SRSF6 Regulates the Alternative Splicing of the Apoptotic Fas Gene by Targeting a Novel RNA Sequence.

Alternative splicing (AS) is a procedure during gene expression that allows the production of multiple mRNAs from a single gene, leading to a larger number of proteins with various functions. The alternative splicing (AS) of Fas (Apo-1/CD95) pre-mRNA can generate membrane-bound or soluble isoforms with pro-apoptotic and anti-apoptotic functions. SRSF6, a member of the Serine/Arginine-rich protein family, plays essential roles in both constitutive and alternative splicing. Here, we identified SRSF6 as an important regulatory protein in Fas AS. The cassette exon inclusion of Fas was decreased by SRSF6-targeting shRNA treatment, but increased by SRSF6 overexpression. The deletion and substitution mutagenesis of the Fas minigene demonstrated that the UGCCAA sequence in the cassette exon of the Fas gene causes the functional disruption of SRSF6, indicating that these sequences are essential for SRSF6 function in Fas splicing. In addition, biotin-labeled RNA-pulldown and immunoblotting analysis showed that SRSF6 interacted with these RNA sequences. Mutagenesis in the splice-site strength alteration demonstrated that the 5' splice-site, but not the 3' splice-site, was required for the SRSF6 regulation of Fas pre-mRNA. In addition, a large-scale RNA-seq analysis using GTEX and TCGA indicated that while SRSF6 expression was correlated with Fas expression in normal tissues, the correlation was disrupted in tumors. Furthermore, high SRSF6 expression was linked to the high expression of pro-apoptotic and immune activation genes. Therefore, we identified a novel RNA target with 5' splice-site dependence of SRSF6 in Fas pre-mRNA splicing, and a correlation between SRSF6 and Fas expression.

Eltrombopag as an Allosteric Inhibitor of the METTL3-14 Complex Affecting the m6A Methylation of RNA in Acute Myeloid Leukemia Cells.

N6A-methyladenosine (m6A) post-transcriptional modification, the most abundant internal RNA modification, is catalyzed by the METTL3-14 methyltransferase complex. Recently, attention has been drawn to the METTL3-14 complex regarding its significant roles in the pathogenesis of acute myeloid leukemia (AML), attracting the potential of novel therapeutic targets for the disease. Herein, we report the identification and characterization of eltrombopag as a selective allosteric inhibitor of the METTL3-14 complex. Eltrombopag exhibited selective inhibitory activity in the most active catalytic form of the METTL3-14 complex by direct binding, and the mechanism of inhibition was confirmed as a noncompetitive inhibition by interacting at a putative allosteric binding site in METTL3, which was predicted by cavity search and molecular docking studies. At a cellular level, eltrombopag displayed anti-proliferative effects in the relevant AML cell line, MOLM-13, in correlation with a reduction in m6A levels. Molecular mechanism studies of eltrombopag using m6A-seq analysis provided further evidence of its cellular function by determining the hypomethylation of leukemogenic genes in eltrombopag-treated MOLM-13 cells and the overlapping of the pattern with those of METTL3-knockdown MOLM-13 cells. In conclusion, eltrombopag was first disclosed as a functional METTL3-14 allosteric inhibitor in AML cells, which could be utilized for the further development of novel anti-AML therapy.

Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling.

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-ß and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.

Global Alternative Splicing Defects in Human Breast Cancer Cells.

Breast cancer is the most frequently occurred cancer type and the second cause of death in women worldwide. Alternative splicing (AS) is the process that generates more than one mRNA isoform from a single gene, and it plays a major role in expanding the human protein diversity. Aberrant AS contributes to breast cancer metastasis and resistance to chemotherapeutic interventions. Therefore, identifying cancer-specific isoforms is the prerequisite for therapeutic interventions intended to correct aberrantly expressed AS events. Here, we performed RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) in breast cancer cells, to identify global breast cancer-specific AS defects. By RT-PCR validation, we demonstrate the high accuracy of RASL-seq results. In addition, we analyzed identified AS events using the Cancer Genome Atlas (TCGA) database in a large number of non-pathological and breast tumor specimens and validated them in normal and breast cancer samples. Interestingly, aberrantly regulated AS cassette exons in cancer tissues do not encode for known functional domains but instead encode for amino acids constituting regions of intrinsically disordered protein portions characterized by high flexibility and prone to be subjected to post-translational modifications. Collectively, our results reveal novel AS errors occurring in human breast cancer, potentially affecting breast cancer-related biological processes.

SRSF9 Regulates Cassette Exon Splicing of Caspase-2 by Interacting with Its Downstream Exon.

Alternative splicing (AS) is an important posttranscriptional regulatory process. Damaged or unnecessary cells need to be removed though apoptosis to maintain physiological processes. Caspase-2 pre-mRNA produces pro-apoptotic long mRNA and anti-apoptotic short mRNA isoforms through AS. How AS of Caspase-2 is regulated remains unclear. In the present study, we identified a novel regulatory protein SRSF9 for AS of Caspase-2 cassette exon 9. Knock-down (KD) of SRSF9 increased inclusion of cassette exon and on the other hand, overexpression of SRSF9 decreased inclusion of this exon. Deletion mutagenesis demonstrated that exon 9, parts of intron 9, exon 8 and exon 10 were not required for the role of SRSF9 in Caspase-2 AS. However, deletion and substitution mutation analysis revealed that AGGAG sequence located at exon 10 provided functional target for SRSF9. In addition, RNA-pulldown mediated immunoblotting analysis showed that SRSF9 interacted with this sequence. Gene ontology analysis of RNA-seq from SRSF9 KD cells demonstrates that SRSF9 could regulate AS of a subset of apoptosis related genes. Collectively, our results reveal a basis for regulation of Caspase-2 AS.

Widespread Alternative Splicing Changes in Metastatic Breast Cancer Cells.

Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types.

Relative strength of 5' splice-site strength defines functions of SRSF2 and SRSF6 in alternative splicing of Bcl-x pre-mRNA.

Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative 5' splice-site selection produces an anti-apoptotic mRNA isoform that includes exon 2b and a pro-apoptotic Bcl-x mRNA isoform that excludes exon 2b. Here we used Bcl-x minigene and identified SRSF2 and SRSF6 as two regulatory factors of 5' splice-site selection of Bcl-x pre-mRNA. We selected binding clusters closer to 5' splice-sites from multiple potential binding sites of SRSF2 and SRSF6 to perform loss of functions analysis through site-directed mutagenesis. Our results demonstrated that these mutations did not abolish regulatory functions of SRSF2 or SRSF6, indicating that a single binding motif or a cluster was not a functional target of these proteins in Bcl-x pre-mRNA splicing. Random deletion mutagenesis did not disrupt the role of SRSF2 and SRSF6. Importantly, mutagenesis of 5' splice-site to a conserved or a weaker score demonstrated that the weaker strength of the target 5' splice-site or higher strength of the other 5' splice-site strength limited the role of SRSF2 and SRSF6 in 5' splice-site activation. [BMB Reports 2021; 54(3): 176-181].

U2AF65-Dependent SF3B1 Function in SMN Alternative Splicing.

Splicing factor 3b subunit 1 (SF3B1) is an essential protein in spliceosomes and mutated frequently in many cancers. While roles of SF3B1 in single intron splicing and roles of its cancer-linked mutant in aberrant splicing have been identified to some extent, regulatory functions of wild-type SF3B1 in alternative splicing (AS) are not well-understood yet. Here, we applied RNA sequencing (RNA-seq) to analyze genome-wide AS in SF3B1 knockdown (KD) cells and to identify a large number of skipped exons (SEs), with a considerable number of alternative 5' splice-site selection, alternative 3' splice-site selection, mutually exclusive exons (MXE), and retention of introns (RI). Among altered SEs by SF3B1 KD, survival motor neuron 2 (SMN2) pre-mRNA exon 7 splicing was a regulatory target of SF3B1. RT-PCR analysis of SMN exon 7 splicing in SF3B1 KD or overexpressed HCT116, SH-SY5Y, HEK293T, and spinal muscular atrophy (SMA) patient cells validated the results. A deletion mutation demonstrated that the U2 snRNP auxiliary factor 65 kDa (U2AF65) interaction domain of SF3B1 was required for its function in SMN exon 7 splicing. In addition, mutations to lower the score of the polypyrimidine tract (PPT) of exon 7, resulting in lower affinity for U2AF65, were not able to support SF3B1 function, suggesting the importance of U2AF65 in SF3B1 function. Furthermore, the PPT of exon 7 with higher affinity to U2AF65 than exon 8 showed significantly stronger interactions with SF3B1. Collectively, our results revealed SF3B1 function in SMN alternative splicing.

Opposite Roles of Tra2ß and SRSF9 in the v10 Exon Splicing of CD44.

CD44 is a transmembrane glycoprotein involved in cell-cell and cell-matrix interactions. Several CD44 protein isoforms are generated in human through alternative splicing regulation of nine variable exons encoding for the extracellular juxta-membrane region. While the CD44 splicing variants have been described to be involved in cancer progression and development, the regulatory mechanism(s) underlying their production remain unclear. Here, we identify Tra2ß and SRSF9 as proteins with opposite roles in regulating CD44 exon v10 splicing. While Tra2ß promotes v10 inclusion, SRSF9 inhibits its inclusion. Mechanistically, we found that both proteins are able to target v10 exon, with GAAGAAG sequence being the binding site for Tra2ß and AAGAC that for SRSF9. Collectively, our data add a novel layer of complexity to the sequential series of events involved in the regulation of CD44 splicing.

hnRNP A1 Regulates Alternative Splicing of Tau Exon 10 by Targeting 3' Splice Sites.

The ratio control of 4R-Tau/3R-Tau by alternative splicing of Tau exon 10 is important for maintaining brain functions. In this study, we show that hnRNP A1 knockdown induces inclusion of endogenous Tau exon 10, conversely, overexpression of hnRNP A1 promotes exon 10 skipping of Tau. In addition, hnRNP A1 inhibits splicing of intron 9, but not intron 10. Furthermore, hnRNP A1 directly interacts with the 3' splice site of exon 10 to regulate its functions in alternative splicing. Finally, gene ontology analysis demonstrates that hnRNP A1-induced splicing and gene expression targets a subset of genes with neuronal function.

Chemical characterization and biological activity data for a novel indirubin derivative, LDD-1819.

This article contains chemical characterization and biological activity data for a novel indirubin derivative, termed LDD-1819. The detailed synthesis procedure and associated NMR data are presented. The concentration-dependent inhibition data of two biological targets, glycogen synthase kinase-3 ß and aurora kinase A are described. The following biological data are also contained in this article: 1) the cellularization of skeletal muscle myotubes by LDD-1819 or two small molecule inhibitors of glycogen synthase kinase-3 ß and aurora kinase A (BIO and reversine) and gene expression data for the myoblast markers Pax-7 and Myf5, 2) Cell viability of hTERT human immortalized fibroblasts, colon cancer cells and breast cancer cells, and 3) Western blotting analysis of full length and cleaved caspse-7, and cleaved poly (ADP-ribose) polymerase (PARP) in hTERT fibroblasts treated with LDD-1819. A schematic diagram of the biological activities of LDD-1819 is also presented. Further interpretation and discussion of these data are provided in the associated research article 'A novel indirubin derivative that increases somatic cell plasticity and inhibits tumorigenicity' (Kim et al., 2019).

Improvement of spinal muscular atrophy via correction of the SMN2 splicing defect by Brucea javanica (L.) Merr. extract and Bruceine D.

BACKGROUND: Spinal muscular atrophy (SMA) is a rare neuromuscular disease and a leading genetic cause of infant mortality. SMA is caused primarily by the deletion of the survival motor neuron 1 (SMN1) gene, which leaves the duplicate gene SMN2 as the sole source of SMN protein. The splicing defect (exon 7 skipping) of SMN2 leads to an insufficient amount of SMN protein. Therefore, correcting this SMN2 splicing defect is considered to be a promising approach for the treatment of SMA. PURPOSE: This study aimed to identify active compounds and extracts from plant resources to rescue SMA phenotypes through the correction of SMN2 splicing. STUDY DESIGN: Of available plant resources, candidates with SMA-related traditional medicine information were selected for screening using a robust luciferase-based SMN2 splicing reporter. Primary hits were further evaluated for their ability to correct the splicing defect and resultant increase of SMN activity in SMA patient-derived fibroblasts. Confirmed hits were finally tested to determine the beneficial effects on the severe Δ7 SMA mouse. METHODS: SMN2 splicing was analyzed using a luciferase-based SMN2 splicing reporter and subsequent RT-PCR of SMN2 mRNAs. SMA phenotypes were evaluated by the survival, body weights, and righting reflex of Δ7 SMA mice. RESULTS: In a screen of 492 selected plant extracts, we found that Brucea javanica extract and its major constituent Bruceine D have SMN2 splicing-correcting activity. Their ability to correct the splicing defect and the resulting increased SMN activity were further confirmed in SMA fibroblasts. Importantly, both B. javanica and Bruceine D noticeably improved the phenotypic defects, especially muscle function, in SMA mice. Reduced expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) contributed to the correction of splicing by B. javanica. CONCLUSION: Our work revealed that B. javanica and Bruceine D correct the SMN2 splicing defect and improve the symptoms of SMA in mice. These resources will provide another possibility for development of a plant-derived SMA drug candidate.

RRM but not the Asp/Glu domain of hnRNP C1/C2 is required for splicing regulation of Ron exon 11 pre-mRNA.

The Ron proto-oncogene is a human receptor for macrophage-stimulating protein (MSP). The exclusion of exon 11 in alternative splicing generates ΔRON protein that is constitutively activated. Heterogenous ribonucleaoprotein (hnRNP) C1/C2 is one of the most abundant proteins in cells. In this manuscript, we showed that both hnRNP C1 and C2 promoted exon 11 inclusion of Ron pre-mRNA and that hnRNP C1 and hnRNP C2 functioned independently but not cooperatively. Moreover, hnRNP C1 stimulated exon 11 splicing through intron 10 activation but not through intron 11 splicing. Furthermore, we showed that, whereas the RRM domain was required for hnRNP C1 function, the Asp/Glu domain was not. In conclusion, hnRNP C1/C2 promoted exon 11 splicing independently by stimulating intron 10 splicing through RRM but not through the Asp/Glu domain. [BMB Reports 2019; 52(11): 641-646].

Activation of Cryptic 3' Splice-Sites by SRSF2 Contributes to Cassette Exon Skipping.

Here we show that the serine/arginine rich splicing factor 2 (SRSF2) promotes cryptic 3' splice-site (3'AG') usage during cassette exon exclusion in survival of motor neuron (SMN2) minigenes. Deletion of the 3'AG' (3'AG'1), its associated branch point (BP') and polypyrimidine tract (PPT') sequences directs SRSF2 to promote a second 3'AG' (3'AG'2) with less conserved associated region for intron splicing. Furthermore, deletion of both 3'AG'1 and 3'AG'2 and their associated sequences triggered usage of a third 3'AG'3 that has very weak associated sequences. Interestingly, when intron splicing was directed to the 3'AG' cryptic splice-sites, intron splicing from the canonical 3'AG splice-site was reduced along with a decrease in cassette exon inclusion. Moreover, multiple SRSF2 binding sites within the intron are responsible for 3'AG' activation. We conclude that SRSF2 facilitates exon exclusion by activating a cryptic 3'AG' and inhibiting downstream intron splicing.

A novel indirubin derivative that increases somatic cell plasticity and inhibits tumorigenicity.

Indirubin-based compounds affect diverse biological processes, such as inflammation and angiogenesis. In this study, we tested a novel indirubin derivative, LDD-1819 (2-((((2Z,3E)-5-hydroxy-5'-nitro-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)ethan-1-aminium chloride) for two major biological activities: cell plasticity and anti-cancer activity. Biological assays indicated that LDD-1819 induced somatic cell plasticity. LDD-1819 potentiated myoblast reprogramming into osteogenic cells and fibroblast reprogramming into adipogenic cells. Interestingly, in an assay of skeletal muscle dedifferentiation, LDD-1819 induced human muscle cellularization and blocked residual proliferative activity to produce a population of mononuclear refractory cells, which is also observed in the early stages of limb regeneration in urodele amphibians. In cancer cell lines, LDD-1819 treatment inhibited cell invasion and selectively induced apoptosis compared to normal cells. In an animal tumor xenograft model, LDD-1819 reduced human cancer cell metastasis in vivo at doses that did not produce toxicity. Biochemical assays showed that LDD-1819 possessed inhibitory activity against glycogen synthase kinase-3ß, which is linked to cell plasticity, and aurora kinase, which regulates carcinogenesis. These results indicate that novel indirubin derivative LDD-1819 is a dual inhibitor of glycogen synthase kinase-3ß and aurora A kinase, and has potential for development as an anti-cancer drug or as a reprogramming agent for cell-therapy based approaches to treat degenerative diseases.

A SMN2 Splicing Modifier Rescues the Disease Phenotypes in an In Vitro Human Spinal Muscular Atrophy Model.

Spinal muscular atrophy (SMA) is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. Only ∼10% of the products of SMN2, a paralogue of SMN1, are functional full-length SMN (SMN-FL) proteins, whereas SMN2 primarily produces alternatively spliced transcripts lacking exon 7. Reduced SMN protein levels in SMA patients lead to progressive degeneration of spinal motor neurons (MNs). In this study, we report an advanced platform based on an SMN2 splicing-targeting approach for SMA drug screening and validation using an SMN2 splicing reporter cell line and an in vitro human SMA model through induced pluripotent stem cell (iPSC) technology. Through drug screening using a robust cell-based luciferase assay to quantitatively measure SMN2 splicing, the small-molecule candidate compound rigosertib was identified as an SMN2 splicing modulator that led to enhanced SMN protein expression. The therapeutic potential of the candidate compound was validated in MN progenitors differentiated from SMA patient-derived iPSCs (SMA iPSC-pMNs) as an in vitro human SMA model, which recapitulated the biochemical and molecular phenotypes of SMA, including lower levels of SMN-FL transcripts and protein, enhanced cell death, and reduced neurite length. The candidate compound exerted strong splicing correction activity for SMN2 and potently alleviated the disease-related phenotypes of SMA iPSC-pMNs by modulating various cellular and molecular abnormalities. Our combined screening platform representing a pMN model of human SMA provides an efficient and reliable drug screening system and is a promising resource for drug evaluation and the exploration of drug modes of action.

Binding of SRSF4 to a novel enhancer modulates splicing of exon 6 of Fas pre-mRNA.

Alternative splicing of exon 6 in Fas pre-mRNA generates a membrane bound pro-apoptotic isoform or soluble anti-apoptotic isoform. SRSF4 is a member of Arginine-Serine rich (SR) protein family. Here we demonstrate that increased SRSF4 expression stimulates exon 6 inclusion, and that reduced SRSF4 expression promotes exon 6 exclusion. We also show that weaker but not stronger 5' splice-site strength of exon 6 abolishes the SRSF4 effects on exon 6 splicing. Furthermore, we identified a novel enhancer on exon 6, on which SRSF4 interacts functionally and physically. Our results illustrate a novel regulatory mechanism of Fas pre-mRNA splicing.

Structural and biochemical insights into inhibition of human primase by citrate.

The eukaryotic primase/polymerase complex synthesizes approximately 107 primers, one per Okazaki fragment, during the replication of mammalian chromosomes, which contain 109 base pairs. Primase catalyzes the synthesis of a short RNA segment to a single-stranded DNA template. Primase is important in DNA replication because no known replicative DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA primer. The primase subcomplex is composed of a small catalytic subunit (p49), and a large accessory subunit (p58). Priming mechanisms remain poorly understood, although large numbers of structures of archaeal and eukaryotic p49 and/or p58 as well as structures of bacterial enzymes have been determined. In this study, we determined the structure of human p49 at 2.2 Šresolution with citrate in its inactive forms. Dibasic citrate was bound at the nucleotide triphosphate (NTP) ß, γ-phosphate binding site through nine hydrogen bonds. We also measured the dissociation constant of citrate and NTPs. We further demonstrated that the p49 activity is regulated by pH and citrate, which was not previously recognized as a key regulator of DNA replication. We propose that the citrate inhibits the primase and regulates DNA replication at the replication fork.

SRSF2 directly inhibits intron splicing to suppresses cassette exon inclusion.

SRSF2, a Serine-Arginine rich (SR) protein, is a splicing activator that mediates exon inclusion and exclusion events equally well. Here we show SRSF2 directly suppresses intron splicing to suppress cassette exon inclusion in SMN premRNA. Through a serial mutagenesis, we demonstrate that a 10 nt RNA sequence surrounding the branch-point (BP), is important for SRSF2-mediated inhibition of cassette exon inclusion through directly interacting with SRSF2. We conclude that SRSF2 inhibits intron splicing to promote exon exclusion. [BMB Reports 2017; 50(8): 423-428].

Effects of PTCs on nonsense-mediated mRNA decay are dependent on PTC location.

The récepteur d'origine nantais (RON) gene is a proto-oncogene that is responsible for encoding the human macrophage-stimulating protein (MSP) 1 receptor. MSP activation induces RON-mediated cell dissociation, migration and matrix invasion. Isoforms of RON that exclude exons 5 and 6 encode the RONΔ160 protein, which promotes cell transformation in vitro and tumor metastasis in vivo. Premature termination codons (PTCs) in exons activate the nonsense-mediated mRNA decay (NMD) signaling pathway. The present study demonstrated that PTCs at various locations in the alternative exons 5 and 6 could induce NMD of the majority of the spliced, or partially spliced, isoforms. However, the isoforms that excluded exon 6 or exons 5 and 6 were markedly increased when produced from mutated minigenes with inserted PTCs. Furthermore, the unspliced isoform of intron 5 was not observed to be decreased by the presence of PTCs. Notably, these effects may be dependent on the location of the PTCs. The current study demonstrated a novel mechanism underlying the regulation of NMD in alternative splicing.