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Adrian R Krainer - One of the best experts on this subject based on the ideXlab platform.

  • An Exonic Splicing Enhancer is required for Splicing of IgM M1-M2 with ΔNΔRS.
    2013
    Co-Authors: Stephanie D. Shaw, Sutapa Chakrabarti, Gourisankar Ghosh, Adrian R Krainer
    Abstract:

    In vitro Splicing of IgM M1-M2 and derivative pre-mRNAs with mutations in the polypyrimidine tract, Exonic Splicing Enhancer, and/or Exonic Splicing silencer: IgM M1-M2 (lanes 1-3), IgMΔE (lanes 4–6), IgMPy↑ (lanes 7–9), IgMPy↑ΔE (lanes 10–12), IgMPTB (lanes 13–15), IgMΔEPTB (lanes 16–18), IgMPy↑PTB (lanes 19–21), and IgMPy↑ΔE PTB (lanes 22–24, mRNA position indicated by asterisk); in S100 alone (lanes 1, 4, 7, 10, 13, 16, 19, and 22), and S100 complemented with 16 pmol of SF2/ASF (lanes 2, 5, 8, 11, 14, 17, 20, and 23), or ΔNΔRS (lanes 3, 6, 9, 12, 15, 18, 21, and 24). The Splicing efficiency is indicated below each lane.

  • exon centric regulation of pyruvate kinase m alternative Splicing via mutually exclusive exons
    Journal of Molecular Cell Biology, 2012
    Co-Authors: Zhenxun Wang, Deblina Chatterjee, Hyun Yong Jeon, Martin Akerman, Matthew Vander G Heiden, Lewis C Cantley, Adrian R Krainer
    Abstract:

    Alternative Splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth. However, the cancer-specific alternative Splicing regulation of PK-M is not completely understood. Here, we demonstrate that PK-M is regulated by reciprocal effects on the mutually exclusive exons 9 and 10, such that exon 9 is repressed and exon 10 is activated in cancer cells. Strikingly, Exonic, rather than intronic, cis-elements are key determinants of PK-M Splicing isoform ratios. Using a systematic sub-Exonic duplication approach, we identify a potent Exonic Splicing Enhancer in exon 10, which differs from its homologous counterpart in exon 9 by only two nucleotides. We identify SRSF3 as one of the cognate factors, and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism. These findings provide mechanistic insights into the complex regulation of alternative Splicing of a key regulator of the Warburg effect, and also have implications for other genes with a similar pattern of alternative Splicing.

  • a positive modifier of spinal muscular atrophy in the smn2 gene
    American Journal of Human Genetics, 2009
    Co-Authors: Thomas W. Prior, Pamela C Snyder, Scott J Bridgeman, Arthur H.m. Burghes, Adrian R Krainer, Kathryn J Swoboda, John T. Kissel
    Abstract:

    Spinal muscular atrophy (SMA) is a common autosomal-recessive motor neuron disease caused by the homozygous loss of the SMN1 gene. A nearly identical gene, SMN2, has been shown to decrease the severity of SMA in a dose-dependent manner. However SMN2 is not the sole phenotypic modifier, because there are discrepant SMA cases in which the SMN2 copy number does not explain the clinical phenotype. This report describes three unrelated SMA patients who possessed SMN2 copy numbers that did not correlate with the observed mild clinical phenotypes. A single base substitution in SMN2, c.859G>C,, was identified in exon 7 in the patients' DNA. We now show that the change creates a new Exonic Splicing Enhancer element and increases the amount of full-length transcripts, thus resulting in the less severe phenotypes. This demonstrates that the c.859G>C substitution is a positive modifier of the SMA phenotype and that not all SMN2 genes are equivalent. We have shown not only that the SMA phenotype is modified by the number of SMN2 genes but that SMN2 sequence variations can also affect the disease severity.

  • distribution of sr protein Exonic Splicing Enhancer motifs in human protein coding genes
    Nucleic Acids Research, 2005
    Co-Authors: Jinhua Wang, Adrian R Krainer, Philip J Smith, Michael Q Zhang
    Abstract:

    Exonic Splicing Enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (http://rulai.cshl.edu/tools/ESE/). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive Splicing and regulated alternative Splicing.

  • disruption of Exonic Splicing Enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of nf1
    Human Mutation, 2004
    Co-Authors: Andrea Zatkova, Adrian R Krainer, Ludwine Messiaen, Ina Vandenbroucke, Rotraud Wieser, Christa Fonatsch, Katharina Wimmer
    Abstract:

    Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of Splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA Splicing mechanisms. Using cDNA-based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional Exonic Splicing Enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild-type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue Splicing of an Enhancer-dependent exon, and found that all seven mutant ESEs had reduced Splicing enhancement activity compared to the wild-type sequences. Our results suggest that the wild-type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense and silent mutations, and to understand the structure-function relationship in the corresponding protein.

Massimo Caputi - One of the best experts on this subject based on the ideXlab platform.

  • a bidirectional sf2 asf and srp40 dependent Splicing Enhancer regulates human immunodeficiency virus type 1 rev env vpu and nef gene expression
    Journal of Virology, 2004
    Co-Authors: Massimo Caputi, Marcel Freund, Susanne Kammler, Corinna Asang, Heiner Schaal
    Abstract:

    The integrated human immunodeficiency virus type 1 (HIV-1) genome is transcribed in a single pre-mRNA that is alternatively spliced into more than 40 mRNAs. We characterized a novel bidirectional Exonic Splicing Enhancer (ESE) that regulates the expression of the HIV-1 env, vpu, rev, and nef mRNAs. The ESE is localized downstream of the vpu-, env-, and nef-specific 3' splice site no. 5. SF2/ASF and SRp40 activate the ESE and are required for efficient 3' splice site usage and binding of the U1 snRNP to the downstream 5' splice site no. 4. U1 snRNP binding to the 5' splice site no. 4 is required for Splicing of the rev and nef mRNAs and to increase expression of the partially spliced env mRNA. Finally, our results indicate that this ESE is necessary for the recruitment of the U1 snRNP to the 5' splice site no. 4, even when the 5' splice site and the U1 snRNA have been mutated to obtain a perfect complementary match. The ESE characterized here is highly conserved in most viral subtypes.

  • sc35 and heterogeneous nuclear ribonucleoprotein a b proteins bind to a juxtaposed Exonic Splicing Enhancer Exonic Splicing silencer element to regulate hiv 1 tat exon 2 Splicing
    Journal of Biological Chemistry, 2004
    Co-Authors: Alan M Zahler, Christian Kroun Damgaard, Jorgen Kjems, Massimo Caputi
    Abstract:

    Abstract Splicing of the human immunodeficiency virus, type 1, primary transcript is highly regulated. Maintaining the proper equilibrium among spliced, unspliced, and partially spliced isoforms is essential for the replication of the virus. Here we characterize a complex cis-acting element located in tat exon 2 that is required for the Splicing regulation of the upstream intron. An Exonic Splicing Enhancer (ESE) and an Exonic Splicing silencer (ESS) are both located within the regulatory element. Heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins bind the ESS to repress Splicing, whereas the SR protein SC35 binds the ESE to activate it. We show that the SC35 and the hnRNP A1 binding sites overlap within the juxtaposed ESE/ESS. We propose that hnRNP A1 binding to the ESS inhibits Splicing of the upstream intron by directly masking the SC35 binding site.

  • a nonsense mutation in the fibrillin 1 gene of a marfan syndrome patient induces nmd and disrupts an Exonic Splicing Enhancer
    Genes & Development, 2002
    Co-Authors: Massimo Caputi, Raymond J Kendzior, Karen L Beemon
    Abstract:

    A nonsense mutation in the fibrillin-1 (FBN1) gene of a Marfan syndrome (MFS) patient induces in-frame exon skipping of FBN1 exon 51. We present evidence, based on both in vivo and in vitro experiments, that the skipping of this exon is due to the disruption of an SC35-dependent Splicing Enhancer within exon 51. In addition, this nonsense mutation induces nonsense-mediated decay (NMD), which degrades the normally spliced mRNA in the patient's cells. In contrast to NMD, skipping of FBN1 exon 51 does not require translation.

  • regulation of fibronectin eda exon alternative Splicing possible role of rna secondary structure for Enhancer display
    Molecular and Cellular Biology, 1999
    Co-Authors: Andres F Muro, Massimo Caputi, Rajalakshmi Pariyarath, Franco Pagani, Emanuele Buratti, Francisco E Baralle
    Abstract:

    The fibronectin primary transcript undergoes alternative Splicing in three noncoordinated sites: the cassette-type EDA and EDB exons and the more complex IIICS region. We have shown previously that an 81-nucleotide region within the EDA exon is necessary for exon recognition and that this region contains at least two Splicing-regulatory elements: a polypurinic Enhancer (Exonic Splicing Enhancer [ESE]) and a nearby silencer element (Exonic Splicing silencer [ESS]). Here, we have analyzed the function of both elements in different cell types. We have mapped the ESS to the nucleotide level, showing that a single base change is sufficient to abolish its function. Testing of the ESE and ESS elements in heterologous exons, individually or as part of the complete EDA regulatory region, showed that only the ESE element is active in different contexts. Functional studies coupled to secondary-structure enzymatic analysis of the EDA exon sequence variants suggest that the role of the ESS element may be exclusively to ensure the proper RNA conformation and raise the possibility that the display of the ESE element in a loop position may represent a significant feature of the exon Splicing-regulatory region.

Thomas Koed Doktor - One of the best experts on this subject based on the ideXlab platform.

  • the deep intronic c 903 469t c mutation in the mtrr gene creates an sf2 asf binding Exonic Splicing Enhancer which leads to pseudoexon activation and causes the cble type of homocystinuria
    Human Mutation, 2010
    Co-Authors: Katerina Homolova, Lisbeth Dahl Schroeder, Thomas Koed Doktor, Petra Zavadakova, Viktor Kozich, Brage S Andresen
    Abstract:

    Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA mis-Splicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5' splice site. Within the pseudoexon we identified an Exonic Splicing Enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in Splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific Splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported.

  • seemingly neutral polymorphic variants may confer immunity to Splicing inactivating mutations a synonymous snp in exon 5 of mcad protects from deleterious mutations in a flanking Exonic Splicing Enhancer
    American Journal of Human Genetics, 2007
    Co-Authors: Lisbeth Dahl Schroeder, Luca Cartegni, Suzette Sorensen, Karsten Nielsen, Thomas Koed Doktor, Thomas J Corydon, Line S Reinert
    Abstract:

    The idea that point mutations in exons may affect Splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial Exonic Splicing Enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed Exonic Splicing silencer (ESS) and is necessary to define a suboptimal 3′ splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on Splicing by itself, it makes Splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on Splicing be evaluated in the context of the relevant haplotype.

  • seemingly neutral polymorphic variants may confer immunity to Splicing inactivating mutations a synonymous snp in exon 5 of mcad protects from deleterious mutations in a flanking Exonic Splicing Enhancer
    American Journal of Human Genetics, 2007
    Co-Authors: Lisbeth Dahl Schroeder, Luca Cartegni, Suzette Sorensen, Karsten Nielsen, Thomas Koed Doktor, Thomas J Corydon, Line S Reinert
    Abstract:

    The idea that point mutations in exons may affect Splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial Exonic Splicing Enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed Exonic Splicing silencer (ESS) and is necessary to define a suboptimal 3′ splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on Splicing by itself, it makes Splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on Splicing be evaluated in the context of the relevant haplotype.

Karsten Nielsen - One of the best experts on this subject based on the ideXlab platform.

  • seemingly neutral polymorphic variants may confer immunity to Splicing inactivating mutations a synonymous snp in exon 5 of mcad protects from deleterious mutations in a flanking Exonic Splicing Enhancer
    American Journal of Human Genetics, 2007
    Co-Authors: Lisbeth Dahl Schroeder, Luca Cartegni, Suzette Sorensen, Karsten Nielsen, Thomas Koed Doktor, Thomas J Corydon, Line S Reinert
    Abstract:

    The idea that point mutations in exons may affect Splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial Exonic Splicing Enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed Exonic Splicing silencer (ESS) and is necessary to define a suboptimal 3′ splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on Splicing by itself, it makes Splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on Splicing be evaluated in the context of the relevant haplotype.

  • seemingly neutral polymorphic variants may confer immunity to Splicing inactivating mutations a synonymous snp in exon 5 of mcad protects from deleterious mutations in a flanking Exonic Splicing Enhancer
    American Journal of Human Genetics, 2007
    Co-Authors: Lisbeth Dahl Schroeder, Luca Cartegni, Suzette Sorensen, Karsten Nielsen, Thomas Koed Doktor, Thomas J Corydon, Line S Reinert
    Abstract:

    The idea that point mutations in exons may affect Splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial Exonic Splicing Enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed Exonic Splicing silencer (ESS) and is necessary to define a suboptimal 3′ splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on Splicing by itself, it makes Splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on Splicing be evaluated in the context of the relevant haplotype.

Luca Cartegni - One of the best experts on this subject based on the ideXlab platform.

  • seemingly neutral polymorphic variants may confer immunity to Splicing inactivating mutations a synonymous snp in exon 5 of mcad protects from deleterious mutations in a flanking Exonic Splicing Enhancer
    American Journal of Human Genetics, 2007
    Co-Authors: Lisbeth Dahl Schroeder, Luca Cartegni, Suzette Sorensen, Karsten Nielsen, Thomas Koed Doktor, Thomas J Corydon, Line S Reinert
    Abstract:

    The idea that point mutations in exons may affect Splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial Exonic Splicing Enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed Exonic Splicing silencer (ESS) and is necessary to define a suboptimal 3′ splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on Splicing by itself, it makes Splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on Splicing be evaluated in the context of the relevant haplotype.

  • seemingly neutral polymorphic variants may confer immunity to Splicing inactivating mutations a synonymous snp in exon 5 of mcad protects from deleterious mutations in a flanking Exonic Splicing Enhancer
    American Journal of Human Genetics, 2007
    Co-Authors: Lisbeth Dahl Schroeder, Luca Cartegni, Suzette Sorensen, Karsten Nielsen, Thomas Koed Doktor, Thomas J Corydon, Line S Reinert
    Abstract:

    The idea that point mutations in exons may affect Splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial Exonic Splicing Enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed Exonic Splicing silencer (ESS) and is necessary to define a suboptimal 3′ splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on Splicing by itself, it makes Splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on Splicing be evaluated in the context of the relevant haplotype.

  • brca2 t2722r is a deleterious allele that causes exon skipping
    American Journal of Human Genetics, 2002
    Co-Authors: James D Fackenthal, Luca Cartegni, Adrian R Krainer, Olufunmilayo I Olopade
    Abstract:

    Patients with a strong family history of breast cancer are often counseled to receive genetic screening for BRCA1 and BRCA2 mutations, the strongest known predictors of breast cancer. A major limitation of genetic testing is the number of inconclusive results due to unclassified BRCA1 and BRCA2 sequence variants. Many known deleterious BRCA1 and BRCA2 mutations affect Splicing, and these typically lie near intron/exon boundaries. However, there are also potential internal Exonic mutations that disrupt functional Exonic Splicing Enhancer (ESE) sequences, resulting in exon skipping. Using previously established sequence matrices for the scoring of putative ESE motifs, we have systematically examined several BRCA2 mutations for potential ESE disruption mutations. These predictions revealed that BRCA2 T2722R (8393C→G), which segregates with affected individuals in a family with breast cancer, disrupts three potential ESE sites. Reverse-transcriptase polymerase chain reaction analysis confirms that this mutation causes exon skipping, leading to an out-of-frame fusion of BRCA2 exons 17 and 19. This represents the first BRCA2 missense mutation shown to be a predicted deleterious protein-truncating mutation and suggests a potentially useful method for determining the clinical significance of a subset of the many unclassified variants in BRCA1 and BRCA2.

  • disruption of an sf2 asf dependent Exonic Splicing Enhancer in smn2 causes spinal muscular atrophy in the absence of smn1
    Nature Genetics, 2002
    Co-Authors: Luca Cartegni, Adrian R Krainer
    Abstract:

    Alteration of correct Splicing patterns by disruption of an Exonic Splicing Enhancer may be a frequent mechanism by which point mutations cause genetic diseases. Spinal muscular atrophy results from the lack of functional survival of motor neuron 1 gene (SMN1), even though all affected individuals carry a nearly identical, normal SMN2 gene. SMN2 is only partially active because a translationally silent, single-nucleotide difference in exon 7 causes exon skipping. Using ESE motif-prediction tools, mutational analysis and in vivo and in vitro Splicing assays, we show that this single-nucleotide change occurs within a heptamer motif of an Exonic Splicing Enhancer, which in SMN1 is recognized directly by SF2/ASF. The abrogation of the SF2/ASF-dependent ESE is the basis for inefficient inclusion of exon 7 in SMN2, resulting in the spinal muscular atrophy phenotype.

  • Exonic Splicing Enhancer motif recognized by human SC35 under Splicing conditions
    2000
    Co-Authors: Hong Xiang Liu, Luca Cartegni, Michael Q Zhang, Shern L. Chew, R. Krainer
    Abstract:

    Exonic Splicing Enhancers (ESEs) are important cis elements required for exon inclusion. Using an in vitro functional selection and amplification procedure, we have identified a novel ESE motif recognized by the human SR protein SC35 under Splicing conditions. The selected sequences are functional and specific: they promote Splicing in nuclear extract or in S100 extract complemented by SC35 but not by SF2/ASF. They can also function in a different Exonic context from the one used for the selection procedure. The selected sequences share one or two close matches to a short and highly degenerate octamer consensus, GRYYcSYR. A score matrix was generated from the selected sequences according to the nucleotide frequency at each position of their best match to the consensus motif. The SC35 score matrix, along with our previously reported SF2/ASF score matrix, was used to search the sequences of two well-characterized Splicing substrates derived from the mouse immunoglobulin M (IgM) and human immunodeficiency virus tat genes. Multiple SC35 high-score motifs, but only two widely separated SF2/ASF motifs, were found in the IgM C4 exon, which can be spliced in S100 extract complemented by SC35. In contrast, multiple high-score motifs for both SF2/ASF and SC35 were found in a variant of the Tat T3 exon (lacking an SC35-specific silencer) whose Splicing can be complemented by either SF2/ASF or SC35. The motif score matrix can help locate SC35-specific Enhancers in natural exon sequences