Types Of Alternative Splicing Research Articles

Comparative Genome-Wide Alternative Splicing Analysis between Preadipocytes and Adipocytes.

Alternative splicing is a ubiquitous regulatory mechanism in gene expression that allows a single gene to generate multiple messenger RNAs (mRNAs). Adipocyte development is regulated by many processes, and recent studies have found that splicing factors also play an important role in adipogenic development. In the present study, we further investigated the differences in selective shearing during different periods of adipocyte differentiation. We identified five alternative splicing types including skipped exon, mutually exclusive exon, Alternative 5' splice site, Alternative 3' splice site, and Retained intron, with skipped exons being the most abundant type of selective shearing. In total, 641 differentially expressed selective shearing genes were obtained, enriched in 279 pathways, from which we selected and verified the accuracy of the sequencing results. Overall, RNA-seq revealed changes in the splicing and expression levels of these new candidate genes between precursor adipocytes and adipocytes, suggesting that they may be involved in adipocyte generation and differentiation.

Cellular senescence induced by down-regulation of PTBP1 correlates with exon skipping of mitochondrial related gene NDUFV3

Abstract As the most prevalent type of alternative splicing in animal cells, exon skipping plays an important role in expanding the diversity of transcriptome and proteome, thereby participating the regulation of diverse physiological and pathological processes such as development, aging and cancer. Cellular senescence serving as an anti-cancer mechanism could also contribute to individual aging. Although the dynamic changes of exon skipping during cellular senescence were revealed, its biological consequence and upstream regulator remain poorly understood. Here, by using human foreskin fibroblasts (HFF) replicative senescence as a model, we discovered that splicing factor PTBP1 was an important contributor for global exon skipping events during senescence. Down-regulated expression of PTBP1 induced senescence-associated phenotypes and related mitochondrial functional changes. Mechanistically, PTBP1 binds to the third exon of mitochondrial complex I subunit coding gene NDUFV3 and protects the exon from skipping. We further confirmed that exon skipping of NDUFV3 correlates with and partially contributes to cellular senescence and related mitochondrial functional changes upon PTBP1 knockdown. Together, we revealed for the first time that mitochondrial related gene NDUFV3 is a new downstream target for PTBP1-regulated exon skipping to mediate cellular senescence and mitochondrial functional changes.

Intron Retention: What that means for the biologist

Introns are non-coding segments within eukaryotic genes that are transcribed along with exons. They are subsequently removed during splicing, and their presence allows for generating diverse mRNA isoforms through the process called alternative splicing. The functional significance of introns going beyond mere "junk DNA" is questionable until recently; scientists identified that these pieces of junk DNA could hang around with the functional mRNA escaping splicing. Intron retention is a type of alternative splicing, a process that occurs during the maturation of messenger RNA (mRNA) in eukaryotic cells. In eukaryotes, genes typically consist of exons (coding regions) and introns (non-coding regions). After transcription, the precursor mRNA (pre-mRNA) undergoes splicing, where introns are removed, and exons are joined together to form the mature mRNA. Intron retention, however, deviates from this typical splicing process. Instead of being entirely removed from the pre-mRNA, the mature mRNA may retain one or more introns. This results in the inclusion of intronic sequences within the final mRNA transcript. The retained introns may contain premature stop codons or regulatory elements that can influence gene expression and protein function. Intron retention is a form of alternative splicing because it generates multiple mRNA isoforms from a single gene. This diversity in mRNA transcripts can produce different protein isoforms with distinct functions or regulatory properties. The regulation of intron retention is complex and involves various cellular factors, including splicing machinery components and RNA-binding proteins. Research on intron retention has revealed its importance in various biological processes, including development, cell growth and differentiation, and diseases like cancer. Aberrant intron retention has been associated with certain genetic disorders and cancers. Understanding the mechanisms and functional consequences of intron retention is crucial for unraveling the complexity of gene regulation and its impact on cellular processes. With an increasing output of data from high throughput assays line RNA Seq, scRNA-Seq, etc., it's high time data scientists look into these signatures in the context of disease pathology. This may well be the tip of the iceberg.

Pan-Cancer Profiling of Intron Retention and Its Clinical Significance in Diagnosis and Prognosis

Alternative splicing can produce transcripts that affect cancer development and thus shows potential for cancer diagnosis and treatment. However, intron retention (IR), a type of alternative splicing, has been studied less in cancer biology research. Here, we generated a pan-cancer IR landscape for more than 10,000 samples across 33 cancer types from The Cancer Genome Atlas (TCGA). We characterized differentially retained introns between tumor and normal samples and identified retained introns associated with survival. We discovered 988 differentially retained introns in 14 cancers, some of which demonstrated diagnostic potential in multiple cancer types. We also inferred a large number of prognosis-related introns in 33 cancer types, and the associated genes included well-known cancer hallmarks such as angiogenesis, metastasis, and DNA mutations. Notably, we discovered a novel intron retention inside the 5′UTR of STN1 that is associated with the survival of lung cancer patients. The retained intron reduces translation efficiency by producing upstream open reading frames (uORFs) and thereby inhibits colony formation and cell migration of lung cancer cells. Besides, the IR-based prognostic model achieved good stratification in certain cancers, as illustrated in acute myeloid leukemia. Taken together, we performed a comprehensive IR survey at a pan-cancer level, and the results implied that IR has the potential to be diagnostic and prognostic cancer biomarkers, as well as new drug targets.

RNA Splicing Factor Mutations Drive Aberrant Canonical and Cryptic Circular RNA Biogenesis in Leukemia

RNA splicing factors (SFs) are recurrently mutated in liquid cancers. Mutations on SFs such as SF3B1, SRSF2, U2AF1, ZRSR2, and the U1 snRNA promote leukemogenesis by driving aberrant pre-mRNA splicing in critical pathways such as DNA damage response (DDR) and cell cycle regulation (CCR). Circular RNAs (circRNAs) are covalently closed RNAs that form via backsplicing ( Fig A). As a novel RNA species, circRNAs are now known to play diverse functional roles in cancer biology. Because backsplicing is a type of alternative splicing, we hypothesized that SF mutations also influence circRNA biogenesis in leukemia. To date, such causal links have not been clearly established. We first performed circRNA-seq on a panel of lymphoid and myeloid cell lines modeling the most common SF mutations ( SF3B1 K700E, SRSF2 P95H, U2AF1 S34F/Q157R, ZRSR2 KO, and U1 snRNA 3A>C). We observed that SF mutations tend to globally upregulate circRNAs with each mutant SF driving its own mutually exclusive set of circRNAs that we have termed SF mutation-associated circRNAs (SF-circRNAs). Each set of SF-circRNAs differs from each other along several features such as average lengths and selection of backsplicing exons. Additionally, while SF-circRNAs were generally mutation-specific, the majority of SF-circRNAs arose from commonly shared genes enriched for the DDR and CCR pathways. These results support a model in which SF mutations drive divergent splicing events convergent on a few pathways as a common mechanism in leukemias. Because SF3B1 is the most recurrently mutated SF in cancers, we concentrated on studying SF3B1 mutation-associated circRNAs (SF3B1-circRNAs). During linear RNA splicing, hotspot mutations on SF3B1 typically promote selection of cryptic 3' splice site (SS). Cryptic 3'SS are weak, inauthentic 3'SS proximal to the canonical site that can be activated for splicing by mutant SF3B1. Reasoning that cryptic 3'SS selection also occurs during backsplicing, we developed a pipeline called CrypticCIRC to call for these events. Through CrypticCIRC, we discovered the existence of cryptic circRNAs that account for nearly 1 in 10 of all SF3B1-circRNAs. Approximately 25% of cryptic circRNAs emanated from de novo 3'SS previously unannotated in the genome. Furthermore, we found that cryptic circRNAs were especially enriched on genes involved in the DDR and CCR pathways, furthering the notion that these pathways are preferentially targeted by SF mutations. Finally, by comparing with linear RNA splicing data, we also observed that some cryptic 3'SS may undergo either aberrant linear splicing or aberrant backsplicing but not both, indicating mutual exclusivity in cryptic splice site selection between linear and backsplicing. Finally, to mechanistically dissect SF-circRNA biology, we focused our study on one candidate circRNA, circZEB1. The parental ZEB1 gene, a master transcription and DDR factor, undergoes both aberrant canonical and cryptic backsplicing at exon 2 with exon 4 to give rise to two circZEB1 isoforms ( Fig B). In our full SF mutant panel, we found that the canonical and cryptic isoforms were consistently upregulated only in SF3B1 mutant cell lines (Nalm-6, K-562, HEK293T, HG-3, and MEC-1); We further confirmed circZeb1 upregulation in mouse models (CD19-Cre Sf3b1 K700E) where the canonical isoform is evolutionarily conserved. These results confirm circZEB1 as a true bona fide SF3B1-circRNA. Focusing on the function of this circRNA, we found that CasRx-mediated knockdown of canonical circZEB1 resulted in increased ZEB1 protein, but not mRNA, expression while overexpression of circZEB1 decreased ZEB1 protein levels. These results show that circZEB1 translationally regulates its parental gene. Finally, consistent with ZEB1's role in genome stability, we also observed a global reduction in γH2AX levels concomitant with circZEB1 ablation, highlighting DDR as a common pathway for SF-circRNAs ( Fig B). Altogether, we demonstrate that SF mutations drive widespread aberrant circRNA biogenesis mainly on DDR and CCR genes. We provide evidence for the existence of cryptic circRNAs as a novel class of circRNAs. We show that circZEB1 promotes DNA damage via regulating ZEB1 translation. Our results collectively highlight circRNAs as an additional regulatory modality alongside linear RNAs through which SF mutations exert function, supporting a unifying mechanism for the pan-cancer impact of SF mutations in leukemia.

Regulation of Flowering Time and Other Developmental Plasticities by 3' Splicing Factor-Mediated Alternative Splicing in Arabidopsis thaliana.

Plants, as sessile organisms, show a high degree of plasticity in their growth and development and have various strategies to cope with these alterations under continuously changing environments and unfavorable stress conditions. In particular, the floral transition from the vegetative and reproductive phases in the shoot apical meristem (SAM) is one of the most important developmental changes in plants. In addition, meristem regions, such as the SAM and root apical meristem (RAM), which continually generate new lateral organs throughout the plant life cycle, are important sites for developmental plasticity. Recent findings have shown that the prevailing type of alternative splicing (AS) in plants is intron retention (IR) unlike in animals; thus, AS is an important regulatory mechanism conferring plasticity for plant growth and development under various environmental conditions. Although eukaryotes exhibit some similarities in the composition and dynamics of their splicing machinery, plants have differences in the 3' splicing characteristics governing AS. Here, we summarize recent findings on the roles of 3' splicing factors and their interacting partners in regulating the flowering time and other developmental plasticities in Arabidopsis thaliana.

N6‐methyladenosine modification changes during the recovery processes for Paulownia witches' broom disease under the methyl methanesulfonate treatment

AbstractPhytoplasmas induce diseases in more than 1000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma has not yet been clarified. N6‐methyladenosine (m6A) is the most common internal modification of the eukaryotic Messenger RNA (mRNA). As one of the species susceptible to phytoplasma infection, the pathogenesis and mechanism of Paulownia has been extensively studied by scholars, but the m6A transcriptome map of Paulownia fortunei (P. fortunei) has not been reported. Therefore, this study aimed to explore the effect of phytoplasma infection on m6A modification of P. fortunei and obtained the whole transcriptome m6A map in P. fortunei by m6A‐seq. The m6A‐seq results of Paulownia witches' broom (PaWB) disease and healthy samples indicate that PaWB infection increased the degree of m6A modification of P. fortunei. The correlation analysis between the RNA‐seq and m6A‐seq data detected that a total of 315 differentially methylated genes were predicted to be significantly differentially expressed at the transcriptome level. Moreover, the functions of PaWB‐related genes were predicted by functional enrichment analysis, and two genes related to maintenance of the basic mechanism of stem cells in shoot apical meristem were discovered. One of the genes encodes the receptor protein kinase CLV2 (Paulownia_LG2G000076), and the other gene encodes the homeobox transcription factor STM (Paulownia_LG15G000976). In addition, genes F‐box (Paulownia_LG17G000760) and MSH5 (Paulownia_LG8G001160) had exon skipping and mutually exclusive exon types of alternative splicing in PaWB‐infected seedling treated with methyl methanesulfonate, and m6A modification was found in m6A‐seq results. Moreover, Reverse Transcription–Polymerase Chain Reaction (RT‐PCR) verified that the alternative splicing of these two genes was associated with m6A modification. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA m6A modification in the process of PaWB. In future studies, we plan to verify genes directly related to PaWB and methylation‐related enzymes in Paulownia to elucidate the pathogenic mechanism of PaWB caused by phytoplasma invasion.

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

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.

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.

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.

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.

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.

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

Whole genome, exon mutation and transcriptomic profiling of acute myeloid leukemia: A case report.

The present study aimed to observe previously unidentified gene mutation and expression profiles associated with acute myeloid leukemia (AML) at the individual level, based on the blood samples of a father-son pair. Genomic DNA and RNA samples from blood serum were collected. Whole-genome sequencing (WGS) and whole-exome sequencing (WES), as well as mRNA sequencing of the son, were performed. For the father's sample, a total of 3,897,164 single nucleotide polymorphisms (SNPs) and 780,834 insertion and deletions (indels) were identified. Regarding amino acid translation, there were 11,316 non-synonymous, 12 stop-loss, 12,033 synonymous, 92 stop-gain SNPs, 63 frameshift insertions, 73 frameshift deletions, 242 non-frameshift insertions, 248 non-frameshift deletions, four stop-gains and two stop-loss for indel variants. Among the AML-related genes that had been previously identified, 14 genes were found in the father's exon region. For WES of the son's DNA, 96,639 SNPs were identified, including 10,504 non-synonymous SNPs. Seven mutant genes were found in sons' exon region compared with 121 AML-related genes. Based on the transcriptomic sequencing, there were 54 differentially expressed mRNAs, including 31 upregulated and 23 downregulated mRNAs. In the exon region, 10,072 SNPs were detected, and different types of alternative splicing in the son's sample were observed. Overall, whole genome, exon mutation and transcriptomic profiling of the present two patients with AML may provide a new insight into the molecular events governing the development of AML.

Comparative Genome-Wide Alternative Splicing Analysis of Longissimus Dorsi Muscles Between Japanese Black (Wagyu) and Chinese Red Steppes Cattle.

Alternative splicing is a ubiquitous regulatory mechanism in gene expression that allows a single gene generating multiple messenger RNAs (mRNAs). Significant differences in fat deposition ability and meat quality traits have been reported between Japanese black cattle (Wagyu) and Chinese Red Steppes, which presented a unique model for analyzing the effects of transcriptional level on marbling fat in livestock. In previous studies, the differentially expressed genes (DGEs) in longissimus dorsi muscle (LDM) samples between Wagyu and other breeds of beef cattle have been reported. In this study, we further investigated the differences in alternative splicing in LDM between Wagyu and Chinese Red Steppes cattle. We identified several alternative splicing types including cassette exon, mutually exclusive exons, alternative 5′ splice site, alternative 3′ splice site, alternative start exon, and intron retention. In total, 115 differentially expressed alternatively spliced genes were obtained, of which 17 genes were enriched in the metabolic pathway. Among the 17 genes, 5 genes, including MCAT, CPT1B, HADHB, SIRT2, and DGAT1, appeared to be the novel spliced candidates that affect the lipid metabolism in cattle. Additionally, another 17 genes were enriched in the Gene Ontology (GO) terms related to muscle development, such as NR4A1, UQCC2, YBX3/CSDA, ITGA7, etc. Overall, altered splicing and expression levels of these novel candidates between Japanese black cattle and Chinese Red Steppes revealed by RNA-seq suggest their potential involvement in the muscle development and fat deposition of beef cattle.

Alternative splicing regulates the α-glucosidase synthesis in Aspergillus neoniger NCIM 1400

Aspergillus neoniger NCIM 1400 whose cell-free fraction was earlier established for transglycosylation activity conferred by α-glucosidase gene (agdA), was subjected to sequence analysis. Preliminary results revealed certain dynamics in the intron splicing mechanism, and to ascertain these molecular events, a detailed study was carried. The electrophoresis results from the cDNA portion (B-fragment) of agdA showed multiple bands, indicating the amplification of one or more fragments. The sequence results of cDNA cloned vector revealed the retention type of alternative splicing in the agdA. The splicing mechanism of agdA in NCIM 1400 was compared to different A. niger strains, which harbours agdA orthologues, using PCR. It was observed that effective intron splicing leads to higher α-glucosidase activity from these selected Aspergillus spp. To explore the dynamics of intron retention in A. neoniger NCIM 1400, time-course analysis of intron retention, enzyme activity, and sugar consumption were carried over a period of 168 h of fungal growth. RT-qPCR results revealed that introns retention was not detected during the initial growth phase when the maltose and its hydrolysed product, glucose were consumed. Here we demonstrate that exhaustion of maltose causes increase in retention of introns in the mRNA transcripts of agdA gene, and this could be the possible mode of regulating this gene.