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Autosomal dominant optic atrophy (ADOA) is an inherited optic neuropathy most frequently associated with OPA1 mutations. Most variants result in haploinsufficiency, and patient cells express roughly half of the normal levels of OPA1 protein. OPA1 is a mitochondrial GTPase that is essential for normal mitochondrial function. We identified and characterized STK-002, an antisense oligonucleotide (ASO) designed to prevent the incorporation of a naturally occurring alternatively spliced nonproductive exon in OPA1. STK-002 dose dependently reduced the inclusion of this exon, and increased OPA1 protein in human cells, including ADOA patient-derived fibroblasts. ADOA patient cells manifest reduced mitochondrial respiration, and treatment with STK-002 improved the parameters of mitochondrial respiratory function in these cells. Since STK-002 increases OPA1 through the wild-type allele, we assessed retinal OPA1 in wild-type cynomolgus monkeys and rabbits after intravitreal administration of STK-002 or a rabbit-specific surrogate. Increased OPA1 protein was produced in retinal tissue in both species at 4 weeks after ASO injection and persisted in monkeys at 8 weeks. STK-002 and enhanced OPA1 immunofluorescence were visualized in retinal ganglion cells of cynomolgus monkeys treated with the ASO. Cumulatively, these data support the progression of STK-002 toward the clinic as the first potential disease-modifying treatment for ADOA.

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RBM10 modulates transcriptome-wide cassette exon splicing. Loss-of-function RBM10 mutations are enriched in thyroid cancers with distant metastases. Analysis of transcriptomes and genes mis-spliced by RBM10 loss showed pro-migratory and RHO/RAC signaling signatures. RBM10 loss increases cell velocity. Cytoskeletal and ECM transcripts subject to exon-inclusion events included vinculin (VCL), tenascin C (TNC) and CD44. Knockdown of the VCL exon inclusion transcript in RBM10-null cells reduced cell velocity, whereas knockdown of TNC and CD44 exon-inclusion isoforms reduced invasiveness. RAC1-GTP levels were increased in RBM10-null cells. Mouse Hras G12V /Rbm1O KO thyrocytes develop metastases that are reversed by RBM10 or by combined knockdown of VCL, CD44 and TNC inclusion isoforms. Thus, RBM10 loss generates exon inclusions in transcripts regulating ECM-cytoskeletal interactions, leading to RAC1 activation and metastatic competency. Moreover, a CRISPR-Cas9 screen for synthetic lethality with RBM10 loss identified NFkB effectors as central to viability, providing a therapeutic target for these lethal thyroid cancers.

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BACKGROUND: X-linked Alport syndrome (XLAS) is an inherited renal disease caused by rare variants of COL4A5 on chromosome Xq22. Many studies have indicated that single nucleotide variants (SNVs) in exons can disrupt normal splicing process of the pre-mRNA by altering various splicing regulatory signals. The male patients with XLAS have a strong genotype-phenotype correlation. Confirming the effect of variants on splicing can help to predict kidney prognosis. This study aimed to investigate whether single nucleotide substitutions, located within three bases at the 5' end of the exons or internal position of the exons in COL4A5 gene, cause aberrant splicing process. METHODS: We analyzed 401 SNVs previously presumed missense and nonsense variants in COL4A5 gene by bioinformatics programs and identified candidate variants that may affect the splicing of pre-mRNA via minigene assays. RESULTS: Our study indicated three of eight candidate variants induced complete or partial exon skipping. Variants c.2678G>C and c.2918G>A probably disturb classic splice sites leading to corresponding exon skipping. Variant c.3700C>T may disrupt splicing enhancer motifs accompanying with generation of splicing silencer sequences resulting in the skipping of exon 41. CONCLUSION: Our study revealed that two missense variants positioned the first nucleotides of the 5' end of COL4A5 exons and one internal exonic nonsense variant caused aberrant splicing. Importantly, this study emphasized the necessity of assessing the effects of SNVs at the mRNA level.

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Targeting alternative exons for therapeutic gain has been achieved in a few instances and potentially could be applied more broadly. The myosin phosphatase (MP) enzyme is a critical hub upon which signals converge to regulate vessel tone. Alternative exon 24 of myosin phosphatase regulatory subunit (Mypt1 E24) is an ideal target as toggling between the two isoforms sets smooth muscle sensitivity to vasodilators such as nitric oxide (NO). This study aimed to develop a gene-based therapy to suppress splicing of Mypt1 E24 thereby switching MP enzyme to the NO-responsive isoform. CRISPR/Cas9 constructs were effective at editing of Mypt1 E24 in vitro; however, targeting of vascular smooth muscle in vivo with AAV9 was inefficient. In contrast, an octo-guanidine conjugated antisense oligonucleotide targeting the 5' splice site of Mypt1 E24 was highly efficient in vivo. It reduced the percent splicing inclusion of Mypt1 E24 from 80% to 10% in mesenteric arteries. The maximal and half-maximal effects occurred at 12.5 and 6.25 mg/kg, respectively. The effect persisted for at least 1 mo without toxicity. This highly effective splice-blocking antisense oligonucleotide could be developed as a novel therapy to reverse vascular dysfunction common to diseases such as hypertension and heart failure.NEW & NOTEWORTHY Alternative exon usage is a major driver of phenotypic diversity in all cell types including smooth muscle. However, the functional significance of most of the hundreds of thousands of alternative exons has not been defined, nor in most cases even tested. If their importance to vascular function were known these alternative exons could represent novel therapeutic targets. Here, we present injection of Vivo-morpholino splice-blocking antisense oligonucleotides as a simple, efficient, and cost-effective method for suppression of alternative exon usage in vascular smooth muscle in vivo.

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BACKGROUND: Gitelman syndrome (GS) is a type of salt-losing tubular disease, most of which is caused by SLC12A3 gene variants, and missense variants account for the majority. Recently, the phenomenon of exon skipping, in which variants disrupt normal pre-mRNA splicing, has been related to a variety of diseases. Therefore, we hypothesize that a certain proportion of SLC12A3 variants can result in disease via interfering with the normal splicing process. METHODS: We analyzed 342 previously presumed SLC12A3 missense variants using bioinformatics programs and identified candidate variants that may alter the splicing of pre-mRNA through minigene assays. RESULTS: Our study revealed that, among ten candidate variants, six variants (c.602G>A, c.602G>T, c.1667C>T, c.1925G>A, c.2548G>C, and c.2549G>C) led to complete or incomplete exon skipping by affecting exonic splicing regulatory elements and/or disturbing canonical splice sites. CONCLUSION: It is worth mentioning that this is the largest study on pre-mRNA splicing of SLC12A3 exonic variants. In addition, our study emphasizes the importance of detecting splicing function at the mRNA level in GS and indicates that minigene analysis is a valuable tool for splicing functional assays of variants in vitro.

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Muscular dystrophy with myositis (mdm) is a naturally occurring mutation in the mouse Ttn gene that results in higher passive stress in muscle fibers and intact muscles compared to wild-type (WT). The goal of this study was to test whether alternative splicing of titin exons occurs in mdm muscles, which contain a small deletion in the N2A-PEVK regions of titin, and to test whether splicing changes are associated with an increase in titin-based passive tension. Although higher levels of collagen have been reported previously in mdm muscles, here we demonstrate alternative splicing of titin in mdm skeletal muscle fibers. We identified Z-band, PEVK, and C-terminus Mex5 exons as splicing hotspots in mdm titin using RNA sequencing data and further reported upregulation in ECM-associated genes. We also treated skinned mdm soleus fiber bundles with trypsin, trypsin + KCl, and trypsin + KCL + KI to degrade titin. The results showed that passive stress dropped significantly more after trypsin treatment in mdm fibers (11 ± 1.6 mN/mm2) than in WT fibers (4.8 ± 1 mN/mm2; p = 0.0004). The finding that treatment with trypsin reduces titin-based passive tension more in mdm than in WT fibers supports the hypothesis that exon splicing leads to the expression of a stiffer and shorter titin isoform in mdm fibers. After titin extraction by trypsin + KCl + KI, mdm fibers (6.7 ± 1.27 mN/mm2) had significantly higher collagen-based passive stress remaining than WT fibers (2.6 ± 1.3 mN/mm2; p = 0.0014). We conclude that both titin and collagen contribute to higher passive tension of mdm muscles.

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Primary distal renal tubular acidosis (dRTA) is a rare tubular disease associated with variants in SLC4A1, ATP6V0A4, ATP6V1B1, FOXⅠ1, or WDR72 genes. Currently, there is growing evidence that all types of exonic variants can alter splicing regulatory elements, affecting the precursor messenger RNA (pre-mRNA) splicing process. This study was to determine the consequences of variants associated with dRTA on pre-mRNA splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 15 candidate variants, 7 variants distributed in SLC4A1 (c.1765C>T, p.Arg589Cys), ATP6V1B1 (c.368G>T, p.Gly123Val; c.370C>T, p.Arg124Trp; c.484G>T, p.Glu162* and c.1102G>A, p.Glu368Lys) and ATP6V0A4 genes (c.322C>T, p.Gln108* and c.1572G>A, p.Pro524Pro) were identified to result in complete or incomplete exon skipping by either disruption of exonic splicing enhancers (ESEs) and generation of exonic splicing silencers, or interference with the recognition of the classic splicing site, or both. To our knowledge, this is the first study on pre-mRNA splicing of exonic variants in the dRTA-related genes. These results highlight the importance of assessing the effects of exonic variants at the mRNA level and suggest that minigene analysis is an effective tool for evaluating the effects of splicing on variants in vitro.

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The reduction of survival motor neuron (SMN) protein causes spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease. Nusinersen is an antisense oligonucleotide, approved by the FDA, which specifically binds to the repressor within SMN2 exon 7 to enhance exon 7 inclusion and augment production of functional SMN protein. Nusinersen is the first new oligonucleotide-based drug targeting the central nervous system for the treatment of SMA. This review of nusinersen will discuss its action mechanism, cellular uptake, trafficking mechanisms, and administration approaches to cross the blood-brain barrier. Furthermore, nusinersen clinical trials will be assessed in terms of pharmacokinetics, tolerability and safety, the clinical outcomes of multiple intrathecal doses, and a discussion on the primary and secondary endpoints.

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The reduction of survival motor neuron (SMN) protein causes spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease. Nusinersen is an antisense oligonucleotide, approved by the FDA, which specifically binds to the repressor within SMN2 exon 7 to enhance exon 7 inclusion and augment production of functional SMN protein. Nusinersen is the first new oligonucleotide-based drug targeting the central nervous system for the treatment of SMA. This review of nusinersen will discuss its action mechanism, cellular uptake, trafficking mechanisms, and administration approaches to cross the blood-brain barrier. Furthermore, nusinersen clinical trials will be assessed in terms of pharmacokinetics, tolerability and safety, the clinical outcomes of multiple intrathecal doses, and a discussion on the primary and secondary endpoints.

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BACKGROUND: Exon splicing is a regulated cellular process in the transcription of protein-coding genes. Technological advancements and cost reductions in RNA sequencing have made quantitative and qualitative assessments of the transcriptome both possible and widely available. RNA-seq provides unprecedented resolution to identify gene structures and resolve the diversity of splicing variants. However, currently available ab initio aligners are vulnerable to spurious alignments due to random sequence matches and sample-reference genome discordance. As a consequence, a significant set of false positive exon junction predictions would be introduced, which will further confuse downstream analyses of splice variant discovery and abundance estimation. RESULTS: In this work, we present a deep learning based splice junction sequence classifier, named DeepSplice, which employs convolutional neural networks to classify candidate splice junctions. We show (I) DeepSplice outperforms state-of-the-art methods for splice site classification when applied to the popular benchmark dataset HS3D, (II) DeepSplice shows high accuracy for splice junction classification with GENCODE annotation, and (III) the application of DeepSplice to classify putative splice junctions generated by Rail-RNA alignment of 21,504 human RNA-seq data significantly reduces 43 million candidates into around 3 million highly confident novel splice junctions. CONCLUSIONS: A model inferred from the sequences of annotated exon junctions that can then classify splice junctions derived from primary RNA-seq data has been implemented. The performance of the model was evaluated and compared through comprehensive benchmarking and testing, indicating a reliable performance and gross usability for classifying novel splice junctions derived from RNA-seq alignment.

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BACKGROUND AND PURPOSE: Retinitis pigmentosa is an important cause of severe visual dysfunction. This study reports a novel splicing mutation in the lecithin retinol acyltransferase (LRAT) gene associated with early onset retinitis pigmentosa and characterizes the effects of this mutation on mRNA splicing and structure. METHODS: Genome-wide linkage analysis followed by dideoxy sequencing of the linked candidate gene LRAT was performed in a consanguineous Pakistani family with autosomal recessive retinitis pigmentosa. In silico prediction and minigene assays were used to investigate the effects of the presumptive splicing mutation. RESULTS: ARRP in this family was linked to chromosome 4q31.21-q32.1 with a maximum LOD score of 5.40. A novel homozygous intronic mutation (NM_004744.4: c.541-15T>G) was detected in LRAT. In silico tools predicted that the AG-creating mutation would activate an intronic cryptic acceptor site, but cloning fragments of wild-type and mutant sequences of LRAT into Exontrap Cloning Vector pET01 and Expression Cloning Vector pCMV-(DYKD4K)-C showed that the primary effect of the sequence change was to weaken the nearby authentic acceptor site and cause exon skipping, with only a small fraction of transcripts utilizing the acceptor site producing the reference transcript. CONCLUSIONS: The c.541-15T>G mutation in LRAT results in aberrant splicing and is therefore predicted to be causal for the early onset retinitis pigmentosa in this family. In addition, this work suggests that minigenes adapted to the specific gene and exon may need to be designed for variants in the first and last exon and intron to mimic the authentic splicing mechanism in vivo.

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The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.

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Understanding differential isoform expression is critical to mechanistically illuminate the biology underlying both normal development and disease states. High-throughput expression profiling analysis of splice variants has thus far been limited by sample requirements and an appropriate platform for quantitation and analysis. Here we describe Affymetrix GeneChip Human Transcriptome Array 2.0, which is employed for comprehensive examination of all known transcript isoforms.

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The exciting world of research with RNAs has to its credit some breakthrough findings that led to newer insights on multiple problems including that of human diseases. After the advent of siRNA, microRNA, and lncRNA, exciting novel molecules called circular RNAs (circRNAs) have been recently described. circRNAs are a class of non-coding RNAs, which are produced by scrambling of exons at the time of splicing. They are primarily produced in the brain region and are naturally present inside the cell. The best known ones so far include a particular type of circRNA namely "circular RNA sponge for miR-7" (ciRS-7 and CDR1as) which is the inhibitor of miR-7 microRNA-known to regulate various diseases like, cancer, neurodegenerative diseases, diabetes, and atherosclerosis. Similarly, another circRNA molecule called circmbl modulates the ratio of linear mRNA by competing with linear muscleblind gene through which it is synthesized. Considering the complex association of these molecules with critical microRNAs and gene families, circRNAs might have important roles in the cause and progression of human diseases. In particular, the multi-factorial nature of neurodegenerative diseases does warrant studies employing novel approaches towards identifying underlying root causes of these ailments. The non-coding RNAs, like circRNAs and microRNAs, could well present a common genetic trigger to multiple factors associated with neurodegenerative diseases. A specific fingerprint of a combination of various marker circRNAs could be explored for early diagnostic purpose as well. Herein, we review the possibility of exploring the role of circRNAs in the context of the central nervous system (CNS) and age-associated neurodegenerative diseases.

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We report a dystrophinopathy patient with an in-frame deletion of DMD exons 45-47, and therefore a genetic diagnosis of Becker muscular dystrophy, who presented with a more severe than expected phenotype. Analysis of the patient DMD mRNA revealed an 82 bp pseudoexon, derived from intron 44, that disrupts the reading frame and is expected to yield a nonfunctional dystrophin. Since the sequence of the pseudoexon and canonical splice sites does not differ from the reference sequence, we concluded that the genomic rearrangement promoted recognition of the pseudoexon, causing a severe dystrophic phenotype. We characterized the deletion breakpoints and identified motifs that might influence selection of the pseudoexon. We concluded that the donor splice site was strengthened by juxtaposition of intron 47, and loss of intron 44 silencer elements, normally located downstream of the pseudoexon donor splice site, further enhanced pseudoexon selection and inclusion in the DMD transcript in this patient.

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Our recent genome-wide association study (GWAS) has identified 105 genome-wide significant SNPs for milk production traits. Of these, one SNP (rs109661298) for milk fat percentage is located within the first intron of the bovine EEF1D gene on BTA14, thus that the EEF1D gene was considered as a novel candidate in dairy cattle. Until now, however, its genomic organization remains undetermined yet and no studies of EEF1D in relation to milk production traits have been reported. To layout the groundwork for the validation of gene function in dairy cattle, we herein investigated its expression pattern in lactating dairy cows. With rapid amplification of 5' cDNA end (5' RACE), two novel alternatively spliced transcript variants of 1202bp and 2195bp were isolated in bovine mammary in lactation, named EEF1Da and EEF1Db (GenBank: KC190039 and KC190038KC190039KC190038). Such two variants contain the different first exon from each other (exon1a vs exon1b: 294bp vs 1287bp) with no overlap and the same remaining 7 exons. Coding sequence similarity between EEF1Da and EEF1Db and three of human EEF1D transcript variants were 85-88%. With semi-quantitative and quantitative real-time RT-PCR, we found that the mRNA level of EEF1Da was similar to the overall EEF1D mRNA and much higher than EEF1Db in the mammary of lactating cows, indicating EEF1Da functions as the dominant transcript variant to encode the EEF1D protein. Tissue expression pattern showed that the mRNA expression of EEF1D and EEF1Da in mammary gland was significantly higher compared with other 7 tissues (P<0.05, P<0.01) with the exception of EEF1D between mammary and lung. Together, our findings present the first report on the alternative splicing of the bovine EEF1D gene and provided basis for further investigation on function validation of EEF1D in dairy cattle.

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In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA) and SGCG (c. (*) 102A/C) genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c. (*) 102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression.

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In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA) and SGCG (c.*102A/C) genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c.*102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression.