Exonic Splicing Enhancer Research Articles

Unique CD44 intronic SNP is associated with tumor grade in breast cancer: a case control study and in silico analysis

BackgroundCD44 encoded by a single gene is a cell surface transmembrane glycoprotein. Exon 2 is one of the important exons to bind CD44 protein to hyaluronan. Experimental evidences show that hyaluronan–CD44 interaction intensifies the proliferation, migration, and invasion of breast cancer cells. Therefore, the current study aimed at investigating the association between specific polymorphisms in exon 2 and its flanking region of CD44 with predisposition to breast cancer.MethodsIn the current study, 175 Iranian female patients with breast cancer and 175 age-matched healthy controls were recruited in biobank, Breast Cancer Research Center, Tehran, Iran. Single nucleotide polymorphisms of CD44 exon 2 and its flanking were analyzed via polymerase chain reaction and gene sequencing techniques. Association between the observed variation with breast cancer risk and clinico-pathological characteristics were studied. Subsequently, bioinformatics analysis was conducted to predict potential exonic splicing enhancer (ESE) motifs changed as the result of a mutation.ResultsA unique polymorphism of the gene encoding CD44 was identified at position 14 nucleotide upstream of exon 2 (A37692→G) by the sequencing method. The A > G polymorphism exhibited a significant association with higher-grades of breast cancer, although no significant relation was found between this polymorphism and breast cancer risk. Finally, computational analysis revealed that the intronic mutation generated a new consensus-binding motif for the splicing factor, SC35, within intron 1.ConclusionsThe current study results indicated that A > G polymorphism was associated with breast cancer development; in addition, in silico analysis with ESE finder prediction software showed that the change created a new SC35 binding site.

A novel mutation in exon 9 of Cullin 3 gene contributes to aberrant splicing in pseudohypoaldosteronism type II.

Pseudohypoaldosteronism type II (PHAII) is a rare renal tubular disease that is inherited in an autosomal dominant manner. Mutations in four genes (WNK1,WNK4,CUL3, and KLHL3) have been identified to be responsible for this disease. Cullin 3 (CUL3) and KLHL3 are subunits of Cullin–RING E3 ubiquitin ligase complexes, and the serine–threonine kinases WNK1 and WNK4 are substrates of this ubiquitin ligase. For CUL3, all mutations associated with PHAII exclusively lead to exon 9 skipping. In this study, we identified a Chinese PHAII kindred caused by a novel synonymous mutation (c.1221A > G p.Glu407Glu) in CUL3, and explored its effects on exon 9 abnormal splicing through an in vitro splicing assay and study of the patients’ RNA. We obtained evidence that this synonymous mutation leads to complete exon 9 skipping, and in silico bioinformatics analysis demonstrated that the CUL3 c.1221A > G mutation might decrease the ratio of exonic splicing enhancers and silencers. This is the first report of PHAII in Chinese patients with a novel CUL3 mutation. Our findings add a novel pathogenic splicing variant to the CUL3 mutational spectrum and provide reference for further research on mechanisms of splicing modulation and development of potential therapeutic reagents for PHAII.

Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice

AbstractSplicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre–mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2-mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2-mutated mice. RNA sequencing of the Srsf2-mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r, Fyn, Gnas, Nsd1, Hnrnpa2b1, and Trp53bp1. Among the mutant Srsf2-associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults.

The mechanisms of a mammalian splicing enhancer.

Exonic splicing enhancer (ESE) sequences are bound by serine & arginine-rich (SR) proteins, which in turn enhance the recruitment of splicing factors. It was inferred from measurements of splicing around twenty years ago that Drosophila doublesex ESEs are bound stably by SR proteins, and that the bound proteins interact directly but with low probability with their targets. However, it has not been possible with conventional methods to demonstrate whether mammalian ESEs behave likewise. Using single molecule multi-colour colocalization methods to study SRSF1-dependent ESEs, we have found that that the proportion of RNA molecules bound by SRSF1 increases with the number of ESE repeats, but only a single molecule of SRSF1 is bound. We conclude that initial interactions between SRSF1 and an ESE are weak and transient, and that these limit the activity of a mammalian ESE. We tested whether the activation step involves the propagation of proteins along the RNA or direct interactions with 3′ splice site components by inserting hexaethylene glycol or abasic RNA between the ESE and the target 3′ splice site. These insertions did not block activation, and we conclude that the activation step involves direct interactions. These results support a model in which regulatory proteins bind transiently and in dynamic competition, with the result that each ESE in an exon contributes independently to the probability that an activator protein is bound and in close proximity to a splice site.

Novel AKT1 mutations associated with cell-cycle abnormalities in gastric carcinoma.

The aim of this study is to identify the AKT1 gene mutation driven pathogenicity in gastric cancer for Mizo population. 50 diffuse-type gastric tumors were analyzed for AKT1 exon 2 and 14 mutations. Cell-cycle aberration was analyzed in the AKT1-mutated samples and the stability of the protein as well as exonic splicing enhancer motifs were examined. The novel mutations, 15553T>A and 25376C>G might affect the exonic splicing enhancers and silencers. Significant decline was observed in the S-phase population in the tumor cells with 15553T>A and 15579G>C mutations suggesting the arrest of G1 phase. The present study is a novel finding of the possible role of AKT1 mutations which might help to identify gastric cancer patients.

CRISPR/Cas9 Deletions in a Conserved Exon of Distal-Less Generates Gains and Losses in a Recently Acquired Morphological Novelty in Flies

Distal-less has been repeatedly co-opted for the development of many novel traits. Here, we document its curious role in the development of a novel abdominal appendage (“sternite brushes”) in sepsid flies. CRISPR/Cas9 deletions in the homeodomain result in losses of sternite brushes, demonstrating that Distal-less is necessary for their development. However, deletions in the coding exon upstream of the homeodomain (Exon 2) produces both losses and gains of brushes. A dissection of Exon 2 reveals that the likely mechanism for gains involves a deletion in an exon-splicing enhancer site that leads to exon-skipping. Such contradictory phenotypes are also observed in butterflies which suggests that deletions in the conserved upstream regions have the potential to generate phenotypic variability in insects that diverged 300 million years ago. Our results demonstrate the importance of Distal-less for the development of a novel abdominal appendage in insects and highlight how site-specific mutations in the same exon can produce contradictory phenotypes.

Depletion of somatic mutations in splicing-associated sequences in cancer genomes

BackgroundAn important goal of cancer genomics is to identify systematically cancer-causing mutations. A common approach is to identify sites with high ratios of non-synonymous to synonymous mutations; however, if synonymous mutations are under purifying selection, this methodology leads to identification of false-positive mutations. Here, using synonymous somatic mutations (SSMs) identified in over 4000 tumours across 15 different cancer types, we sought to test this assumption by focusing on coding regions required for splicing.ResultsExon flanks, which are enriched for sequences required for splicing fidelity, have ~ 17% lower SSM density compared to exonic cores, even after excluding canonical splice sites. While it is impossible to eliminate a mutation bias of unknown cause, multiple lines of evidence support a purifying selection model above a mutational bias explanation. The flank/core difference is not explained by skewed nucleotide content, replication timing, nucleosome occupancy or deficiency in mismatch repair. The depletion is not seen in tumour suppressors, consistent with their role in positive tumour selection, but is otherwise observed in cancer-associated and non-cancer genes, both essential and non-essential. Consistent with a role in splicing modulation, exonic splice enhancers have a lower SSM density before and after controlling for nucleotide composition; moreover, flanks at the 5’ end of the exons have significantly lower SSM density than at the 3’ end.ConclusionsThese results suggest that the observable mutational spectrum of cancer genomes is not simply a product of various mutational processes and positive selection, but might also be shaped by negative selection.

Genetic and molecular risk factors within the newly identified primate‐specific exon of the SAP97/DLG1 gene in the 3q29 schizophrenia‐associated locus

The synapse-associated protein 97/discs, large homolog 1 of Drosophila (DLG1) gene encodes synaptic scaffold PDZ proteins interacting with ionotropic glutamate receptors including the N-methyl-D-aspartate type glutamate receptor (NMDAR) that is presumed to be hypoactive in brains of patients with schizophrenia. The DLG1 gene resides in the chromosomal position 3q29, the microdeletion of which confers a 40-fold increase in the risk for schizophrenia. In the present study, we performed genetic association analyses for DLG1 gene using a Japanese cohort with 1808 schizophrenia patients and 2170 controls. We detected an association which remained significant after multiple comparison testing between schizophrenia and the single nucleotide polymorphism (SNP) rs3915512 that is located within the newly identified primate-specific exon (exon 3b) of the DLG1 gene and constitutes the exonic splicing enhancer sequence. When stratified by onset age, although it did not survive multiple comparisons, the association was observed in non-early onset schizophrenia, whose onset-age selectivity is consistent with our recent postmortem study demonstrating a decrease in the expression of the DLG1 variant in early-onset schizophrenia. Although the present study did not demonstrate the previously reported association of the SNP rs9843659 by itself, a meta-analysis revealed a significant association between DLG1 gene and schizophrenia. These findings provide a valuable clue for molecular mechanisms on how genetic variations in the primate-specific exon of the gene in the schizophrenia-associated 3q29 locus affect its regulation in the glutamate system and lead to the disease onset around a specific stage of brain development.

Mutation-Based Therapy for Duchenne Muscular Dystrophy: Antisense Treatment Arrives in the Clinic.

Duchenne muscular dystrophy (DMD) arises from mutations in the dystrophin gene. The dystrophin gene is composed of 79 exons, and the majority of mutations in DMD are deletions, often spanning multiple exons.1 In 2016, the US Food and Drug Administration (FDA) granted accelerated approval for eteplirsen (Exondys51), an antisense oligonucleotide compound designed to block exon 51 of dystrophin to restore the reading frame in patients with DMD with specific mutations (Figure, A).2 This treatment is directed at ≈10% to 15% of patients with DMD (≈1500 treatment-eligible individuals). The approval of eteplirsen is game changing for the field of molecular gene correction. However, its approval was viewed as controversial because of the unconventional clinical trial data and limited efficacy. Figure. Eteplirsen (Exondys51)-mediated reading-frame correction of Duchenne muscular dystrophy (DMD) mutations. A, Top , Schematic of the dystrophin protein and its domains. ABD1 indicates actin binding domain; CR, cysteine rich domain; CT, C-terminal domain; and H, hinge regions. The spectrin-like repeats are numbered. The red bar indicates the approximate exon 51 targeting region. Middle , Schematic showing eteplirsen-mediated reading-frame correction of a DMD frameshift mutation. The normal dystrophin locus from exons 41 to 52 is shown, indicating the reading frame of each exon. Many patients with DMD have variable-sized deletions spanning exons 47 to 50, disrupting the reading frame (dashed blue line). Eteplirsen (red box) is an antisense oligonucleotide that binds to exon-splicing enhancer sequences in exon 51, causing its exclusion from mRNA. Because exon 51 is excluded, exons 46 (or 47, 48, and 49) joins to exon 52 (solid blue line) to restore an open reading frame. B , Antisense oligonucleotides have complementary sequences to those within an exon, in this case exon 51. Chemical modifications to the antisense oligonucleotides permit the double-stranded hybrid …

A Genetic Variant of ASCT2 Hampers In Vitro RNA Splicing and Correlates with Human Longevity.

Given the role of amino acid regulation for physiological and pathological cell proliferation, we investigated whether the variability of solute carrier family 1, member 5 (SLC1A5, namely ASCT2), encoding for ASCT2 protein, a major glutamine transporter, is related to longevity. A total of 607 differently aged unrelated individuals, 351 very old subjects (≥85 years, range 85-106 years, mean age 93.82 ± 4.44 years) and 256 younger controls (<85 years, range 64-84 years, mean age 73.60 ± 5.70 years) were analyzed. Single-nucleotide polymorphisms (SNPs) were selected by a tagging SNP approach prioritizing those most likely to be of functional relevance. Genotyping was carried out using iPLEX Gold Genotyping Assay (Sequenom MassARRAY), and a logistic regression analysis was used to examine the associations between genotypes and the longevity phenotype. Age-associated variants were predicted to be involved in the alteration of an exonic splicing enhancer (ESE) site by Human Splicing Finder (HSF) v.3. Minigene constructs containing the alleles associated with longevity allowed the assessment of the functional effects of the polymorphisms. Two polymorphisms were found associated with human longevity (rs3027958 and rs1644343). Indeed, the major alleles of both SNPs positively influence the probability to become long-lived. In vitro assays suggested that the minor allele of rs3027958 alters an ESE site, thus inducing intron retention. We provide evidence about the functional role of intronic variants of the ASCT2 gene in hampering the splicing process, and the effect of these variants on survival, confirming the crucial role played by this amino acid transporter in modulating longevity.

Overcoming imatinib resistance conferred by the BIM deletion polymorphism in chronic myeloid leukemia with splice-switching antisense oligonucleotides.

Many tyrosine kinase-driven cancers, including chronic myeloid leukemia (CML), are characterized by high response rates to specific tyrosine kinase inhibitors (TKIs) like imatinib. In East Asians, primary imatinib resistance is caused by a deletion polymorphism in Intron 2 of the BIM gene, whose product is required for TKI-induced apoptosis. The deletion biases BIM splicing from exon 4 to exon 3, generating splice isoforms lacking the exon 4-encoded pro-apoptotic BH3 domain, which impairs the ability of TKIs to induce apoptosis. We sought to identify splice-switching antisense oligonucleotides (ASOs) that block exon 3 but enhance exon 4 splicing, and thereby resensitize BIM deletion-containing cancers to imatinib. First, we mapped multiple cis-acting splicing elements around BIM exon 3 by minigene mutations, and found an exonic splicing enhancer acting via SRSF1. Second, by a systematic ASO walk, we isolated ASOs that corrected the aberrant BIM splicing. Eight of 67 ASOs increased exon 4 levels in BIM deletion-containing cells, and restored imatinib-induced apoptosis and TKI sensitivity. This proof-of-principle study proves that resistant CML cells by BIM deletion polymorphism can be resensitized to imatinib via splice-switching BIM ASOs. Future optimizations might yield a therapeutic ASO as precision-medicine adjuvant treatment for BIM-polymorphism-associated TKI-resistant CML and other cancers.

Mutation in an exonic splicing enhancer site causing chronic granulomatous disease

In a male patient suffering from X-linked chronic granulomatous disease (CGD) we found a c.389G>T mutation in exon 5 of the CYBB gene. We have analyzed why 95% of the transcripts of this gene lacked exon 5, leading to a frameshift and premature termination codon. The mutation was located in a region comprising three putative exonic splicing enhancer binding sites, for SRSF1, SRFS2 and SRFS6, according to the ESEfinder Tool (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi). With the Analyser Splice Tool we calculated the probability of skipping of exon 5 in CYBB mRNA, and by means of Sroogle the number of putative binding motifs for splicing enhancer and splicing silencer proteins (http://astlab.tau.ac.il/index.php). These analyses clarify why this exon was skipped in the majority of the mRNA. The normally spliced transcript contains an amino acid change p.Arg130Leu. This poorly expressed transcript gives rise to a protein with low expression but presumably normal activity, leading to a respiratory burst activity in the patient's neutrophils of about 15% of normal.

Abstract 1396: Role of epigenetic reader and alternative mRNA splicing variant MBD2 variant 2 in triple-negative breast cancer

Abstract Background: Among all breast cancer patients, those diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst prognosis, in part attributable to the fact that TNBC lacks targets for effective molecularly-targeted therapies. The concept that EGFR inhibitor drugs could be used as a targeted treatment against TNBC has been put forth because roughly 50% of TNBC express high levels of EGFR. However clinical trials targeting EGFR did not significantly improve patient outcomes. Our recent work offers a potential explanation as to why EGFR inhibitors failed and supports an innovative therapeutic approach combining an EGFR tyrosine kinase inhibitor with the novel antioxidant biologic, CAT-SKL. Our data indicate that inhibition of cancer stem cell-like cells depends on antioxidant-induced downregulation of an alternative mRNA splice variant of the methyl-CpG binding domain 2 gene, MBD2_v2. Objective and Methods: The purpose of the present study was to evaluate the importance of MBD2_v2 in TNBC and better understand how CAT-SKL regulates its expression. Using TNBC cell lines we investigated the effects of MBD2_v2 expression on tumor initiation capacity. Furthermore we studied MBD2_v2 mRNA levels in an in vivo tumor xenograph mouse model of TNBC and in human TNBC samples using an existing database. Then by RNA-seq analysis we assessed how CAT-SKL regulates MBD2_v2 expression in TNBC cells through differential gene expression. Results: The results support that MBD2_v2 expression is regulated by redox signaling in TNBC cells, which is tightly linked to TNBC cell tumor initiation capacity. Furthermore, MBD2_v2 was upregulated in tumors harvested from overweight mice, which also displayed increased tumor take rate, suggesting that a pro-inflammatory tumor microenvironment could play a role in promoting MBD2_v2 expression. In addition, RNA-seq analysis identified an overrepresentation of mRNA splicing factors with expression downregulated by CAT-SKL treatment of TNBC cells. From this gene set two splicing co-factors are highlighted: SRRM1 and CPSF3. These co-factors are critical for splicing factor function, 3’ end formation and polyadenylation; and both have been linked via protein-protein interaction to a relevant exonic splicing enhancer site in exon 3 of the MBD2 _v2 transcript. Finally, according to the Kaplan Meier Plotter database high MBD2_v2 levels in BC were associated with shorter relapse free survival. Conclusion: Our investigation sheds light on the relevance of MBD2_v2 expression in TNBC, and how a select antioxidant regulates its expression. The application of which holds promise of a novel, targeted therapeutic modality for TNBC, the subtype with the worst prognosis and highest need for treatment options. Citation Format: Emily A. Girsch, Bin Bao, Cristina Mitrea, Gregory Dyson, Julie Boerner, Lisa Polin, Stan R. Terlecky, Aliccia Bollig-Fischer. Role of epigenetic reader and alternative mRNA splicing variant MBD2 variant 2 in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1396. doi:10.1158/1538-7445.AM2017-1396

Abstract 4281: Identification of putative cancer susceptibility genes through whole exome sequencing of a family presenting hereditary colorectal cancer

Abstract Germline mutations in known colorectal cancer (CRC) predisposing genes accounts for less than half of the genetic cause of hereditary CRC cases. The absence of mutations in the remaining patients suggests that other yet unknown CRC predisposing genes might exist. However, recently studies with large cohorts probably discounts the existence of major unknown genes and it is thought that rare or even family private variants and genes may contribute to familial CRC. Thus, the aim of our study was to identify novel CRC susceptibility genes by whole exome sequencing (WES) of a family presenting hereditary CRC. The studied family fulfilled Amsterdam I criteria and appears to segregate a dominant cancer syndrome with a high risk of colon and rectal cancer, with four affected members in three generations. The index patient’s tumor was microsatellite stable and positive for the four mismatch repair proteins at immunohistochemistry. Genetic testing of the proband was negative for the following genes: MLH1, MSH2, MSH6, PMS2, PMS1, TP53, CHEK2. We performed WES in three family members: two with rectal cancer (onset 26 and 56 years) and one with colon cancer (onset 63 years). Using a functional-based variant prioritization, we identified six novel or very rare (MAF&amp;lt;0.002) candidate variants segregating with the phenotype. Two variants were loss-of-function stop codon variants in LSG1 and RRH genes. Three variants were missense (ARID1A, MC1R and SPEN), and three were synonymous (KIT, POLE and XAB2), all occurring in cancer-related genes. The identified KIT variant (c.1983C&amp;gt;A) is located 8 base pairs from the 3’ end of exon 13, and is predicted it to disrupt an exonic splicing enhancer by in silico tools (ESR finder and Human Splice Finder), possibly affecting the splice of this gene. Pyrosequencing of the KIT variant in tumor cDNA revealed an allelic imbalance between mutated and wild type alleles, with the mutated allele being three times more expressed. Transcript analysis using a customized heteroduplex enrichment-based method identified a novel KIT transcript in the index patient’s tumor, presenting an exon skipping event of exon 12. The resulting transcript (c.1775_1879del) is predicted to produce an in-frame deletion of 35 amino acids (p.Gly592_Pro627delinsAla) at the beginning of the protein kinase domain. The functional impact of this transcript and its relevance for CRC predisposition will be further evaluated. Moreover, experiments evaluating allelic imbalance, loss of heterozygosity and alternative spliced transcripts for the other identified variants are ongoing. Our results provide a set of putative CRC predisposing genes and underpin that WES of highly informative CRC families, ascertained on the basis of highly selected phenotypes and followed by functional validation, are likely to continue to prove an effective strategy for gene discovery. Citation Format: Giovana T. Torrezan, Anna Smirnova, Felipe C. Silva, Erika M. Santos, Renan Valieris, Jorge E. de Souza, Samuel Aguiar, Benedito M. Rossi, Sandro J. de Souza, Dirce M. Carraro. Identification of putative cancer susceptibility genes through whole exome sequencing of a family presenting hereditary colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4281. doi:10.1158/1538-7445.AM2017-4281

Analysis of Competing HIV-1 Splice Donor Sites Uncovers a Tight Cluster of Splicing Regulatory Elements within Exon 2/2b.

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.

Splicing-Correcting Therapy for SMA

Spinal muscular atrophy (SMA) is caused by deficiency of SMN protein, which is crucial for spliceosome subunits biogenesis. Most SMA patients have SMN1 deletions, leaving SMN2 as sole SMN source; however, a C→T substitution converts an exonic-splicing enhancer (ESE) to a silencer (ESS), causing frequent exon7 skipping in SMN2 pre-mRNA and yielding a truncated protein. Antisense treatment to SMN2 intron7-splicing silencer (ISS) improves SMN expression and motor function. To view this Bench to Bedside, open or download the PDF.

PTBP1: A candidate gene for intellectual disability

Intellectual disability (ID) is a common heterogeneous disease. Many genes have been implicated in the etiology of ID, yet gene discovery continues at a fast pace. In this study, we investigated the genetic cause of ID in a consanguineous Arab family. For that purpose we utilized autozygosity mapping, candidate gene testing, and whole exome sequencing to find the causative mutation. We identified a synonymous mutation, c.708C>T, in the polypyrimidine tract binding protein 1 (PTBP1) gene that fully segregated with the ID phenotype; Mutationtaster predicted the mutation to be disease causing. Analysis of the mutation revealed its effect on an exonic splice enhancer suggesting a change in the potential binding to its target sequence. This mutation was not found in 514 ethnically matched chromosomes and only one allele harbored the same mutation out of 120,782 alleles in the Exome Aggregation Consortium (ExAC) database. PTBP1 plays a major role in splicing related to neuronal differentiation; its repressive activity regulates activation of a brain specific alternative splicing program. This is the first study to implicate a PTBP1 mutation in human disease.

A common polymorphism decreases LRP1 mRNA stability and is associated with increased plasma factor VIII levels

The low-density lipoprotein receptor-related protein 1 (LRP1) gene is associated with increased levels of plasma factor VIII (FVIII). We aimed to explore eight functional genetic LRP1 variants for their potential roles in regulating FVIII levels and acute ischemic stroke (AIS). This genetic association study enrolled 192 patients with AIS and 134 controls. There were no significant differences in the genetic frequency of the eight functional single-nucleotide polymorphisms (SNPs) between the control and AIS groups. However, while analyzing the association between the eight SNPs and plasma FVIII levels, subjects with T/T genotype of rs1800137 (vs. CC+CT) were found to be associated with higher FVIII levels (23.5IU/dL; 95% confidence interval, 7.4–39.5IU/dL; P=0.0044) after adjusting for age, gender, estimated glomerular filtration rate, O blood type, inflammatory state, and body mass index.An analysis of the mRNA stability and abundance was designed and performed using minigene system transfected into HepG2 cells to assess the possible differences in mRNA stabilities between rs1800137 CC (rs1800137C) and TT (rs1800137T) genotypes. Site-directed mutagenesis revealed that rs1800137T accounts for the observed decrease in mRNA stability. The SNP rs1800137, located in exon 8, has been identified as an exon-splicing enhancer in silico. However, alternative splicing of LRP1 without inclusion of exon 8 was not identified. In transfected HepG2 cells, cycloheximide slowed down the degradation of the rs1800137T-containing minigene. These results demonstrate that synonymous SNP rs1800137 can lead to increased plasma FVIII levels due to decreased mRNA stability via translation-dependent mRNA degradation associated with codon optimality.

Identified of a novel cis-element regulating the alternative splicing of LcDREB2

Alternative splicing (AS) is an important gene regulation mechanism in plants. Despite the widespread use of AS in plant gene expression regulation, the identification of the cis-elements involved in the AS mechanism is rarely reported in plants. To explore the regulation mechanism of the AS of LcDREB2, a DREB2 ortholog from Sheepgrass (Leymus chinensis), the genomic sequences of LcDREB2 and its homologs in Poaceae were aligned, and six mutations were introduced in the conserved sequence of LcDREB2. By analyzing the distinct transcript patterns of the LcDREB2 mutants in transgenic Oryza sativa, a novel cis-element that affected the AS of LcDREB2 was identified as Exonic Splicing Enhancer 1 (ESE1). In addition, five serine-arginine rich (SR) proteins were confirmed to interact with ESE1 by electrophoretic mobility shift assay (EMSA). To further explore the expression regulation mechanism of the DREB subfamily, phylogenetic analysis of DREB2 paralogous genes was performed. The results strongly supported the hypothesis that AS is conserved in Poaceae plants and that it is an evolutionary strategy for the regulation of the functional expression of genes. The findings and methods of our study will promote a substantial step forward in understanding of the plant AS regulation mechanism.

An NS-segment exonic splicing enhancer regulates influenza A virus replication in mammalian cells

Influenza virus utilizes host splicing machinery to process viral mRNAs expressed from both M and NS segments. Through genetic analysis and functional characterization, we here show that the NS segment of H7N9 virus contains a unique G540A substitution, located within a previously undefined exonic splicing enhancer (ESE) motif present in the NEP mRNA of influenza A viruses. G540A supports virus replication in mammalian cells while retaining replication ability in avian cells. Host splicing regulator, SF2, interacts with this ESE to regulate splicing of NEP/NS1 mRNA and G540A substitution affects SF2–ESE interaction. The NS1 protein directly interacts with SF2 in the nucleus and modulates splicing of NS mRNAs during virus replication. We demonstrate that splicing of NEP/NS1 mRNA is regulated through a cis NEP-ESE motif and suggest a unique NEP-ESE may contribute to provide H7N9 virus with the ability to both circulate efficiently in avian hosts and replicate in mammalian cells.