Alternative splicing in disease

Human pyruvate dehydrogenase (PDH)-complex deficiency is an inborn error of metabolism that is extremely heterogeneous in its presentation and clinical course. In a study of 14 patients (7 females and 7 males), we have found a mutation in the coding region of the E1 alpha gene in all 14 patients. Two female patients had the same 7-bp deletion at nt 927; another female patient had a 3-bp deletion at nt 931. Another female patient was found to have a deletion of exon 6 in her cDNA. Two other female patients were found to have insertions, one of 13 bp at nt 981 and one of 46 bp at nucleotide 1078. Two male patients were found to have a 4-bp insertion at nucleotide 1163. The remaining six patients all had missense mutations. A male patient and a female patient both had an A1133G mutation. The other missense mutations were C214T, C615A, and C787G (two patients). Five of these mutations are novel mutations, five have been previously reported in other patients, and two were published obser...

Nucleic acid therapeutics are witnessing an impressive acceleration in recent years. They work through multiple mechanisms of action, including downregulation of gene expression and modulation of RNA splicing. While several drugs based on the former mechanism have been approved, few target the latter, despite the promise of RNA splicing modulation. To improve our ability to discover novel RNA splicing-modulating therapies, we developed HCS-Splice, a robust cell-based High-Content Screening (HCS) assay. By implementing the use of a two-colour (GFP/RFP) fluorescent splicing reporter plasmid, we developed a versatile, effective, rapid, and robust high-throughput strategy for the identification of potent splicing-modulating molecules. The HCS-Splice strategy can also be used to functionally confirm splicing mutations in human genetic disorders or to screen drug candidates. As a proof-of-concept, we introduced a dementia-related splice-switching mutation in Microtubule-Associated Protein...

The matricellular protein Cysteine rich 61 (Cyr61) displays a remarkable diversity of multiple cellular functions involved in significant physiologic and pathologic processes. Cyr61 is known as an important player in tumor progression, promoting neovascularization and metastasis. Our prior investigations elucidated an oxygen-dependent Cyr61 alternative splicing process characterized by retention of its intron 3, regulating its biological function in a hypoxia-driven on/off switch mechanism. In this work, we identified extracellular acidosis as a potent inducer for altered Cyr61 alternative splicing pattern regulating Cyr61 expression. Intriguingly, splicing factor hTRA2-beta1 displayed an opposite effect on Cyr61 expression. Nuclear hTRA2-beta1 protein expression was found markedly reduced under acidic conditions. In keeping with these conclusions, we show that hTRA2-beta1 can specifically bind a 'GAAG' motif in Cyr61 exon 3 RNA, that the splicing factor displays acidosis-dependent protein localization in cellular compartments, and shRNA-mediated hTRA2-beta1 knock-down triggers the same effects on Cyr61 alternative splicing like acidosis or hypoxia. Our findings strongly support the hypothesis of a specific regulation of Cyr61 expression by hTRA2-beta1.

Nucleic acid therapeutics are witnessing an impressive acceleration in recent years. They work through multiple mechanisms of action, including downregulation of gene expression and modulation of RNA splicing. While several drugs based on the former mechanism have been approved, few target the latter, despite the promise of RNA splicing modulation. To improve our ability to discover novel RNA splicing-modulating therapies, we developed HCS-Splice, a robust cell-based High-Content Screening (HCS) assay. By implementing the use of a two-colour (GFP/RFP) fluorescent splicing reporter plasmid, we developed a versatile, effective, rapid, and robust high-throughput strategy for the identification of potent splicing-modulating molecules. The HCS-Splice strategy can also be used to functionally confirm splicing mutations in human genetic disorders or to screen drug candidates. As a proof-of-concept, we introduced a dementia-related splice-switching mutation in Microtubule-Associated Protein...

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase C␦ (PKC␦), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKC␦, PKC␦VIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKC␦VIII via utilization of a downstream 5 splice site of exon 10 on PKC␦ pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKC␦VIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKC␦VIII alternative splicing. To identify the cis-elements involved in 5 splice site selection we cloned a minigene, which included PKC␦ exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5 splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKC␦ minigene promoted selection of 5 splice site II. Mutation of the SC35 binding site in the PKC␦ minigene abolished RA-mediated utilization of 5 splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKC␦ exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKC␦ pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKC␦VIII. Vitamin A, an important micronutrient and its active metabolite all-trans-retinoic acid (RA) 2 influence a broad range of physiological and pathological processes in the embryonic central nervous system and in the mature brain. Protein kinase C (PKC), a serine/threonine kinase family, consists of 11 isoforms and their splice variants and is involved in the regulation of cellular differentiation, growth, and apoptosis (2). Protein kinase C␦, a member of the novel PKC subfamily, is implicated

The CFTR splicing mutation 3849 ؉ 10 kb C 3 T creates a novel donor site 10 kilobases (kb) into intron 19 of the gene and is one of the more common splicing mutations that causes cystic fibrosis (CF). It has an elevated prevalence among patients with atypically mild disease and normal sweat electrolytes and is especially prominent in Ashkenazi Jews. This class of splicing mutations, reported in several genes, involves novel splice sites activated deep within introns while leaving wild-type splice elements intact. CFTR cDNA constructs that modeled the 3849 ؉ 10 kb C 3 T mutation were expressed in 3T3 mouse fibroblasts and in CFT1 human tracheal and C127 mouse mammary epithelial cells. In all three cell types, aberrant splicing of CFTR pre-mRNA was comparable to that reported in vivo in CF patients. Treatment of the cells with 2-O-methyl phosphorothioate oligoribonucleotides antisense toward the aberrant donor and acceptor splice sites or to the retained exon-like sequence, disfavored aberrant splicing and enhanced normal processing of CFTR pre-mRNA. This antisense-mediated correction of splicing was dose-and sequencedependent and was accompanied by increased production of CFTR protein that was appropriately glycosylated. Antisense-mediated correction of splicing in a mutation-specific context represents a potential gene therapy modality with applicability to many inherited disorders.

Hutchinson-Gilford progeria (HGPS), a rare and severe developmental disorder characterized by features recalling premature aging, and restrictive dermopathy (RD), a neonatal lethal genodermatosis, have recently been identified as being primary or secondary "laminopathies." These are heterogeneous disorders due to altered function of lamins A/C or related proteins. In physiological conditions, mature lamin A is obtained through a series of post-translational processing steps performed on a protein precursor, prelamin A. The major pathophysiological mechanism involved in progeria is an aberrant splicing of pre-mRNAs issued from the LMNA gene, due to a de novo heterozygous point mutation, leading to the production and accumulation of truncated lamin A precursors. Aberrant splicing of prelamin A pre-mRNAs causing the production of more extensively truncated precursors is involved in the allelic disease restrictive dermopathy. Other restrictive dermopathy cases are due to the inactivation of a key enzyme involved in the maturation of lamin A precursors (ZMPSTE24). In functional terms, all these conditions share the same pathophysiological basis: intranuclear accumulation of lamin A precursors, which cannot be fully processed (due to primary or secondary events) and exert toxic, dominant negative effects on nuclear homeostasis. Most other laminopathies are due to autosomal dominant LMNA point mutations inferred to cause single amino acid substitutions. In any case, the impact of these mutations on pre-mRNA splicing has rarely been assessed. These disorders affect different tissues and organs, mainly including bone, skin, striated muscles, adipose tissue, vessels, and peripheral nerves in isolated or combined fashions, giving rise to syndromes whose severity ranges from mild to perinatally lethal. In this chapter we review the structure and functions of lamins A/C in physiological and pathological conditions, describe their known or putative roles, namely, in the pathogenesis of HGPS and RD in relation to existing animal models, and envisage possible targeted therapeutic strategies on the basis of recent research results. Progress in Molecular and Subcellular Biology Philippe Jeanteur (Ed.

Pyruvate dehydrogenase (PDH) complex deficiency is a major cause of lactic acidosis and Leigh's encephalomyelopathies in infancy and childhood, resulting in early death in the majority of patients. Most of the molecular defects have been localized in the coding regions of the E1α PDH gene. Recently, we identified a novel mutation of the E1α PDH gene in a patient with an encephalopathy and lactic acidosis. This mutation, located downstream of exon 7, activates a cryptic splice donor and leads to the retention of intronic sequences. Here, we demonstrate that the mutation results in an increased binding of the SR protein SC35. Consistently, ectopic overexpression of this splicing factor enhanced the use of the cryptic splice site, whereas small interfering RNA-mediated reduction of the SC35 protein levels in primary fibroblasts from the patient resulted in the almost complete disappearance of the aberrantly spliced E1α PDH mRNA. Our findings open the exciting prospect for a novel t...

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 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 hu...

Expression of the entire complement of human immunodeficiency virus type 1 (HIV-1) viral proteins depends on the competing activities of viral RNA splicing and export into the cytoplasm by Rev. To investigate the possibility that modulation of viral RNA metabolism may alter Rev function, we analyzed the impact of multiple SR proteins on both processes. While overexpression of several of the SR factors altered splicing of HIV-1 env mRNA, they had disparate effects on Rev function that varied with the cell line used. Subsequent examination of exon splicing enhancer (ESE) and/or silencer (ESS) deletions suggests that the effects of the SR proteins on Rev function are not mediated through interaction with these elements. However, analysis of the deletions did indicate that the ESE and/or ESS does have significant effects on Rev function, with deletion of the ESS augmenting the magnitude of the response to Rev and deletion of the ESE significantly reducing it. In situ hybridization and r...

The relatively limited number of human protein encoding genes highlights the importance of the diversity generated at the level of the mRNA transcripts. As alternative RNA splicing plays a key role in mediating this diversity, it becomes critical to develop the tools and platforms that will deliver quantitative information on the specific expression levels associated with splice isoforms. This chapter describes the constraints generated by this global transcriptome analysis and the state-of-the-art techniques and products available to the scientific community.

We previously described a novel homozygous point mutation (FGB c.115-600A4G) located deep within intron 1 of the fibrinogen beta gene (FGB), as a likely cause of afibrinogenemia. While this was the only mutation detected, its pathological mechanism was unclear. Here we show the mutation causes the inclusion of a 50-bp cryptic exon by creating a consensus heptad motif recognized by the spliceosome recruiting protein pre-mRNA splicing factor (SF2)/arginine/serine-rich alternative splicing factor (ASF) splicing factor 2/alternative splicing factor (SF2/ASF). Translation of the aberrant mRNA would result in truncation of the Bb chain, preventing fibrinogen synthesis. Selective introduction of a second mutation into the enhancer motif abolished the SF2/ASF binding motif and re-established normal pre-mRNA splicing. Subsequent introduction of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) into transfected cells containing the mutant construct blocked the protein-RNA interaction and successfully restored normal splicing ($50% at 2 lM and $90% at 10 lM). The molecular characterization of this case has revealed a unique disease mechanism, shown the importance of screening for deep intronic mutations, and provided evidence that antisense gene therapy is potentially practical for the treatment of diseases caused by this class of mutation.

We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.

Previous studies showed that short term exposure of cells to high glucose destabilized protein kinase C (PKC) βII mRNA, whereas PKCβI mRNA levels remained unaltered. Because PKCβ mRNAs share common sequences other than the PKCβII exon encoding a different ...

SC35 belongs to the family of SR proteins that regulate alternative splicing in a concentration-dependent manner in vitro and in vivo. We previously reported that SC35 is expressed through alternatively spliced mRNAs with differing 3¢ untranslated sequences and stabilities. Here, we show that overexpression of SC35 in HeLa cells results in a signi®cant decrease of endogenous SC35 mRNA levels along with changes in the relative abundance of SC35 alternatively spliced mRNAs. Remarkably, SC35 leads to both an exon inclusion and an intron excision in the 3¢ untranslated region of its mRNAs. In vitro splicing experiments performed with recombinant SR proteins demonstrate that SC35, but not ASF/SF2 or 9G8, speci®cally activates these alternative splicing events. Interestingly, the resulting mRNA is very unstable and we present evidence that mRNA surveillance is likely to be involved in this instability. SC35 therefore constitutes the ®rst example of a splicing factor that controls its own expression through activation of splicing events leading to expression of unstable mRNA.

Two half-brothers and their mother had symptomatic pyruvate dehydrogenase complex deficiency. The infants had severe congenital lactic acidosis, seizures, and apneic spells and died at the ages of 3 and 4 months. The mother was less symptomatic with mental retardation, truncal ataxia, and dysarthria. The residual pyruvate dehydrogenase activities in cultured skin fibroblasts from the 2 infants and their mother were 7, 15, and 10% of control values. Immunoblot analysis showed negligible amounts of Ela and E1P subunits of the complex. Northern blot analysis for the Ela subunit showed normal results. In the 2 sons, complementary DNA sequence analysis revealed a cytosine to thymine mutation in exon 4, resulting in a change of arginine 127 to tryptophan in the E l a subunit. Restriction enzyme analysis of the polymerase chain reaction product representing exon 4 of the E l a gene revealed that the mother was a heterozygote. Complementary DNA restriction analysis and methylation analysis of the X chromosome DXS255 loci revealed skewed activation of the mutant allele, consistent with the deficient pyruvate dehydrogenase activity in the mother's fibroblasts. The milder maternal phenotype is consistent with variable X-inactivation patterns in different organs of female heterozygotes.

Two half-brothers and their mother had symptomatic pyruvate dehydrogenase complex deficiency. The infants had severe congenital lactic acidosis, seizures, and apneic spells and died at the ages of 3 and 4 months. The mother was less symptomatic with mental retardation, truncal ataxia, and dysarthria. The residual pyruvate dehydrogenase activities in cultured skin fibroblasts from the 2 infants and their mother were 7, 15, and 10% of control values. Immunoblot analysis showed negligible amounts of Ela and E1P subunits of the complex. Northern blot analysis for the Ela subunit showed normal results. In the 2 sons, complementary DNA sequence analysis revealed a cytosine to thymine mutation in exon 4, resulting in a change of arginine 127 to tryptophan in the E l a subunit. Restriction enzyme analysis of the polymerase chain reaction product representing exon 4 of the E l a gene revealed that the mother was a heterozygote. Complementary DNA restriction analysis and methylation analysis of the X chromosome DXS255 loci revealed skewed activation of the mutant allele, consistent with the deficient pyruvate dehydrogenase activity in the mother's fibroblasts. The milder maternal phenotype is consistent with variable X-inactivation patterns in different organs of female heterozygotes.

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase C␦ (PKC␦), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKC␦, PKC␦VIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKC␦VIII via utilization of a downstream 5 splice site of exon 10 on PKC␦ pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKC␦VIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKC␦VIII alternative splicing. To identify the cis-elements involved in 5 splice site selection we cloned a minigene, which included PKC␦ exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5 splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKC␦ minigene promoted selection of 5 splice site II. Mutation of the SC35 binding site in the PKC␦ minigene abolished RA-mediated utilization of 5 splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKC␦ exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKC␦ pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKC␦VIII. Vitamin A, an important micronutrient and its active metabolite all-trans-retinoic acid (RA) 2 influence a broad range of physiological and pathological processes in the embryonic central nervous system and in the mature brain. Protein kinase C (PKC), a serine/threonine kinase family, consists of 11 isoforms and their splice variants and is involved in the regulation of cellular differentiation, growth, and apoptosis (2). Protein kinase C␦, a member of the novel PKC subfamily, is implicated

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase C␦ (PKC␦), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKC␦, PKC␦VIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKC␦VIII via utilization of a downstream 5 splice site of exon 10 on PKC␦ pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKC␦VIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKC␦VIII alternative splicing. To identify the cis-elements involved in 5 splice site selection we cloned a minigene, which included PKC␦ exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5 splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKC␦ minigene promoted selection of 5 splice site II. Mutation of the SC35 binding site in the PKC␦ minigene abolished RA-mediated utilization of 5 splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKC␦ exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKC␦ pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKC␦VIII. Vitamin A, an important micronutrient and its active metabolite all-trans-retinoic acid (RA) 2 influence a broad range of physiological and pathological processes in the embryonic central nervous system and in the mature brain. Protein kinase C (PKC), a serine/threonine kinase family, consists of 11 isoforms and their splice variants and is involved in the regulation of cellular differentiation, growth, and apoptosis (2). Protein kinase C␦, a member of the novel PKC subfamily, is implicated

Mutations in the E3-binding protein component of pyruvate dehydrogenase complex have been demonstrated in a few cases of Leigh syndrome. We report that two mutations previously detected in the E3-binding protein cDNA are the consequence of splicesite mutations. Both involved a single base substitution in the conserved dinucleotides of splice junctions, one leading to skipping of an exon and the other, to activation of a cryptic site. Our findings add to the understanding of molecular basis of E3-binding protein deficiency and indicate yet again the high frequency of splicing mutations in this gene.

Pyruvate dehydrogenase (PDH) complex de®ciency, a common cause of congenital lactic acidosis, is mostly due to mutations in the X-linked gene coding for the E1a subunit of the complex. We have studied two unrelated girls presenting a static encephalopathy with spastic quadriplegia, microcephaly and seizures and in one girl, hypocalcaemia, a new ®nding in PDH complex de®ciency. PDH de®ciency was diagnosed in adolescence and both girls had low PDH complex activity in muscle but normal amounts of all subunits on Western blotting, and a normal lactate/pyruvate ratio in blood and CSF. Mutation analysis of the E1a gene at the cDNA or DNA level revealed an arginine to histidine substitution at amino acid position 288 (R288H) in the girl with hypocalcaemia and a 12 bp insertion, predicting a four amino acid duplication at the cterminal end of the protein in the second girl. They both carried a normal and a mutated E1a gene and X-inactivation studies showed skewed patterns. Conclusion Mutation identi®cation in pyruvate dehydrogenase complex de®ciency remains important especially for the determination of the recurrence risk and for reliable genetic counselling in couples with an aected child.

Point mutations frequently cause genetic diseases by disrupting the correct pattern of pre-mRNA splicing. The effect of a point mutation within a coding sequence is traditionally attributed to the deduced change in the corresponding amino acid. However, some point mutations can have much more severe effects on the structure of the encoded protein, for example when they inactivate an exonic splicing enhancer (ESE), thereby resulting in exon skipping. ESEs also appear to be especially important in exons that normally undergo alternative splicing. Different classes of ESE consensus motifs have been described, but they are not always easily identified. ESEfinder (http://exon.cshl.edu/ESE/) is a web-based resource that facilitates rapid analysis of exon sequences to identify putative ESEs responsive to the human SR proteins SF2/ASF, SC35, SRp40 and SRp55, and to predict whether exonic mutations disrupt such elements.

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 hu...