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BackgroundPathogenic variants of RUNX2, a gene that encodes an osteoblast-specific transcription factor, have been shown as the cause of Cleidocranial dysplasia (CCD), which is a rare hereditary skeletal and dental disorder with dominant mode of inheritance and a broad range of clinical variability. Due to the relative lack of clinical complications resulting in CCD, the medical diagnosis of this disorder is challenging, which leaves it underdiagnosed.MethodsIn this study, nine healthy and affected members of an Iranian family were investigated. PCR and sequencing of all exons and exon-intron boundaries of runt-related transcription factor 2 (RUNX2; NM_001024630) gene was performed on proband. Co-segregation analysis was conducted in the other family members for the identified variant. Additionally, a cohort of 100 Iranian ethnicity-matched healthy controls was screened by Amplification Refractory Mutation System PCR method.ResultsThe novel splice site variant (c.860-2A>G), which was identified in the intron 6 of RUNX2 gene, co-segregated with the disease in the family, and it was absent in healthy controls. Pathogenicity of this variant was determined by several software, including , human splicing finder, which predicts the formation or disruption of splice donor sites, splice acceptor sites, exonic splicing silencer sites, and exonic splicing enhancer sites. In silico analysis predicted this novel variant to be disease causing.ConclusionThe identified variant is predicted to have an effect on splicing, which leads to exon skipping and producing a truncated protein via introducing a premature stop codon.

Purpose Existing data do not explain the reason why some individuals homozygous for the hypomorphic FECH allele develop erythropoietic protoporphyria (EPP) while the majority are completely asymptomatic. This study aims to identify novel possible genetic variants contributing to this variable phenotype. Methods High-throughput resequencing of the FECH gene, qualitative analysis of RNA, and quantitative DNA methylation examination were performed on a cohort of 72 subjects. Results A novel deep intronic variant was found in four homozygous carriers developing a clinically overt disease. We demonstrate that this genetic variant leads to the insertion of a pseudo-exon containing a stop codon in the mature FECH transcript by the abolition of an exonic splicing silencer site and the concurrent institution of a new methylated CpG dinucleotide. Moreover, we show that the hypomorphic FECH allele is linked to a single haplotype of about 20 kb in size that encompasses three noncoding variants that were previously associated with expression quantitative trait loci (eQTLs). Conclusion This study confirms that intronic variants could explain the variability in the clinical manifestations of EPP. Moreover, it supports the hypothesis that the control of the FECH gene expression can be mediated through a methylation-dependent modulation of the precursor messenger RNA (pre-mRNA) splicing pattern.

Plants perceive environmental light conditions and optimize their growth and development accordingly by regulating gene activity at multiple levels. Photoreceptors are important for light sensing and downstream gene regulation. Phytochromes, red/far-red light receptors, are believed to regulate light-responsive alternative splicing, but little is known about the underlying mechanism. Alternative splicing is primarily regulated by transacting factors, such as splicing regulators, and by cis-acting elements in precursor mRNA. In the moss Physcomitrella patens, we show that phytochrome 4 (PpPHY4) directly interacts with a splicing regulator, heterogeneous nuclear ribonucleoprotein F1 (PphnRNP-F1), in the nucleus to regulate light-responsive alternative splicing. RNA sequencing analysis revealed that PpPHY4 and PphnRNP-F1 coregulate 70% of intron retention (IR) events in response to red light. A repetitive GAA motif was identified to be an exonic splicing silencer that controls red light-responsive IR. Biochemical studies indicated that PphnRNP-F1 is recruited by the GAA motif to form RNA-protein complexes. Finally, red light elevates PphnRNP-F1 protein levels via PpPHY4, increasing levels of IR. We propose that PpPHY4 and PphnRNP-F1 regulate alternative splicing through an exonic splicing silencer to control splicing machinery activity in response to light.

Noncoding RNAs are increasingly promising drug targets yet ligand design is hindered by a paucity of methods that reveal driving factors in selective small molecule : RNA interactions, particularly given the difficulties of high-resolution structural characterization. HIV RNAs are excellent model systems for method development given their targeting history, known structure-function relationships, and the unmet need for more effective treatments. Herein we report a strategy combining synthetic diversification, profiling against multiple RNA targets, and predictive cheminformatic analysis to identify driving factors for selectivity and affinity of small molecules for distinct HIV RNA targets. Using this strategy, we discovered improved ligands for multiple targets and the first ligands for ESSV, an exonic splicing silencer critical to replication. Computational analysis revealed guiding principles for future designs and a predictive cheminformatics model of small molecule : RNA binding. These methods are expected to facilitate progress toward selective targeting of disease-causing RNAs.

BACKGROUND The CYP17A1 gene encodes for cytochrome P450 enzyme CYP17A1, which is involved with the steroidogenic pathway including mineralocorticoids. The CYP17A1 polymorphisms might affect enzyme activity, then leading to a state of mineralocorticoid 11-deoxycorticosterone excess characterized by hypertension, suppressed plasma renin activity, and low aldosterone concentrations. The aim of this study was to investigate the contribution of CYP17A1 polymorphisms in inducing the susceptibility to essential hypertension among the Southwest Han Chinese population. MATERIAL AND METHODS Eight single nucleotide polymorphisms of CYP17A1 were genotyped in a case-control study for samples by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS The polymorphisms rs11191548 and rs4919687 were significantly associated with hypertension risk, which was confirmed by systolic and diastolic blood pressure distribution analyses between different genotype groups, and these two polymorphisms were found in linkage disequilibrium. The rs4919687 polymorphism was estimated to cause the destruction of exonic splicing silencer (ESR and Motif 3) sites and to transform the transcription factor AREB6 binding site, respectively, in the bioinformatics analyses. The haplotypes rs4919686A-rs3740397G -rs4919687C-rs743572C-rs11191548C and rs4919686A-rs3740397G-rs4919687T-rs743572C- rs11191548T were found to be susceptible to essential hypertension. CONCLUSIONS Our findings suggest that the CYP17A1 polymorphisms could be a genetic risk factor for essential hypertension among the Yunnan Han Chinese population, which would have implications for the treatment of this complex disorder.

Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.

Bombyx mori doublesex (Bmdsx) functions as a double-switch gene in the final step of the sex-determination cascade in the silkworm Bombyx mori. The P-element somatic inhibitor (PSI) protein in B. mori interacts with Bmdsx pre-mRNA in CE1 as an exonic splicing silencer to promote male-specific splicing of Bmdsx. However, the character of the interaction between BmPSI and Bmdsx pre-mRNA remains unclear. Electrophoretic mobility shift assay (EMSA) results showed that the four KH_1 motifs in BmPSI are all essential for the binding, especially the former two KH_1 motifs. Three active sites (I116, L127, and IGGI) in the KH_1 motif were found to be necessary for the binding through EMSA, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The PSI homologous protein in S. litura (SlPSI) was purified and the binding of SlPSI and CE1 was verified. Compared with BmPSI, the mutant SlPSI proteins of I116 and IGGI lost their ability to bind to CE1. In conclusion, the binding of PSI and dsx pre-mRNA are generally conserved in both B. mori and S. litura. These findings provide clues for sex determination in Lepidoptera.

Previously, dominant partial interferon-gamma receptor 1 (IFN-g-R1) susceptibility to environmental mycobacteria was found with IFNGR1 deletions or premature stop. Our aim was to search for IFNGR1 variants in patients with mycobacterial osteoarticular lesions. Biopsies from the patients were examined for acid-fast bacilli, inflammatory cell infiltration, and mycobacterial niacin. Mycobacterial rRNA was analyzed using a target-amplified rRNA probe test. Peripheral-blood-leukocyte genomic DNA was isolated from 19 patients using the QIAamp DNA Mini Kit, and all IFNGR1 exons were sequenced using an ABIPRISM 3130 device. After the discovery of an exon 5 variant, a Polish newborn population sample (n = 100) was assayed for the discovered variant. Splice sites and putative amino acid interactions were analyzed. All patients tested were positive for mycobacteria; one was heterozygous for the IFNGR1 exon 5 single-nucleotide-missense substitution (g.20746A>G, p.Ile183Val). No other variant was found. The splice analysis indicated the creation of an exonic splicing silencer, and alternatively, molecular graphics indicated that the p.Ile183Val might alter beta-strand packing (loss of van der Waals contacts; Val183/Pro205), possibly altering the IFN-g-R1/IFN-g-R2 interaction. The probability of non-deleterious variant was estimated as

Here we present a detailed analysis of the alternative splicing regulation of human CD46, which generates different isoforms with distinct functions. CD46 is a ubiquitous membrane protein that protects host cells from complement and plays other roles in immunity, autophagy, and cell adhesion. CD46 deficiency causes an autoimmune disorder, and this protein is also involved in pathogen infection and cancer. Before this study, the mechanisms of CD46 alternative splicing remained unexplored even though dysregulation of this process has been associated with autoimmune diseases. We proved that the 5′ splice sites of CD46 cassette exons 7 and 8 encoding extracellular domains are defined by noncanonical mechanisms of base pairing to U1 small nuclear RNA. Next we characterized the regulation of CD46 cassette exon 13, whose inclusion or skipping generates different cytoplasmic tails with distinct functions. Using splicing minigenes, we identified multiple exonic and intronic splicing enhancers and silencers that regulate exon 13 inclusion via trans-acting splicing factors like PTBP1 and TIAL1. Interestingly, a common splicing activator such as SRSF1 appears to repress CD46 exon 13 inclusion. We also report that expression of CD46 mRNA isoforms is further regulated by non-sense-mediated mRNA decay and transcription speed. Finally, we successfully manipulated CD46 exon 13 inclusion using antisense oligonucleotides, opening up opportunities for functional studies of the isoforms as well as for therapeutics for autoimmune diseases. This study provides insight into CD46 alternative splicing regulation with implications for its function in the immune system and for genetic disease. MOE (Min. of Education, S’pore) Published version

Most genes mutated in microcephaly patients are expressed ubiquitously, and yet the brain is the only major organ compromised in most patients. Why the phenotype remains brain specific is poorly understood. In this study, we used in vitro differentiation of human embryonic stem cells to monitor the effect of a point mutation in kinetochore null protein 1 (KNL1; CASC5), identified in microcephaly patients, during in vitro brain development. We found that neural progenitors bearing a patient mutation showed reduced KNL1 levels, aneuploidy, and an abrogated spindle assembly checkpoint. By contrast, no reduction of KNL1 levels or abnormalities was observed in fibroblasts and neural crest cells. We established that the KNL1 patient mutation generates an exonic splicing silencer site, which mainly affects neural progenitors because of their higher levels of splicing proteins. Our results provide insight into the brain-specific phenomenon, consistent with microcephaly being the only major phenotype of patients bearing KNL1 mutation., Simons Foundation. Postdoctoral Fellowship, International Rett Syndrome Foundation (Postdoctoral Fellowship), Brain & Behavior Research Foundation (Young Investigator Grant), National Institutes of Health (U.S.) (grant HD 045022), National Institutes of Health (U.S.) (grant R37-CA084198), National Institutes of Health (U.S.) (grant 1U19AI131135-01)

Mutations affecting specific splicing regulatory elements offer suitable models to better understand their interplay and to devise therapeutic strategies. Here we characterize a meaningful splicing model in which numerous Hemophilia B-causing mutations, either missense or at the donor splice site (5′ss) of coagulation F9 exon 2, promote aberrant splicing by inducing the usage of a strong exonic cryptic 5′ss. Splicing assays with natural and artificial F9 variants indicated that the cryptic 5′ss is regulated, among a network of regulatory elements, by an exonic splicing silencer (ESS). This finding and the comparative analysis of the F9 sequence across species showing that the cryptic 5′ss is always paralleled by the conserved ESS support a compensatory mechanism aimed at minimizing unproductive splicing. To recover splicing we tested antisense oligoribonucleotides masking the cryptic 5′ss, which were effective on exonic changes but promoted exon 2 skipping in the presence of mutations at the authentic 5′ss. On the other hand, we observed a very poor correction effect by small nuclear RNA U1 (U1snRNA) variants with increased or perfect complementarity to the defective 5′ss, a strategy previously exploited to rescue splicing. Noticeably, the combination of the mutant-specific U1snRNAs with antisense oligonucleotides produced appreciable amounts of correctly spliced transcripts (from 0 to 20–40%) from several mutants of the exon 2 5′ss. Based on the evidence of an altered interplay among ESS, cryptic and the authentic 5′ss as a disease-causing mechanism, we provide novel experimental insights into the combinatorial correction activity of antisense molecules and compensatory U1snRNAs.

The correct splicing of precursor-mRNA depends on the actual splice sites plus exonic and intronic regulatory elements recognized by the splicing machinery. Surprisingly, an increasing number of examples reveal that exonic mutations disrupt the binding of splicing factors to these sequences or generate new splice sites or regulatory elements, causing disease. This contradicts the general assumption that missense mutations disrupt protein function and that synonymous mutations are merely polymorphisms. Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused mainly by mutations in the PKD1 gene. Recently, we analyzed a substantial number of PKD1 missense or synonymous mutations to further characterize their consequences on pre-mRNA splicing. Our results showed that one missense and 2 synonymous mutations induce significant defects in pre-mRNA splicing. Thus, it appears that aberrant splicing as a result of exonic mutations is a previously unrecognized cause of ADPKD.

Objective The objective of this study was to investigate the genetic etiology of the X-linked disorder “Hypomyelination of Early Myelinating Structures” (HEMS). Methods We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients’ fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus–Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing.

Pre-mRNA molecules in humans contain mostly short internal exons flanked by longer introns. To explain the removal of such introns, exon recognition instead of intron recognition has been proposed. We studied this exon definition using designer exons (DEs) made up of three prototype modules of our own design: an exonic splicing enhancer (ESE), an exonic splicing silencer (ESS), and a Reference Sequence (R) predicted to be neither. Each DE was examined as the central exon in a three-exon minigene. DEs made of R modules showed a sharp size dependence, with exons shorter than 14 nt and longer than 174 nt splicing poorly. Changing the strengths of the splice sites improved longer exon splicing but worsened shorter exon splicing, effectively displacing the curve to the right. For the ESE we found, unexpectedly, that its enhancement efficiency was independent of its position within the exon. For the ESS we found a step-wise positional increase in its effects; it was most effective at the 3′ end of the exon. To apply these results quantitatively, we developed a biophysical model for exon definition of internal exons undergoing cotranscriptional splicing. This model features commitment to inclusion before the downstream exon is synthesized and competition between skipping and inclusion fates afterward. Collision of both exon ends to form an exon definition complex was incorporated to account for the effect of size; ESE/ESS effects were modeled on the basis of stabilization/destabilization. This model accurately predicted the outcome of independent experiments on more complex DEs that combined ESEs and ESSs.

Background & aims Wilson disease is an inborn error of metabolism caused by abnormalities of the copper-transporting protein-encoding gene ATP7B. Recently, the phenomenon of exon skipping, in which exonic mutations result in abnormal splicing, has been associated with various diseases. The present study investigated the splicing defects of the ATP7B exonic variants identified in a cohort of 44 patients with Wilson disease. Method All patients were analysed for ATP7B gene by direct sequencing or multiplex ligation-dependent probe amplification analysis. To identify the potential pathogenicity of the candidate mutations that may induce exon skipping, both in vivo RT-PCR analysis using RNA from peripheral leukocytes and in vitro functional splicing by minigene construction were conducted. Results The patterns of inheritance of the mutations in ATP7B identified in 44 patients exhibited homozygotes (7 patients), compound heterozygotes (32 patients) and heterozygotes (5 patients). In all patients, we detected 25 different ATP7B mutations, including 17 missenses, 1 frameshift, 3 nonsenses, 2 exonic deletions and 2 splicing alteration. In these mutations, 4 mutations have not been previously described in the literature or entered in human genome mutation database. Furthermore, we identified synonymous mutation c.4014T>A and missense mutation R919G caused exon skipping in the ATP7B mRNA transcript. Conclusion Our results suggest that aberrant exon skipping associated to putative splicing enhancer disruption and silencer creation is one previously unrecognized mechanism in Wilson disease. What is more, the multiplex ligation-dependent probe amplification assay for the detection of exon deletions may be valuable in individuals with clinical Wilson disease diagnosis where one or no mutation has been identified by sequencing.

Background D antigen is one of the most clinically significant blood group antigens. Variation of the RHD gene can cause weak D or partial D phenotypes. While most variations are missense substitutions with amino acid changes, those without are called “silent” or “synonymous” substitutions. Synonymous substitutions often have little effect on the protein, not altering the phenotype. However, effect on splicing can affect end-product protein. We report a new synonymous variation, RHD 1056C>G, that resulted in weak D phenotype, and predicted its effect with various in silico methods. Methods Serologic testing of the D antigen with full sequencing of the RHD gene was done. Human Splice Finder was used to predict the effect of this variation, and validation of this method was done with all known RHD variations reported in the literature. Results RHD 1056C>G was predicted to cause the formation of an exonic splicing silencer (ESS) site. The creation of new ESS site potentially inhibits the splicing event, resulting alteration of splicing. This is similar to remodeling of splice acceptor or donor site, as this kind of deep exonic variation could affect the D antigen's quality or quantity. This is in concordance with serologic results, which showed only delayed weak agglutination to anti-D reagents. Conclusions The analytic methods we applied showed good correlation with the actual phenotype, along with concordant results when analyzing other known variants reported in the literature. We conclude that RHD 1056C>G results in serologic weak D phenotype.

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3′splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.

HIV-1 is able to express multiple protein types and isoforms from a single 9 kb mRNA transcript. These proteins are also expressed at particular stages of viral development, and this is achieved through the control of alternative splicing and the export of these transcripts from the nucleus. The nuclear export is controlled by the HIV protein Rev being required to transport incompletely spliced and partially spliced mRNA from the nucleus where they are normally retained. This implies a close relationship between the control of alternate splicing and the nuclear export of mRNA in the control of HIV-1 viral proliferation. This review discusses both the processes. The specificity and regulation of splicing in HIV-1 is controlled by the use of specific splice sites as well as exonic splicing enhancer and exonic splicing silencer sequences. The use of these silencer and enhancer sequences is dependent on the serine arginine family of proteins as well as the heterogeneous nuclear ribonucleoprotein family of proteins that bind to these sequences and increase or decrease splicing. Since alternative splicing is such a critical factor in viral development, it presents itself as a promising drug target. This review aims to discuss the inhibition of splicing, which would stall viral development, as an anti-HIV therapeutic strategy. In this review, the most recent knowledge of splicing in human immunodeficiency viral development and the latest therapeutic strategies targeting human immunodeficiency viral splicing are discussed.

The HIV-1 accessory protein Vif is essential for viral replication by counteracting the host restriction factor APOBEC3G (A3G), and balanced levels of both proteins are required for efficient viral replication. Noncoding exons 2/2b contain the Vif start codon between their alternatively used splice donors 2 and 2b (D2 and D2b). For vif mRNA, intron 1 must be removed while intron 2 must be retained. Thus, splice acceptor 1 (A1) must be activated by U1 snRNP binding to either D2 or D2b, while splicing at D2 or D2b must be prevented. Here, we unravel the complex interactions between previously known and novel components of the splicing regulatory network regulating HIV-1 exon 2/2b inclusion in viral mRNAs. In particular, using RNA pulldown experiments and mass spectrometry analysis, we found members of the heterogeneous nuclear ribonucleoparticle (hnRNP) A/B family binding to a novel splicing regulatory element (SRE), the exonic splicing silencer ESS2b, and the splicing regulatory proteins Tra2/SRSF10 binding to the nearby exonic splicing enhancer ESE2b. Using a minigene reporter, we performed bioinformatics HEXplorer-guided mutational analysis to narrow down SRE motifs affecting splice site selection between D2 and D2b. Eventually, the impacts of these SREs on the viral splicing pattern and protein expression were exhaustively analyzed in viral particle production and replication experiments. Masking of these protein binding sites by use of locked nucleic acids (LNAs) impaired Vif expression and viral replication. IMPORTANCE Based on our results, we propose a model in which a dense network of SREs regulates vif mRNA and protein expression, crucial to maintain viral replication within host cells with varying A3G levels and at different stages of infection. This regulation is maintained by several serine/arginine-rich splicing factors (SRSF) and hnRNPs binding to those elements. Targeting this cluster of SREs with LNAs may lead to the development of novel effective therapeutic strategies.

The c.2101A>G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism. Through ATR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6±1% of correct transcripts only) depends on the poor exon 9 definition (47±4% in the ATRwt context), because the change was ineffective when the weak 5' or the 3' splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101A nucleotide is conserved across species, and the SS-causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101A>C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28±7% of correct transcripts). The observation that an antisense oligonucleotide (AONATR) targeting the c.2101A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1ATR) designed to perfectly base-pair the weak 5'ss, rescued exon inclusion (63±3%) in the ATRSS-allele. Most importantly, upon lentivirus-mediated delivery, the U1ATR partially rescued ATR mRNA splicing (from ~19% to ~54%) and protein (from negligible to ~6%) in embryonic fibroblasts derived from humanized ATRSS mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101A>G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome.

The MLH1 and MSH2 genes in DNA mismatch repair are important in the pathogenesis of gastrointestinal cancer. Recent studies of normal and alternative splicing suggest that the deleterious effects of missense mutations may in fact be splicing-related when they are located in exonic splicing enhancers (ESEs) or exonic splicing silencers (ESSs). In this study, we used ESE-finder and FAS-ESS software to analyze the potential ESE/ESS motifs of the 114 missense mutations detected in the two genes in East Asian gastrointestinal cancer patients. In addition, we used the SIFT tool to functionally analyze these mutations. The amount of the ESE losses (68) was 51.1% higher than the ESE gains (45) of all the mutations. However, the amount of the ESS gains (27) was 107.7% higher than the ESS losses (13). In total, 56 (49.1%) mutations possessed a potential exonic splicing regulator (ESR) error. Eighty-one mutations (71.1%) were predicted to be deleterious with a lower tolerance index as detected by the Sorting Intolerant from Tolerant (SIFT) tool. Among these, 38 (33.3%) mutations were predicted to be functionally deleterious and possess one potential ESR error, while 18 (15.8%) mutations were predicted to be functionally deleterious and exhibit two potential ESR errors. These may be more likely to affect exon splicing. Our results indicated that there is a strong correlation between missense mutations in MLH1 and MSH2 genes detected in East Asian gastrointestinal cancer patients and ESR motifs. In order to correctly understand the molecular nature of mutations, splicing patterns should be compared between wild-type and mutant samples.

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3’ splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping., Author Summary HRAS was the first human proto-oncogene reported and p.Gly12Val in codon 12 the first oncogenic mutation described. Somatic mutations in HRAS are important drivers in cancer, and germline mutations cause Costello syndrome. Until now, it has been believed that the severity of mutations located in the HRAS codon 12/13 mutational hot spot sequence is exclusively determined by the activity of the encoded proteins. Here we show that mutations in exon 2 of the HRAS proto-oncogene can have a previously unrecognized effect on splicing efficiency and thereby determine HRAS activity. We report a patient with Costello syndrome (CS), who despite having the classical, severe, oncogenic p.Gly12Val mutation in HRAS has a mild clinical phenotype. We show that this is due to his specific sequence change, c.35_36GC>TG, encoding p.Gly12Val, which disrupts an ESE fundamental for efficient splicing of exon 2. We have explored this in detail and were able to show that HRAS exon 2 is a weak exon, which is dependent on the balance between positive and negative splicing regulatory factors, like SRSF2 and hnRNPF/H in order to be properly included. We show that different mutations in the mutational hot spot in HRAS codon 12 and 13 (c.34-39) affect splicing of HRAS differently, suggesting that this mechanism may also influence their occurrence in CS and their oncogenic potential in somatic cancers. Finally, we show that blocking access to the fundamental ESE, located in HRAS c.34-39, using a splice switching oligonucleotide (SSOs), causes exon 2 skipping, abolishes production of functional HRAS protein and halts proliferation of cancer cells. This shows a previously unknown weakness of the HRAS proto-oncogene, namely that exon 2 is weakly defined and therefore is a suitable target for SSO-based therapy, thereby pointing towards a potential new direction for therapeutic targeting of RAS in anti-cancer treatment.

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3' acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.

The Δ6 desaturase, encoded by FADS2, plays a crucial role in omega-3 and omega-6 fatty acid synthesis. These fatty acids are essential components of the central nervous system, and they act as precursors for eicosanoid signaling molecules and as direct modulators of gene expression. The polypyrimidine tract binding protein (PTB or hnRNP I) is a splicing factor that regulates alternative pre-mRNA splicing. Here, PTB is shown to bind an exonic splicing silencer element and repress alternative splicing of FADS2 into FADS2 AT1. PTB and FADS2AT1 were inversely correlated in neonatal baboon tissues, implicating PTB as a major regulator of tissue-specific FADS2 splicing. In HepG2 cells, PTB knockdown modulated alternative splicing of FADS2, as well as FADS3, a putative desaturase of unknown function. Omega-3 fatty acids decreased by nearly one half relative to omega-6 fatty acids in PTB knockdown cells compared with controls, with a particularly strong decrease in eicosapentaenoic acid (EPA) concentration and its ratio to arachidonic acid (ARA). This is a rare demonstration of a mechanism specifically altering the cellular omega-3 to omega-6 fatty acid ratio without any change in diet/media. These findings reveal a novel role for PTB, regulating availability of membrane components and eicosanoid precursors for cell signaling.

Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non-small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.

PKU is caused by mutations in PAH. A c.30C>G synonymous variation in exon 1, previously reported as neutral, was observed in two patients. The variation creates a GGG triplet, which is part of several exonic splicing silencer (ESS) motifs. Because the 5'-splice site of PAH exon 1 is intrinsically weak and therefore could be responsive to a new flanking ESS, we hypothesized that c.30C>G could cause aberrant mRNA splicing. We demonstrate that c.30C>G causes aberrant mRNA splicing in two different reporter minigenes, and that this is abolished if a preexisting flanking GGG triplet is disrupted. GGG triplets are part of the consensus motif bound by splicing-inhibitory hnRNPH proteins and we observed a dramatic increase in hnRNPH binding to c.30C>G PAH RNA. We conclude that c.30C>G creates a hnRNPH-binding ESS, which can disrupt mRNA splicing. A disease-causing mutation in HEXB, which has previously been associated with exon skipping in patients also creates a GGG triplet. We show that the mutant HEXB motif causes exon skipping of a reporter minigene and that this is also influenced by a flanking GGG triplet. We suggest that aberrant splicing caused by creation/abolishment of GGG triplets located together with a preexisting flanking GGG triplet, may be an underreported cause of human disease. It is important to recognize that exonic sequence changes may disrupt mRNA splicing. This is particularly important in PAH, since PKU patients harboring such mutations are unlikely to respond to therapy with 6R-tetrahydrobiopterin (BH(4)), despite the fact that the genetic code indicates otherwise.

T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.

Alternative splicing is a main component of protein diversity, and aberrant splicing is known to be one of the main causes of genetic disorders such as cancer. Many statistical and computational approaches have identified several major factors that determine the splicing event, such as exon/intron length, splice site strength, and density of splicing enhancers or silencers. These factors may be correlated with one another and thus result in a specific type of splicing, but there has not been a systematic approach to extracting comprehensible association patterns. Here, we attempted to understand the decision making process of the learning machine on intron retention event. We adopted a hybrid learning machine approach using a random forest and association rule mining algorithm to determine the governing factors of intron retention events and their combined effect on decision-making processes. By quantifying all candidate features into five category values, we enhanced the understandability of generated rules. The interesting features found by the random forest algorithm are that only the adenine- and thymine-based triplets such as ATA, TTA, and ATT, but not the known intronic splicing enhancer GGG triplet is shown the significant features. The rules generated by the association rule mining algorithm also show that constitutive introns are generally characterized by high adenine- and thymine-based triplet frequency (level 3 and above), 3' and 5' splice site scores, exonic splicing silencer scores, and intron length, whereas retained introns are characterized by low-level counterpart scores.

hnRNP A1 binds to RNA in a cooperative manner. Initial hnRNP A1 binding to an exonic splicing silencer at the 3′ end of human immunodeficiency virus type 1 (HIV-1) tat exon 3, which is a high-affinity site, is followed by cooperative spreading in a 3′-to-5′ direction. As hnRNP A1 propagates toward the 5′ end of the exon, it antagonizes binding of a serine/arginine-rich (SR) protein to an exonic splicing enhancer, thereby inhibiting splicing at that exon's alternative 3′ splice site. tat exon 3 and the preceding intron of HIV-1 pre-mRNA can fold into an elaborate RNA secondary structure in solution, which could potentially influence hnRNP A1 binding. We report here that hnRNP A1 binding and splicing repression can occur on an unstructured RNA. Moreover, hnRNP A1 can effectively unwind an RNA hairpin upon binding, displacing a bound protein. We further show that hnRNP A1 can also spread in a 5′-to-3′ direction, although when initial binding takes place in the middle of an RNA, spreading preferentially proceeds in a 3′-to-5′ direction. Finally, when two distant high-affinity sites are present on the same RNA, they facilitate cooperative spreading of hnRNP A1 between the two sites.

In addition to normal alternative splicing events (those that take place in untreated cells), there are also those that are inducible in response to environmental stimuli. We previously reported that the alternative splicing of p53-inducible gene 3 (PIG3) exon 4 is modulated in response to UV radiation. Here, we investigate the mechanisms mediating the alternative splicing of this exon. Through the use of various minigene constructs our results reveal that numerous factors influence the normal alternative splicing of PIG3 exon 4, and that these ultimately affect its UV-inducible alternative splicing. Included among these are sequence elements located within exon 4 itself as well as adjacent sequences required for intron definition (the pyrimidine tract, 3'- and 5'-splice sites). Within exon 4, we identified a novel hnRNP A1-dependent exonic splicing silencer (ESS) whose mutation inhibited the alternative splicing of a PIG3 minigene. Although previously implicated in the UV-inducible alternative splicing of other transcripts, RNAi-mediated silencing of hnRNP A1/A2 or hSlu7 did not prevent the UV-enhancement of exon 4 skipping. Overall, our results suggest that numerous factors contribute to the normal alternative splicing of PIG3 exon 4 and that UV-inducible increases in this process require that the splicing of this exon be maintained in a sufficiently weakened state under normal conditions.

Multiple sclerosis is a demyelinating neurodegenerative disease with a strong genetic component. Previous genetic risk studies have failed to identify consistently linked regions or genes outside of the major histocompatibility complex on chromosome 6p. We describe allelic association of a polymorphism in the gene encoding the interleukin 7 receptor α chain ( IL7R ) as a significant risk factor for multiple sclerosis in four independent family-based or case-control data sets (overall P = 2.9 × 10−7). Further, the likely causal SNP, rs6897932, located within the alternatively spliced exon 6 of IL7R, has a functional effect on gene expression. The SNP influences the amount of soluble and membrane-bound isoforms of the protein by putatively disrupting an exonic splicing silencer.

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.

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 high-affinity 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.

Functional annotation of SNPs (as generated by HapMap (http://www.hapmap.org) for instance) is a major challenge. SNPs that lead to single amino acid substitutions, stop codons, or frameshift mutations can be readily interpreted, but these represent only a fraction of known SNPs. Many SNPs are located in sequences of splicing relevance-the canonical splice site consensus sequences, exonic and intronic splice enhancers or silencers (exonic splice enhancer [ESE], intronic splice enhancer [ISE], exonic splicing silencer [ESS], and intronic splicing silencer [ISS]), and others. We propose using sets of matching DNA and complementary DNA (cDNA) as a screening method to investigate the potential splice effects of SNPs in RT-PCR experiments with tissue material from genotyped sources. We have developed a software solution (SNPSplicer; http://www.ikmb.uni-kiel.de/snpsplicer) that aids in the rapid interpretation of such screening experiments. The utility of the approach is illustrated for SNPs affecting the donor splice sites (rs2076530:A>G, rs3816989:G>A) leading to the use of a cryptic splice site and exon skipping, respectively, and an exonic splice enhancer SNP (rs2274987:C/T), leading to inclusion of a new exon. We anticipate that this methodology may help in the functional annotation of SNPs in a more high-throughput fashion.

The inhibition of glucose-6-phosphate dehydrogenase (G6PD) expression by arachidonic acid occurs by changes in the rate of pre-mRNA splicing. Here, we have identified a cis-acting RNA element required for regulated splicing of G6PD mRNA. Using transfection of G6PD RNA reporter constructs into rat hepatocytes, the cis-acting RNA element involved in this regulation was localized to nucleotides 43-72 of exon 12 in the G6PD mRNA. In in vitro splicing assays, RNA substrates containing exon 12 were not spliced. In contrast, RNA substrates containing other regions (exons 8 and 9 or exons 10 and 11) of the G6PD mRNA were efficiently spliced. Furthermore, exon 12 can inhibit splicing when substituted for other exons in RNA substrates that are readily spliced. This activity of the exon 12 regulatory element suggests that it is an exonic splicing silencer. Consistent with its activity as a splicing silencer, spliceosome assembly was inhibited on RNA substrates containing exon 12 compared with RNAs representing other regions of the G6PD transcript. Elimination of nucleotides 43-72 of exon 12 did not restore splicing of exon 12-containing RNA; thus, the 30-nucleotide element may not be exclusively a silencer. The binding of heterogeneous nuclear ribonucleoproteins K, L, and A2/B1 from both HeLa and hepatocyte nuclear extracts to the element further supports its activity as a silencer. In addition, SR proteins bind to the element, consistent with the presence of enhancer activity within this sequence. Thus, an exonic splicing silencer is involved in the inhibition of splicing of a constitutively spliced exon in the G6PD mRNA.

Precursor messenger RNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles in a stepwise process. The spliceosome's dynamic nature suggests the potential for regulation at numerous points along the assembly pathway; however, thus far, naturally occurring regulation of splicing has only been found to influence a small subset of spliceosomal intermediates. Here we report that the exonic splicing silencer (ESS1) that represses splicing of PTPRC (encoding CD45) exon 4 does not function by the typical mechanism of inhibiting binding of U1 or U2 small nuclear ribonucleoproteins (snRNPs) to the splice sites. Instead, a U1-, U2- and ATP-dependent complex forms across exon 4 that is required for inhibiting progression to the U4-U6-U5 tri-snRNP-containing B complex. Such inhibition represents a new mechanism for splicing regulation and suggests that regulation can probably occur at many of the transitions along the spliceosome assembly pathway.

Correct splice site recognition is critical in pre-mRNA splicing. We find that almost all of a diverse panel of exonic splicing silencer (ESS) elements alter splice site choice when placed between competing sites, consistently inhibiting use of intron-proximal 5' and 3' splice sites. Supporting a general role for ESSs in splice site definition, we found that ESSs are both abundant and highly conserved between alternative splice site pairs and that mutation of ESSs located between natural alternative splice site pairs consistently shifted splicing toward the intron-proximal site. Some exonic splicing enhancers (ESEs) promoted use of intron-proximal 5' splice sites, and tethering of hnRNP A1 and SF2/ASF proteins between competing splice sites mimicked the effects of ESS and ESE elements, respectively. Further, we observed that specific subsets of ESSs had distinct effects on a multifunctional intron retention reporter and that one of these subsets is likely preferred for regulation of endogenous intron retention events. Together, our findings provide a comprehensive picture of the functions of ESSs in the control of diverse types of splicing decisions.