• Genomics & Informatics
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• v.14 no.4
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• pp.211-215
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• 2016

The alteration of alternative splicing patterns has an effect on the quantification of functional proteins, leading to phenotype variation. The splicing quantitative trait locus (sQTL) is one of the main genetic elements affecting splicing patterns. Here, we report the results of genome-wide sQTLs across 141 strains of Arabidopsis thaliana with publicly available next generation sequencing datasets. As a result, we found 1,694 candidate sQTLs in Arabidopsis thaliana at a false discovery rate of 0.01. Furthermore, among the candidate sQTLs, we found 25 sQTLs that overlapped with the list of previously examined trait-associated single nucleotide polymorphisms (SNPs). In summary, this sQTL analysis provides new insight into genetic elements affecting alternative splicing patterns in Arabidopsis thaliana and the mechanism of previously reported trait-associated SNPs.

• Tuberculosis and Respiratory Diseases
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• v.79 no.2
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• pp.85-90
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• 2016

Background: Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. Methods: RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. Results: RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. Conclusion: In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

• BMB Reports
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• v.40 no.1
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• pp.15-21
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• 2007

Breast cancer is the most common malignancy among women, and mutations in the BRCA1 gene produce increased susceptibility to these malignancies in certain families. In this study, the forward 1-13 exons of breast cancer associated gene BRCA1 were cloned from breast cancer cell line ZR-75-30 by RT-PCR method. Sequence analysis showed that nine BRCA1 splice forms were isolated and characterized, compared with wild-type BRCA1 gene, five splice forms of which were novel. These splice isoforms were produced from the molecular mechanism of 5' and 3' alternative splicing. All these splice forms deleting exon 11b and the locations of alternative splicing were focused on two parts:one was exons 2 and 3, and the other was exons 9 and 10. These splice forms accorded with GT-AG rule. Most these BRCA1 splice variants still kept the original reading frame. Western blot analysis indicated that some BRCA1 splice variants were expressed in ZR-75-30 cell line at the protein level. In addition, we confirmed the presence of these new transcripts of BRCA1 gene in MDA-MB-435S, K562, Hela, HLA, HIC, H9, Jurkat and human fetus samples by RT-PCR analysis. These results suggested that breast cancer associated gene BRCA1 may have unexpectedly a large number of splice variants. We hypothesized that alternative splicing of BRCA1 possibly plays a major role in the tumorigenesis of breast and/or ovarian cancer. Thus, the identification of cancer-specific splice forms will provide a novel source for the discovery of diagnostic or prognostic biomarkers and tumor antigens suitable as targets for therapeutic intervention.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.2153-2158
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• 2014

Beclin 1 is a key factor for initiation and regulation of autophagy, which is a cellular catabolic process involved in tumorigenesis. To investigate the role of alternative splicing of Beclin1 in the regulation of autophagy in leukemia cells, Beclin1 mRNA from 6 different types of cell lines and peripheral blood mononuclear cells from 2 healthy volunteers was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants were analyzed by DNA sequencing. A transcript variant of Beclin 1 gene carrying a deletion of exon 11, which encoded a C-terminal truncation of Beclin 1 isoform, was found. The alternative isoform was assessed by bioinformatics, immunoblotting and subcellular localization. The results showed that this variable transcript is generated by alternative 3' splicing, and its translational product displayed a reduced activity in induction of autophagy by starvation, indicating that the spliced isoform might function as a dominant negative modulator of autophagy. Our findings suggest that the alternative splicing of Beclin 1 might play important roles in leukemogenesis regulated by autophagy.

• BMB Reports
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• v.49 no.8
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• pp.405-413
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• 2016

Tau proteins, which stabilize the structure and regulate the dynamics of microtubules, also play important roles in axonal transport and signal transduction. Tau proteins are missorted, aggregated, and found as tau inclusions under many pathological conditions associated with neurodegenerative disorders, which are collectively known as tauopathies. In the adult human brain, tau protein can be expressed in six isoforms due to alternative splicing. The aberrant splicing of tau pre-mRNA has been consistently identified in a variety of tauopathies but is not restricted to these types of disorders as it is also present in patients with non-tau proteinopathies and RNAopathies. Tau mis-splicing results in isoform-specific impairments in normal physiological function and enhanced recruitment of excessive tau isoforms into the pathological process. A variety of factors are involved in the complex set of mechanisms underlying tau mis-splicing, but variation in the cis-element, methylation of the MAPT gene, genetic polymorphisms, the quantity and activity of spliceosomal proteins, and the patency of other RNA-binding proteins, are related to aberrant splicing. Currently, there is a lack of appropriate therapeutic strategies aimed at correcting the tau mis-splicing process in patients with neurodegenerative disorders. Thus, a more comprehensive understanding of the relationship between tau mis-splicing and neurodegenerative disorders will aid in the development of efficient therapeutic strategies for patients with a tauopathy or other, related neurodegenerative disorders.

• BMB Reports
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• v.54 no.3
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• pp.176-181
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• 2021

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
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• v.55 no.7
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• pp.348-353
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• 2022

Gastrointestinal cancer is associated with a high mortality rate. Here, we report that the splice variant of NRP/B contributes to tumorigenic activity in highly malignant gastric cancer through dissociation from the tumor repressor, HDAC5. NRP/B mRNA expression is significantly higher in the human gastric cancer tissues than in the normal tissues. Further, high levels of both the NRP/B splice variant and Lgr5, but not the full-length protein, are found in highly tumorigenic gastric tumor cells, but not in non-tumorigenic cells. The loss of NRP/B markedly inhibits cell migration and invasion, which reduces tumor formation in vivo. Importantly, the inhibition of alternative splicing increases the levels of NRP/B-1 mRNA and protein in AGS cells. The ectopic expression of full-length NRP/B exhibits tumor-suppressive activity, whereas NRP/B-2 induces the noninvasive human gastric cancer cells tumorigenesis. The splice variant NRP/B-2 which loses the capacity to interact with tumor repressors promoted oncogenic activity, suggesting that the BTB/POZ domain in the N-terminus has a crucial role in the suppression of gastric cancer. Therefore, the regulation of alternative splicing of the NRP/B gene is a potential novel target for the treatment of gastrointestinal cancer.

• Molecules and Cells
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• v.46 no.1
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• pp.48-56
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• 2023

Genomic information stored in the DNA is transcribed to the mRNA and translated to proteins. The 3' untranslated regions (3'UTRs) of the mRNA serve pivotal roles in post-transcriptional gene expression, regulating mRNA stability, translation, and localization. Similar to DNA mutations producing aberrant proteins, RNA alterations expand the transcriptome landscape and change the cellular proteome. Recent global analyses reveal that many genes express various forms of altered RNAs, including 3'UTR length variants. Alternative polyadenylation and alternative splicing are involved in diversifying 3'UTRs, which could act as a hidden layer of eukaryotic gene expression control. In this review, we summarize the functions and regulations of 3'UTRs and elaborate on the generation and functional consequences of 3'UTR diversity. Given that dynamic 3'UTR length control contributes to phenotypic complexity, dysregulated 3'UTR diversity might be relevant to disease development, including cancers. Thus, 3'UTR diversity in cancer could open exciting new research areas and provide avenues for novel cancer theragnostics.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.10
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• pp.1471-1477
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• 2017

Objective: Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase (AXL) gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form) in equine skeletal muscle to gain insight(s) into the role of each alternative transcript during exercise. Methods: We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR), and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR). Results: Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D) prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3) and immunoglobin (Ig) domain was different between two alternative isoforms. Conclusion: It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an $NF-{\kappa}B$ signaling pathway. Further study is necessary to uncover biological function(s) and significance of the alternative splicing isoforms in race horse skeletal muscle.

• Molecules and Cells
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• v.41 no.8
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• pp.733-741
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• 2018

Mutations in spliceosome components have been implicated in carcinogenesis of various types of cancer. One of the most frequently found is U2AF1 S34F missense mutation. Functional analyses of this mutation have been largely limited to hematological malignancies although the mutation is also frequently seen in other cancer types including lung adenocarcinoma (LUAD). We examined the impact of knockdown (KD) of wild type (wt) U2AF1 and ectopic expression of two splice variant S34F mutant proteins in terms of alternative splicing (AS) pattern and cell cycle progression in A549 lung cancer cells. We demonstrate that induction of distinct AS events and disruption of mitosis at distinct sub-stages result from KD and ectopic expression of the mutant proteins. Importantly, when compared with the splicing pattern seen in LUAD patients with U2AF1 S34F mutation, ectopic expression of S34F mutants but not KD was shown to result in common AS events in several genes involved in cell cycle progression. Our study thus points to an active role of U2AF1 S34F mutant protein in inducing cell cycle dysregulation and mitotic stress. In addition, alternatively spliced genes which we describe here may represent novel potential markers of lung cancer development.