of mild to severe anemia (reviewed in ref. 6).With the emergence of high-throughput sequencing technologies, splice site mutations in specific transcripts have been identified as common contributors to neuromuscular disorders, metabolic disorders, cancers, leukemias, deafness, and blindness, among other disorders (reviewed in ref. 4). Retinitis pigmentosa, the most prevalent form of inherited blindness in adults, represents one such disease that is primarily the consequence of mutations in splice sites of vision-relevant transcripts or splicing factors responsible for their recognition (reviewed in ref. 7). Neurofibromatosis type I, a disease characterized by tumors of nerve tissue, is an inherited disorder in which nearly 30% of the documented mutations disrupt neurofibromin 1 (NF1) splice sites (reviewed in ref. 8). Despite etiologic progress, the relationships between disease-causing pre-mRNA splice s...

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“…S4A). As reported previously (30), nearly all of the detected RP2 mRNAs composing the WT Py tract included the central exon (exon 4) (Fig. S4B).…”

Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Structure-guided U2AF ⁶⁵ variant improves recognition and splicing of a defective pre-mRNA

Agrawal

McLaughlin

Jenkins

et al. 2014

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

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show abstract

“…RNA splicing requires a complex interplay of multiple RNA-binding proteins that are equipped with domains to bind sequence motifs on single stranded RNA to ensure accurate determination of exon recognition. In silico interrogation of the wild-type pre-mRNA segment derived from the 132-bp mutation-rich intronic sequence between exons 12 and 13 for accessible splicing factor binding sites resulted in identification of multiple potential binding sites for the RNA-binding proteins hnRNP-E2/ PCBP, hnRNP-I/PTB, and hnRNP-L, three members of the heterogenous nuclear ribonucleoprotein family of splicing factors that act as global regulators of alternative splicing and are abundantly expressed in infant leukemias (40)(41)(42)(43)(44)(45)(46)(47)(48)(49) (Fig. 2A.1).…”

Section: Resultsmentioning

confidence: 99%

CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia

Uckun

Goodman

et al. 2010

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

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Here, we report that primary leukemic cells from infants with newly diagnosed B-precursor leukemia express a truncated and functionally defective CD22 coreceptor protein that is unable to transmit apoptotic signals because it lacks most of the intracellular domain, including the key regulatory signal transduction elements and all of the cytoplasmic tyrosine residues. Expression of this structurally and functionally abnormal CD22 protein is associated with a very aggressive in vivo growth of patients' primary leukemia cells causing disseminated overt leukemia in SCID mice. The abnormal CD22 coreceptor is encoded by a profoundly aberrant mRNA arising from a splicing defect that causes the deletion of exon 12 (c.2208-c.2327) (CD22ΔE12) and results in a truncating frameshift mutation. The splicing defect is associated with multiple homozygous mutations within a 132-bp segment of the intronic sequence between exons 12 and 13. These mutations cause marked changes in the predicted secondary structures of the mutant CD22 pre-mRNA sequences that affect the target motifs for the splicing factors hnRNP-L, PTB, and PCBP that are up-regulated in infant leukemia cells. Forced expression of the mutant CD22ΔE12 protein in transgenic mice perturbs B-cell development, as evidenced by B-precursor/B-cell hyperplasia, and corrupts the regulation of gene expression, causing reduced expression levels of several genes with a tumor suppressor function. We further show that CD22ΔE12-associated unique gene expression signature is a discriminating feature of newly diagnosed infant leukemia patients. These striking findings implicate CD22ΔE12 as a previously undescribed pathogenic mechanism in human B-precursor leukemia.

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Design and In Vitro Use of Antisense Oligonucleotides to Correct Pre-mRNA Splicing Defects in Inherited Retinal Dystrophies

Garanto

Collin

2017

Retinal Gene Therapy

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Antisense oligonucleotides (AONs) are small molecules able to bind to the pre-mRNA and modulate splicing. The increasing amount of intronic mutations leading to pseudoexon insertion in genes underlying inherited retinal dystrophies (IRDs) has highlighted the potential of AONs as a therapeutic tool for these disorders. Here we describe how to design and test AON molecules in vitro in order to correct pre-mRNA splicing defects involved in IRDs.

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Identification of an Intronic Single-Point Mutation inRP2as the Cause of Semidominant X-linked Retinitis Pigmentosa

Cited by 21 publications

References 24 publications

Structure-guided U2AF ⁶⁵ variant improves recognition and splicing of a defective pre-mRNA

Structure-guided U2AF ⁶⁵ variant improves recognition and splicing of a defective pre-mRNA

CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia

Design and In Vitro Use of Antisense Oligonucleotides to Correct Pre-mRNA Splicing Defects in Inherited Retinal Dystrophies

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Identification of an Intronic Single-Point Mutation inRP2as the Cause of Semidominant X-linked Retinitis Pigmentosa

Cited by 21 publications

References 24 publications

Structure-guided U2AF 65 variant improves recognition and splicing of a defective pre-mRNA

Structure-guided U2AF 65 variant improves recognition and splicing of a defective pre-mRNA

CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia

Design and In Vitro Use of Antisense Oligonucleotides to Correct Pre-mRNA Splicing Defects in Inherited Retinal Dystrophies

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Product

Resources

About

Structure-guided U2AF ⁶⁵ variant improves recognition and splicing of a defective pre-mRNA

Structure-guided U2AF ⁶⁵ variant improves recognition and splicing of a defective pre-mRNA