Types Of Splicing Research Articles

Evidence for the role of transcription factors in the co-transcriptional regulation of intron retention

BackgroundAlternative splicing is a widespread regulatory phenomenon that enables a single gene to produce multiple transcripts. Among the different types of alternative splicing, intron retention is one of the least explored despite its high prevalence in both plants and animals. The recent discovery that the majority of splicing is co-transcriptional has led to the finding that chromatin state affects alternative splicing. Therefore, it is plausible that transcription factors can regulate splicing outcomes.ResultsWe provide evidence for the hypothesis that transcription factors are involved in the regulation of intron retention by studying regions of open chromatin in retained and excised introns. Using deep learning models designed to distinguish between regions of open chromatin in retained introns and non-retained introns, we identified motifs enriched in IR events with significant hits to known human transcription factors. Our model predicts that the majority of transcription factors that affect intron retention come from the zinc finger family. We demonstrate the validity of these predictions using ChIP-seq data for multiple zinc finger transcription factors and find strong over-representation for their peaks in intron retention events.ConclusionsThis work opens up opportunities for further studies that elucidate the mechanisms by which transcription factors affect intron retention and other forms of splicing.AvailabilitySource code available at https://github.com/fahadahaf/chromir

Transcriptomic Analysis Reveals mRNA and Alternative Splicing Events in Ovine Skeletal Muscle Satellite Cells during Proliferation and Differentiation

Skeletal muscle satellite cells (SMSCs), which are highly multifunctional muscle-derived stem cells, play an essential role in myogenesis and regeneration. Here, the transcriptional profile of SMSCs during proliferation and differentiation were constructed using the RNA-Seq method. A total of 1954 differentially expressed genes (DEGs) and 1092 differentially alternative splicing genes (DAGs) were identified including 1288 upregulated genes as well as 666 downregulated genes. GO and KEGG analyses showed that the DEGs and DAGs were enriched in the MAPK (mitogen-activated protein kinase) signaling pathway, the PI3K-Akt (phosphatidylinositol-tris-phosphate kinase 3/protein kinase B) signaling pathway, the Wnt signaling pathway, and the Ras signaling pathway. In total, 1479 alternative splice events (AS) were also identified during SMSC proliferation and differentiation. Among them, a unique AS event was the major per-mRNA splicing type, and SE was the predominant splicing pattern. Furthermore, transcription factors with AS were scanned during SMSC differentiation such as myocyte enhancer factor-2C (MEF2C) and the nuclear receptor subfamily 4 group A member 2 (NR4A2). Our results imply that MEF2C and NR4A2 can interact, and we speculate that NR4A2 and MEF2C might regulate the myogenesis of ovine SMSCs through interaction. Together, our study provides useful information on the transcriptional regulation of SMSCs during proliferation and differentiation at the transcriptional level, and provides a valuable resource for understanding the molecular mechanism of myogenesis and muscle development.

Molecular mechanism that underlies cotton response to salt and drought stress revealed by complementary transcriptomic and iTRAQ analyses

Drought and salinity severely retard cotton growth and development, resulting in a substantial reduction in global cotton production. To investigate the molecular mechanisms underlying plant response, we compared the transcriptomic and proteomic expression profiles of cotton seedlings exposed to drought or salt stress using RNA-sequencing and iTRAQ sequencing, respectively, to gain a comprehensive understanding of the common and specific responses of cotton plants to salt and drought stress. The complexity of transcriptional and post-transcriptional regulations of salt and drought stress-responsive mechanisms was manifested most obviously by the tenuous yet intertwined relationships between the number of transcripts and the profile of protein expression. Intron retention was found to be the most prevalent alternative splicing type, suggesting a significant regulatory function at the transcriptional level. Enrichment analysis revealed that the majority of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were involved in the MAPK signaling pathway, phenylpropanoid biosynthesis, plant hormone signal transduction, and flavonoid biosynthesis. In particular, a number of MAPK cascade genes, including GhMEKK36, GhMAPKK3, GhMAPKK4, GhMAPK1, and GhMAPK5 were prominently upregulated by both the drought and salt stress treatments, and the functional role of these genes was investigated by ectopic expression in transgenic Arabidopsis, which exhibited significantly enhanced stress tolerance. This study advances our understanding of the molecular mechanisms that underpin cotton responses to drought and salt stress, offering crucial insights for further elaboration of the genes and pathways that are involved in cotton drought and salt tolerance.

PABPN1 prevents the nuclear export of an unspliced RNA with a constitutive transport element and controls human gene expression via intron retention.

Intron retention is a type of alternative splicing where one or more introns remain unspliced in a polyadenylated transcript. Although many viral systems are known to translate proteins from mRNAs with retained introns, restriction mechanisms generally prevent export and translation of incompletely spliced mRNAs. Here, we provide evidence that the human nuclear poly(A)-binding protein, PABPN1, functions in such restrictions. Using a reporter construct in which nuclear export of an incompletely spliced mRNA is enhanced by a viral constitutive transport element (CTE), we show that PABPN1 depletion results in a significant increase in export and translation from the unspliced CTE-containing transcript. Unexpectedly, we find that inactivation of poly(A)-tail exosome targeting by depletion of PAXT components had no effect on export and translation of the unspliced reporter mRNA, suggesting a mechanism largely independent of nuclear RNA decay. Interestingly, a PABPN1 mutant selectively defective in stimulating poly(A) polymerase elongation strongly enhanced the expression of the unspliced, but not of intronless, reporter transcripts. Analysis of RNA-seq data also revealed that PABPN1 controls the expression of many human genes via intron retention. Notably, PABPN1-dependent intron retention events mostly affected 3'-terminal introns and were insensitive to PAXT and NEXT deficiencies. Our findings thus disclose a role for PABPN1 in restricting nuclear export of intron-retained transcripts and reinforce the interdependence between terminal intron splicing, 3' end processing, and polyadenylation.

Effect of confining stirrups and bar gap in improving bond behavior of glass fiber reinforced polymer (GFRP) bar lap splices in RC beams

Fiber-reinforced polymer (FRP) bars are becoming a popular replacement for steel rebars that are vulnerable to corrosion, especially under harsh corrosive environments. Despite several advantages for using FRP rebars, the bond between FRP bars and surrounding concrete has been a major concern that affects the behavior of FRP-reinforced structural members. The experimental program of this study comprises testing eight full-scale glass FRP (GFRP) reinforced beams under two-point loads. There were two control beams and six beams having rebar lap splices. The beams were reinforced using 2ϕ12 GFRP bars. The first control had continuous GFRP bars, and the 2nd control had GFRP lap splice conforming to the relevant ACI code. The lap splice was 65 times the bar diameter in six beams having lap splice. The contact lap and non-contact laps were used along with the variable spacing of shear stirrups in the lap zone (50, 100 mm, ∞, i.e., no shear stirrups). The lap splice, designed conforming to the ACI code, was found to be very conservative. The reduction in shear stirrups’ spacing in the lap zone and the gap between lapped bars was found to enhance the bond performance of GFRP bars by up to 31 %. The authors’ previous models for predicting development length and bond are found to show good predictions for the experimental results of the present study as well. Analytical models are developed for predicting with sufficient precision the peak load for GFRP reinforced beams with the two types of lap splices (non-contact and contact).

Numerical modeling on shear performance of braced cold-formed steel composite walls

In this paper, the specific influence of diagonal braces on the hysteretic behaviors, including lateral stiffness, yield load, and load-bearing capacity, of braced composite cold-formed steel (CFS) walls have been investigated through the combination of previous experiment results and numerical analysis. A detailed modeling method using connector function in ABAQUS for simulating single screw connection has been introduced. Furthermore, the finite element models based on experimental specimens have been constructed, an excellent match of finite element analysis and experimental results are yielded to prepare for parametric analysis; five significant parameters, vertical load, panel splice type, screw spacing, section thickness of end studs, and diagonal braces, were considered in parametric analysis to in-depth investigate the hysteretic behavior of walls. Finally, some valued conclusions expected to be benefited engineering applications have been summarized.

Landscape of exitrons in gastric cancer.

SummaryBackgroundExitron is a new type of non-canonical alternative splicing. Accumulating evidence implies exitron may have pathological function and contribute to another source of anti-tumor immunogenicity in various cancers. Its role in gastric cancer remains poorly understood. Large-scale, multi-omics analysis could comprehensively characterize the landscape of exitrons in gastric cancer, reveal undiscovered mechanism and hopefully identify molecular biomarkers for predicting immunotherapy response.MethodsWe collected datasets from five studies for analysis. RNA sequencing was used for exitron identification. Somatic mutations were detected by whole exome sequencing. Neopeptides were confirmed by proteome mass spectrometry.Findings42174 gastric cancer-specific exitrons (GCSEs) were identified in 632 patients. GCSEs were clinically relevant to gender, age, Lauren type, tumor stage and prognosis. Tissue specificity test and pathogenic exitron prediction revealed their unique functional impact. GCSEs were mutually exclusive with mutations and demonstrated both unique and complementary function against TP53 mutation in gastric cancer. We further established splicing regulatory network to reveal upstream regulation of exitron splicing. We also evaluated the immunogenicity and diagnostic potential of GCSEs. Evidence of GCSEs-derived neopeptide expression was validated by whole proteome mass spectrometry. PD-1 and Siglecs were significantly increased in high neoantigen load patients. But exitron-related biomarkers failed to predict immunotherapy response, possibly due to small sample size and insufficient sequencing depth.InterpretationThe present study provided a comprehensive multidimensional landscape of gastric cancer exitrons and underscores insights into underexplored mechanism in gastric cancer pathology.FundingThe Guangdong Provincial Key Laboratory of Precision Medicine for Gastroinstestinal Cancer (2020B121201004), the Guangdong Provincial Major Talents Project (No. 2019JC05Y361) and National Natural Science Foundation of China (grant number:82172960 and 81872013).

ScanExitronLR: characterization and quantification of exitron splicing events in long-read RNA-seq data.

Exitron splicing is a type of alternative splicing where coding sequences are spliced out. Recently, exitron splicing has been shown to increase proteome plasticity and play a role in cancer. Long-read RNA-seq is well suited for quantification and discovery of alternative splicing events; however, there are currently no tools available for the detection and annotation of exitrons in long-read RNA-seq data. Here, we present ScanExitronLR, an application for the characterization and quantification of exitron splicing events in long-reads. From a BAM alignment file, reference genome and reference gene annotation, ScanExitronLR outputs exitron events at the individual transcript level. Outputs of ScanExitronLR can be used in downstream analyses of differential exitron splicing. In addition, ScanExitronLR optionally reports exitron annotations such as truncation or frameshift type, nonsense-mediated decay status and Pfam domain interruptions. We demonstrate that ScanExitronLR performs better on noisy long-reads than currently published exitron detection algorithms designed for short-read data. ScanExitronLR is freely available at https://github.com/ylab-hi/ScanExitronLR and distributed as a pip package on the Python Package Index. Supplementary data are available at Bioinformatics online.

Splicing of branchpoint-distant exons is promoted by Cactin, Tls1 and the ubiquitin-fold-activated Sde2.

Intron diversity facilitates regulated gene expression and alternative splicing. Spliceosomes excise introns after recognizing their splicing signals: the 5′-splice site (5′ss), branchpoint (BP) and 3′-splice site (3′ss). The latter two signals are recognized by U2 small nuclear ribonucleoprotein (snRNP) and its accessory factors (U2AFs), but longer spacings between them result in weaker splicing. Here, we show that excision of introns with a BP-distant 3′ss (e.g. rap1 intron 2) requires the ubiquitin-fold-activated splicing regulator Sde2 in Schizosaccharomyces pombe. By monitoring splicing-specific ura4 reporters in a collection of S. pombe mutants, Cay1 and Tls1 were identified as additional regulators of this process. The role of Sde2, Cay1 and Tls1 was further confirmed by increasing BP–3′ss spacings in a canonical tho5 intron. We also examined BP-distant exons spliced independently of these factors and observed that RNA secondary structures possibly bridged the gap between the two signals. These proteins may guide the 3′ss towards the spliceosome's catalytic centre by folding the RNA between the BP and 3′ss. Orthologues of Sde2, Cay1 and Tls1, although missing in the intron-poor Saccharomyces cerevisiae, are present in intron-rich eukaryotes, including humans. This type of intron-specific pre-mRNA splicing appears to have evolved for regulated gene expression and alternative splicing of key heterochromatin factors.

The alternative 3' splice site of GPNMB may promote neuronal survival after neonatal hypoxic-ischemic encephalopathy injury.

This study aimed to decipher the effect of glycoprotein nonmetastatic melanoma protein B(GPNMB) on neonatal hypoxic-ischemic encephalopathy (NHIE) and its potential molecular mechanism. The hypoxic-ischemic (HI) model was established in 7-day-old rats, and then, Zea-Longa scores and Nissl staining were performed to measure brain damage post-HI. In addition, gene sequencing was used to detect the differential expression genes (DEGs), and then, Gene Ontologyand Kyoto Encyclopedia of Genes and Genomes databases were used to determine the function of DEGs. Furthermore, an oxygen-glucose deprivation (OGD) model was developed in SY5Y cells and human fetal neurons, and then, the level of GPNMB was verified by quantitative real-time polymerase chain reaction. In addition, methyl thiazolyl tetrazolium and cell counting kit-8 assays were applied after GPNMB interference. Finally, the alternative splicing of GPNMB expression was analyzed using Splice Grapher software. The results indicated that HI induced marked neurological impairment and neuron injury in rats. Also, GPNMB was the most obviously upregulated gene in DEGs. Additionally, GPNMB was upregulated significantly in SY5Y and fetal neurons after OGD, and GPNMB-si promoted an increase in cell viability and number. Moreover, we found that the GPNMB alternative splicing type was the Alternative 3' splice site, with the alternative splicing site in 143382985:143404102. Herein, GPNMB promotes a crucial regulatory mechanism with alternative splicing for neuronal survival after NHIE.

A comprehensive study of alternative splicing in malignant pleural mesothelioma identifies potential therapeutic targets in a new cluster with poor survival.

BackgroundMalignant pleural mesothelioma (MPM) is one of the most aggressive tumors with few effective treatments worldwide. It has been suggested that alternative splicing at the transcriptome level plays an indispensable role in MPM.MethodsWe analyzed the splicing profile of 84 MPM patients from the TCGA cohort by using seven typical splicing types. We classified MPM patients based on their splicing status and conducted a comprehensive analysis of the correlation between the splicing classification and clinical characteristics, genetic variation, pathway changes, immune heterogeneity, and potential therapeutic targets.ResultsThe expression of the alternative splicing regulator SRPK1 is significantly higher in MPM tissues than in normal tissues, and correlates with poor survival. SRPK1 deficiency promotes MPM cell apoptosis and inhibits cell migration in vitro. We divided the MPM patients into four clusters based on their splicing profile and identified two clusters associated with the shortest (cluster 3) and longest (cluster 4) survival time. We present the different gene signatures of each cluster that are related to survival and splicing. Comprehensive analysis of data from the GDSC and TCGA databases revealed that cluster 3 MPM patients could respond well to the small‐molecule inhibitor CHIR‐99021, a small‐molecule inhibitor of GSK‐3.ConclusionWe performed unsupervised clustering of alternative splicing data from 84 MPM patients from the TCGA database and identified a cluster associated with the worst prognosis that was sensitive to a GSK‐3 inhibitor.

Genome-wide alternative splicing profile in the posterior kidney of brown trout (Salmo trutta) during proliferative kidney disease

BackgroundThe cnidarian myxozoan parasite Tetracapsuloides bryosalmonae causes chronic proliferative kidney disease (PKD) in salmonids. This parasite is a serious threat to wild and cultured salmonids. T. bryosalmonae undergoes intra-luminal sporogonic development in the kidney of brown trout (Salmo trutta) and the viable spores are released via urine. We investigated the alternative splicing pattern in the posterior kidney of brown trout during PKD.ResultsRNA-seq data were generated from the posterior kidney of brown trout collected at 12 weeks post-exposure to T. bryosalmonae. Subsequently, this data was mapped to the brown trout genome. About 153 significant differently expressed alternatively spliced (DEAS) genes, (delta PSI = 5%, FDR P-value < 0.05) were identified from 19,722 alternatively spliced events. Among the DEAS genes, the least and most abundant alternative splicing types were alternative 5′ splice site (5.23%) and exon skipping (70.59%), respectively. The DEAS genes were significantly enriched for sodium-potassium transporter activity and ion homeostasis (ahcyl1, atp1a3a, atp1a1a.1, and atp1a1a.5). The protein-protein interaction network analysis enriched two local network clusters namely cation transporting ATPase C-terminus and Sodium/potassium ATPase beta chain cluster, and mixed inclusion of Ion homeostasis and EF-hand domain cluster. Furthermore, the human disease-related salmonella infection pathway was significantly enriched in the protein-protein interaction network.ConclusionThis study provides the first baseline information about alternative splicing in brown trout during PKD. The generated data lay a foundation for further functional molecular studies in PKD - brown trout infection model. The information generated from the present study can help to develop therapeutic strategies for PKD in the future.

In situ hybridization assay for circular RNA visualization based on padlock probe and rolling circle amplification

Circular RNAs (circRNAs) are a class of noncoding RNAs generated by a specific type of RNA alternative splicing called backsplicing through various mechanisms. Recently, thousands of circRNAs have been identified by high-throughput RNA sequencing technologies and bioinformatics analysis. However, the functions of the majority have not been fully elucidated yet. Different tools, such as in situ hybridization, can help visualize the spatial temporal distribution of circRNA molecules, thus assisting the understanding of their biological and physiological functions. Here, we present a simple and straightforward method based on padlock probe hybridization and rolling circle amplification (RCA) for in situ detection of circRNAs. We compared our method with the commercially available BaseScope assay for the detection of Cdr1as in the mouse brain tissue. The result showed that the two methods have achieved comparable detection efficiency, thus demonstrating our padlock probe assay as an alternative yet simple circRNA in situ detection method for the research community.

The Prognostic Significance of Spliceosomal Proteins for Patients with Glioblastoma

Glioblastoma (GBM) is considered one of the most aggressive human cancers. Earlier, our group have demonstrated that alternative RNA splicing plays an important role in the regulation of the GBM phenotype. To continue this study, we analyzed the type of RNA splicing and the expression levels of the spliceosomal genes in a large number of tumor tissue samples and patient-derived GBM sphere lines. We demonstrated that the expression level of splicing factors allows dividing GBM patients into groups with different survival prognosis and also reflects the phenotype of the tumor. In addition, we identified the alternative splicing events that may regulate the GBM phenotype. Finally, we for the first time compared the expression profiles of the spliceosomal genes in different regions of the same tumor and identified splicing factors whose expression most significantly correlates with GBM patients’ survival. Aforementioned data emphasize the important role of pre-mRNA splicing in GBM progression.

Accepting multiple splicing systems

We introduce an accepting splicing system based on a type of splicing, multiple splicing, which has never considered so far for accepting systems. This type of splicing differs from the usual operation in that several (not necessarily distinct) rules can be applied simultaneously to the same string. We first consider accepting multiple splicing systems where the number of splicing sites is a predefined constant. We prove that this model is computationally complete, if the constant is 2, by simulating a 2-tag system. Moreover, we show that the simulation is time-complexity preserving, and discuss also the descriptional complexity of the accepting splicing system given by our construction. We then consider the accepting multiple splicing systems where the number of sites has either an upper bound or a lower bound. The computational power of these systems is also investigated. We finally discuss some open problems.

Chemerin effect on transcriptome of the porcine endometrium during implantation determined by RNA-sequencing†.

It is well known that the body's metabolism and reproduction are closely related. Chemerin (CHEM) is one of many biologically active proteins secreted by the adipose tissue involved in the regulation of the energy homeostasis of the organism. In the present study, RNA-sequencing was performed to investigate the differentially expressed genes (DEGs), long non-coding RNAs (lncRNAs), and alternatively spliced (AS) transcripts in the cultured porcine endometrium exposed to chemerin for 24hours (CHEM; 400ng/mL) collected during the implantation period (15-16days of gestation). High-throughput sequencing of transcriptomes was performed on the Illumina NovaSeq 6000 platform (Illumina, USA). In the current study, among all 130 DEGs, 58 were upregulated and 72 were downregulated in the CHEM-treated group. DEGs were assigned to 73 functional annotations. Twelve identified lncRNAs indicated a difference in the expression profile after CHEM administration. Additionally, we detected 386 differentially AS events encompassed 274 protein-coding genes and 2 lncRNAs. All AS events were divided into five alternative splicing types: alternative 3' splice site (A3SS), 5' splice site (A5SS), mutually exclusive exons (MXE), retention intron (RI), and skipping exon (SE). Within all AS events, we identified 42 A3SS, 43 A5SS, 53 MXE, 9 RI, and 239 SE. In summary, CHEM affects the transcriptomic profile of the porcine endometrium, controlling the expression of numerous genes, including those involved in the cell migration and adhesion, angiogenesis, inflammation, and steroidogenesis. It can be assumed that CHEM may be an important factor for a proper course of gestation and embryo development.

Photo forgery detection using RGB color model permutations

ABSTRACT A detection of fake photos is a serious concern since general users can easily create with mobile apps and computer software. In this paper, a novel method that can detect fake photos accurately is proposed. RGB color model permutations are considered and non-decimated shift-invariant wavelet transform is applied. The proposed method extracts features using the Markov process and texture operator based on co-occurrence in both the spatial and frequency domains. The feature vector dimension is reduced by using an infinite feature selection algorithm and feature selection provides quality features to improve a detection accuracy and reduce a classification model training time. The experimental analysis is performed on four photo forgery datasets and demonstrated the accuracy of the proposed scheme is outstanding for both types of forgery, splicing and copy-move when compared with previous forgery detection schemes.

The Identification of Prognostic and Metastatic Alternative Splicing in Skin Cutaneous Melanoma.

Skin cutaneous melanoma (SKCM) is a type of highly invasive cancer originated from melanocytes. It is reported that aberrant alternative splicing (AS) plays an important role in the neoplasia and metastasis of many types of cancer. Therefore, we investigated whether ASEs of pre-RNA have such an influence on the prognosis of SKCM and the related mechanism of ASEs in SKCM. The RNA-seq data and ASEs data for SKCM patients were obtained from the TCGA and TCGASpliceSeq database. The univariate Cox regression revealed 1265 overall survival-related splicing events (OS-SEs). Screened by Lasso regression, 4 OS-SEs were identified and used to construct an effective prediction model (AUC: .904), whose risk score was proved to be an independent prognostic factor. Furthermore, Kruskal–Wallis test and Mann–Whitney–Wilcoxon test showed that an aberrant splicing type of aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2) regulated by CDC-like kinase 1 (CLK1) was associated with the metastasis and stage of SKCM. Besides, the overlapped signal pathway for AIMP2 was galactose metabolism identified by the co-expression analysis. External database validation also confirmed that AIMP2, CLK1, and the galactose metabolism were associated with the metastasis and stage of SKCM patients. ChIP-seq and ATAC-seq methods further confirmed the transcription regulation of CLK1, AIMP2, and other key genes, whose cellular expression was detected by Single Cell Sequencing. In conclusion, we proposed that CLK1-regulated AIMP2-78704-ES might play a critical role in the tumorigenesis and metastasis of SKCM via galactose metabolism. Besides, we established an effective model with MTMR14-63114-ES, URI1-48867-ES, BATF2-16724-AP, and MED22-88025-AP to predict the metastasis and prognosis of SKCM patients.

Aberrant splicing events caused by insertion of genes of interest into expression vectors.

Background: Expression of genes of interest from plasmids or lentiviral vectors is one of the most common tools in molecular and gene therapy. Aberrant splicing between the inserted gene of interest and downstream vector sequence has not been systematically analyzed.Methods: Formation of aberrant fusion transcripts and proteins was detected by RT-PCR, sequencing, Western blotting and mass spectrometry. Bioinformatic analysis was performed to identify all human and mouse genes prone to vector-dependent aberrant splicing. Selected genes were experimentally validated.Results: When we expressed human FACI in cultured cells, an aberrant splicing event was found to occur between FACI transcript and downstream plasmid sequence through one exon-exon junction in FACI that accidentally contributes a splice donor site. To explore whether this could be a general phenomenon, we searched the whole human and mouse genomes for protein-coding genes that harbor an exon-exon junction resembling a splice donor site. Almost all genes prone to this type of aberrant splicing were identified. A total of 17 genes among the hits were randomly selected for experimental validation. RT-PCR and sequencing results verified that 13 genes were aberrantly spliced on the identified exon-exon junctions. In addition, all 17 genes were aberrantly spliced on their V5 tag sequence. Aberrant fusion protein expression from all 17 genes was validated by immunoblotting. Aberrant splicing was prevented by recoding the V5 tag or the splice sites.Conclusions: Our study revealed an unexpectedly high frequency of vector-dependent aberrant splicing events. Aberrant formation of the resulting fusion proteins could undermine the accuracy of gain-of-function studies and might cause potential side effects when the therapeutic gene is expressed in vivo. Our work has implications in improving vector construction and epitope tagging for gene expression and therapy.

Temporal Modulation of Differential Alternative Splicing in HaCaT Human Keratinocyte Cell Line Chronically Exposed to Arsenic for up to 28 Wk.

Background:Chronic arsenic exposure via drinking water is associated with an increased risk of developing cancer and noncancer chronic diseases. Pre-mRNAs are often subject to alternative splicing, generating mRNA isoforms encoding functionally distinct protein isoforms. The resulting imbalance in isoform species can result in pathogenic changes in critical signaling pathways. Alternative splicing as a mechanism of arsenic-induced toxicity and carcinogenicity is understudied.Objective:This study aimed to accurately profile differential alternative splicing events in human keratinocytes induced by chronic arsenic exposure that might play a role in carcinogenesis.Methods:Independent quadruplicate cultures of immortalized human keratinocytes (HaCaT) were maintained continuously for 28 wk with 0 or sodium arsenite. RNA-sequencing (RNA-Seq) was performed with poly(A) RNA isolated from cells harvested at 7, 19, and 28 wk with subsequent replicate multivariate analysis of transcript splicing (rMATS) analysis to detect and quantify differential alternative splicing events. Reverse transcriptase-polymerase chain reaction (RT-PCR) for selected alternative splicing events was performed to validate RNA-Seq predictions. Functional enrichment was performed by gene ontology (GO) analysis of the differential alternative splicing event data set at each time point.Results:At least 600 differential alternative splicing events were detected at each time point tested, comprising all the five main types of alternative splicing and occurring in both open reading frames (ORFs) and untranslated regions (UTRs). Based on functional relevance ELK4, SHC1, and XRRA1 were selected for validation of predicted alternative splicing events at 7 wk by RT-PCR. Densitometric analysis of RT-PCR data corroborated the rMATS predicted alternative splicing for all three events. Protein expression validation of the selected alternative splicing events was challenging given that very few isoform-specific antibodies are available. GO analysis demonstrated that the enriched terms in differential alternatively spliced mRNAs changed dynamically with the time of exposure. Notably, RNA metabolism and splicing regulation pathways were enriched at the 7-wk time point, when the greatest number of differentially alternatively spliced mRNAs are detected. Our preliminary proteomic analysis demonstrated that the expression of the canonical isoforms of the splice regulators DDX42, RMB25, and SRRM2 were induced upon chronic arsenic exposure, corroborating the splicing predictions.Discussion:These results using cultures of HaCaT cells suggest that arsenic exposure disrupted an alternative splice factor network and induced time-dependent genome-wide differential alternative splicing that likely contributed to the changing proteomic landscape in arsenic-induced carcinogenesis. However, significant challenges remain in corroborating alternative splicing data at the proteomic level. https://doi.org/10.1289/EHP9676