Types Of Alternative Splicing Events Research Articles

A c.726C>G (p.Tyr242Ter) nonsense mutation-associated with splicing alteration (NASA) of WDR45 gene underlies β-propeller protein-associated neurodegeneration (BPAN)

Neurodegeneration with brain iron accumulation (NBIA) is a clinically and genetically heterogeneous disease characterized by increased iron deposition in the basal ganglia and progressive degeneration of the nervous system in adulthood. However, in early childhood, there were no characteristic features to perform early diagnosis. In our study, a female child exhibited global developmental delay, intellectual disability, and febrile seizure without other distinct clinical phenotypes. Through whole exome sequencing (WES), a de novo nonsense mutation (c.726C>G, p.Tyr242Ter) of WDR45 gene was identified in this child. She was finally diagnosed as β‐propeller protein-associated neurodegeneration (BPAN), one of the recently identified subtypes of NBIA. This mutation could act as a premature stop codon (PSC) which rendered the mutated transcripts to be degraded by nonsense-mediated mRNA decay (NMD), leading to decreased levels of PSC-containing mRNAs. Additionally, through mini-gene splicing assays, this mutation could result in an unprecedented novel transcript with the exon 9 of WDR45 excluded by nonsense-associated splicing alteration (NASA). Transcriptome sequencing (RNA-seq) on total RNAs from PBMCs of the trio revealed three types of alternative splicing events in the patient. Further research implied that downregulation of iron transport genes (TFRC, TFR2, SCARA5) might be the underlying mechanism for the iron accumulation in patients with deficient WDR45. This is the first report about NASA happening in WDR45. It implies that nonsense mutations approximal to splicing sites could affect the disease pathogenesis through more than one molecular mechanism and should be taken into consideration when conducting genetic counseling.

Alternative Splicing under Cold Stress in Paper Mulberry

The paper mulberry is a commonly found tree species with a long history of cultivation. It also serves as a crucial case study for understanding how woody plants adapt to low temperatures. Under cold treatment, we observed a substantial number of alternative splicing (AS) genes, showcasing the intricate landscape of AS events. We have detected all seven types of AS events, with the alternative 3′ splice site (A3) having the most. We observed that many genes that underwent differential AS were significantly enriched in starch and sucrose metabolism and circadian rhythm pathways. Moreover, a considerable proportion of differentially spliced genes (DSGs) also showed differential expression, with 20.38% and 25.65% under 12 h and 24 h cold treatments, respectively. This suggests a coordinated regulation between gene AS and expression, playing a pivotal role in the paper mulberry’s adaptation to cold stress. We further investigated the regulatory mechanisms of AS, identifying 41 serine/arginine-rich (SR) splicing factors, among which 11 showed differential expression under cold treatment, while 29 underwent alternative splicing. Additionally, genes undergoing AS displayed significantly higher DNA methylation levels under cold stress, while normal splicing (non-AS) genes exhibited relatively lower methylation levels. These findings suggest that methylation may play an important role in governing gene AS. Finally, our research will provide useful information on the role of AS in the cold acclimation tolerance of the paper mulberry.

MkcDBGAS: a reference-free approach to identify comprehensive alternative splicing events in a transcriptome.

Alternative splicing (AS) is an essential post-transcriptional mechanism that regulates many biological processes. However, identifying comprehensive types of AS events without guidance from a reference genome is still a challenge. Here, we proposed a novel method, MkcDBGAS, to identify all seven types of AS events using transcriptome alone, without a reference genome. MkcDBGAS, modeled by full-length transcripts of human and Arabidopsis thaliana, consists of three modules. In the first module, MkcDBGAS, for the first time, uses a colored de Bruijn graph with dynamic- and mixed- kmers to identify bubbles generated by AS with precision higher than 98.17% and detect AS types overlooked by other tools. In the second module, to further classify types of AS, MkcDBGAS added the motifs of exons to construct the feature matrix followed by the XGBoost-based classifier with the accuracy of classification greater than 93.40%, which outperformed other widely used machine learning models and the state-of-the-art methods. Highly scalable, MkcDBGAS performed well when applied to Iso-Seq data of Amborella and transcriptome of mouse. In the third module, MkcDBGAS provides the analysis of differential splicing across multiple biological conditions when RNA-sequencing data is available. MkcDBGAS is the first accurate and scalable method for detecting all seven types of AS events using the transcriptome alone, which will greatly empower the studies of AS in a wider field.

Differentially expressed transcripts study during pregnancy and postpartum anestrus of yak (Bos grunniens)

Background: Yak is the main livestock species in the plateau area, and its reproductive performance is low, usually two years or three years. A very few of yaks recover within a certain period of time after delivery and smoothly enter the next estrous cycle, while most of them enter the postpartum anestrus and show no estrus performance. However, the key biological factors and influencing mechanisms that cause postpartum anestrus in yaks are not clear. Objective: To study the expression of differential transcripts in ovaries of yak during pregnancy and postpartum anestrus. Methods: Each three yaks in pregnancy and anestrus under natural grazing conditions in Haiyan County, Qinghai Province were selected and slaughtered, and their ovaries were collected and sent to Biomarker Technologies. Oxford Nanopore Technologies single-molecule real-time electrical signal sequencing technology was used to perform full-length transcriptome sequencing. Astalavista software was used to identify the types of alternative splicing events in yak estrus and pregnancy, and TAPIS pipeline was used to identify alternative polyadenylation. Results: The results showed that there were 1751 differentially expressed transcripts (DETs) between pregnancy and anestrus in yak, of which 808 were upregulated and 943 were downregulated. GO analysis showed that the biological processes of DETs were mainly reproductive, reproductive and rhythmic processes. KEGG analysis showed that the DET cell junction-related adhesion junction protein (β-catenin) and amino terminal kinase (JNK) were involved in FAs (local adhesion). Phosphatidylinositol-3-kinase (PI3K) is involved in the PI3K/AKT/mTOR signaling pathway. Circadian rhythm output cycle failure (Clock) and brain and muscle tissue aromatic hydrocarbon receptor nuclear transporter-like protein 1 (Bmal1) are involved in circadian rhythm signaling pathway. Conclusion: This study found that β-catenin, JNK, PI3K, Clock and Bmal1 were closely related to postpartum anestrus in yak.

#6118 ALTERNATIVE SPLICING IN MECHANICALLY STRETCHED PODOCYTES AS A MODEL OF GLOMERULAR HYPERTENSION

Abstract Background and Aims It has been shown that mRNA splicing plays a relevant role in disease pathophysiology. However, the possible role of alternative splicing (AS) in hypertensive nephropathy (HN) has not been investigated. The purpose of the Sys_CARE (Systems Medicine Investigation of AS in Cardiac and Renal Diseases) project is to investigate the possible implication of AS events in HN development and progression. Method Differentiated murine podocytes were seeded on a flexible silicone membrane, and mechanically stretched using the Stretchy apparatus (NIPOKA GmbH, Greifswald). Cells were stretched for 3 days under low and high stretch conditions with a frequency of 0.5 Hz. The mRNA and proteins were isolated and analyzed using RNA_Seq and LC-MS/MS techniques. Enrichment analysis identified proteins which were classified according to the related biological processes, molecular function and cellular components. Splicing and transcript expression were evaluated with bioinformatical tools (rMATS, leafcutter, Whippet, MAJIQ, IsoformSwitchAnalyzeR). Results Proteomic analysis of cultured podocytes revealed significant different expression levels for 135 proteins (54 increased and 81 decreased) and 424 proteins (195 increased and 229 decreased) under low and high stretch conditions, respectively, compared to unstretched conditions. Interestingly, most of the proteins with decreased intensity upon stretch are cytoskeleton and actin-binding proteins. In contrast, the up-regulated proteins are more associated in clusters affecting mRNA processing and splicing. By RNA_Seq we identified over 1000 different splicing events including all types of alternative splicing events. We screened for candidates that showed an alternative splicing event in multiple tools, were found in the proteomics, were podocyte-specific, or showed altered expression in glomerulopathies such as diabetic nephropathy. In this regard, we found an isoform switch of Myl6 and Shroom3 after mechanical stretch. Conclusion Using mechanically stretched podocytes as a model of HN, we found significant up- and down-regulated proteins. In addition, by RNA_Seq we identified an isoform switch of Myl6 and Shroom3 that might be essential for the development of HN.

Dysregulation of the chromatin environment leads to differential alternative splicing as a mechanism of disease in a human model of autism spectrum disorder.

Autism spectrum disorder (ASD) affects 1 in 44 children. Chromatin regulatory proteins are overrepresented among genes that contain high risk variants in ASD. Disruption of the chromatin environment leads to widespread dysregulation of gene expression, which is traditionally thought of as a mechanism of disease pathogenesis associated with ASD. Alternatively, alterations in chromatin dynamics could also lead to dysregulation of alternative splicing, which is understudied as a mechanism of ASD pathogenesis. The anticonvulsant valproic acid (VPA) is a well-known environmental risk factor for ASD that acts as a class I histone deacetylase inhibitor. However, the precise molecular mechanisms underlying defects in human neuronal development associated with exposure to VPA are understudied. To dissect how VPA exposure and subsequent chromatin hyperacetylation influence molecular signatures involved in ASD pathogenesis, we conducted RNA sequencing (RNA-seq) in human cortical neurons that were treated with VPA. We observed that differentially expressed genes (DEGs) were enriched for mRNA splicing, mRNA processing, histone modification and metabolism related gene sets. Furthermore, we observed widespread increases in the number and the type of alternative splicing events. Analysis of differential transcript usage (DTU) showed that exposure to VPA induces extensive alterations in transcript isoform usage across neurodevelopmentally important genes. Finally, we find that DEGs and genes that display DTU overlap with known ASD-risk genes. Altogether, these findings suggest that, in addition to differential gene expression, changes in alternative splicing correlated with alterations in the chromatin environment could act as an additional mechanism of disease in ASD.

Mis-Splicing Due to Somatic U2AF2 Mutations in Myeloid Neoplasms

Background Recent genetic studies have identified frequent mutations affecting a number of genes encoding RNA splicing factors (SDs) in myelodysplastic syndromes (MDS) and related myeloid neoplasms (MNs). Among these, most frequently mutation are SF3B1, SRSF2, U2AF1, and ZRSR2, whose functions have been intensively studied. By contrast, U2AF2, another SF, is also recurrently mutated in MNs. U2AF2 is a component of the U2 auxiliary factor that forms a heterodimer with U2AF1 for the recognition of the 3’ splice site (3'SS). U2AF2 contains a sequence-specific RNA-binding region with two RNA recognition motifs and identifies polypyrimidine (Py) tract signals of nascent transcripts. However, compared to other splicing factor mutations, U2AF2 mutations are much rare, preventing the detailed analysis of their role in leukemogenesis. Methods To characterize the role of U2AF2 mutations, we systematically analyzed mutation spectrum in 6,369 with different MNs using targeted-capture sequencing. We also performed RNA sequencing of cKit(+) bone marrow cells from 6 U2AF2 mutated MDS cases along with 52 MDS patients without common splicing factor mutations and 25 healthy individuals. THP1 and HL60 leukemia cells with exogenous expression of wildtype or mutant U2AF2 were established to evaluate the splicing response. We also used clustered regularly interspaced short palindromic repeats /CRISPR-associated protein-9 nuclease (CRISPR/cas9) to introduce the p.190_195del mutation to U2AF2 in K562 and MOLM-13 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant U2AF2. Results In total, U2AF2 mutations were found in 34 (0.53%) of 6,369 MN cases, of which 29 had the recurrent p.190_195del mutation, the median age of U2AF2 mutations was 55 years (range 31-79) and 55% were diagnosed with MDS, 31% with CMML and 7% with AML. U2AF2-mutated patients showed a male predominance (90%). The most frequently co-mutated gene was ASXL1 with 52%, followed by SETBP1 (35%) STAG2 (21%),RUNX1 (21%), CSF3R, NRAS, DNMT3A, NF1, PTPN11, ETV6, and STAT3. To investigate the effects of the U2AF2 p.190_195del mutation on RNA splicing, we analyzed transcriptome sequencing, followed by the rMATS bioinformatics pipeline to determine alternative splicing (AS) events . Various types of AS events were identified, including skipped exons (SEs), alternative 5’ ss exons (A5SSs), alternative 3’ ss exons (A3SSs), retained introns (RIs), and mutually exclusive exons (MXEs). Among these, the skipping exons are the most frequent in both U2AF2-mutant primary MDS cases and cell lines. To explore these alternative splicing results in more detail, we next examined differentially skipped exons. Unsupervised uniform manifold approximation and projection (UMAP) analysis based on the standardized splicing ratio of skipping exons segregated U2AF2-mutated patients from other MDS cases without common splicing factor mutations and healthy individuals. To prioritize mutant U2AF2-induced alterative splicing events, we intersected significant skipping exons across 2 datasets: MDS patient samples with and without U2AF2 p.190_195del mutations, and MDS patient samples with U2AF2 p.190_195del mutations and healthy individuals. The differential skipping exon usages and differential gene expression profiles enables both consensus sequence analysis and pathway/gene-set enrichment analysis. Conclusions Our study revealed that the U2AF2 p.190_195del regulated aberrant alternative splicing facilitated MDS progression through perturbations in splice isoforms. A better understanding of the U2AF2 p.190_195del and its critical specific changes will provide novel insights into disease pathophysiology and potentially inform future treatment decisions.

Comprehensive Transcriptome Analysis Reveals Sex-Specific Alternative Splicing Events in Zebrafish Gonads.

Alternative splicing is an important way of regulating gene functions in eukaryotes. Several key genes involved in sex determination and gonadal differentiation, such as nr5a1 and ddx4, have sex-biased transcripts between males and females, suggesting a potential regulatory role of alternative splicing in gonads. Currently, the sex-specific alternative splicing events and genes have not been comprehensively studied at the genome-wide level in zebrafish. In this study, through global splicing analysis on three independent sets of RNA-seq data from matched zebrafish testes and ovaries, we identified 120 differentially spliced genes shared by the three datasets, most of which haven’t been reported before. Functional enrichment analysis showed that the GO terms of mRNA processing, mRNA metabolism and microtubule-based process were strongly enriched. The testis- and ovary-biased alternative splicing genes were identified, and part of them (tp53bp1, tpx2, mapre1a, kif2c, and ncoa5) were further validated by RT-PCR. Sequence characteristics analysis suggested that the lengths, GC contents, and splice site strengths of the alternative exons or introns may have different influences in different types of alternative splicing events. Interestingly, we identified an unexpected high proportion (over 70%) of non-frameshift exon-skipping events, suggesting that in these cases the two protein isoforms derived from alternative splicing may both have functions. Furthermore, as a representative example, we found that the alternative splicing of ncoa5 causes the loss of a conserved RRM domain in the short transcript predominantly produced in testes. Our study discovers novel sex-specific alternative splicing events and genes with high reliabilities in zebrafish testes and ovaries, which would provide attractive targets for follow-up studies to reveal the biological significances of alternative splicing events and genes in sex determination and gonadal differentiation.

Transcriptome Analyses Reveal Essential Roles of Alternative Splicing Regulation in Heat-Stressed Holstein Cows.

Heat stress (HS) severely impacts the productivity and welfare of dairy cows. Investigating the biological mechanisms underlying HS response is crucial for developing effective mitigation and breeding strategies. Therefore, we evaluated the changes in milk yield, physiological indicators, blood biochemical parameters, and alternative splicing (AS) patterns of lactating Holstein cows during thermoneutral (TN, N = 19) and heat-stress (HS, N = 17) conditions. There was a significant (p < 0.05) decline in milk yield as physiological indicators increased after exposure to natural HS conditions. The levels of eight out of 13 biochemical parameters of HS were also significantly altered in the presence of HS (p < 0.05). These results demonstrate that HS negatively influences various biological processes of Holstein cows. Furthermore, we investigated AS events based on the RNA-seq data from blood samples. With HS, five common types of AS events were generally increased by 6.7–38.9%. A total of 3470 AS events corresponding to 3143 unique genes were differentially alternatively spliced (DSGs) (p-adjusted < 0.05) between TN and HS groups. The functional annotation results show that the majority of DSGs are involved in mRNA splicing and spliceosomal complex, followed by enrichment in immune and metabolic processes. Eighty-seven out of 645 differentially expressed genes (DEGs) (fold change ≥ 1.5 and false discovery rate < 0.05) overlapped with DSGs. Further analyses showed that 20 of these genes were significantly enriched for the RNA splicing, RNA binding, and RNA transport. Among them, two genes (RBM25 and LUC7L3) had strong interrelation and co-expression pattern with other genes and were identified as candidate genes potentially associated with HS responses in dairy cows. In summary, AS plays a crucial role in changing the transcriptome diversity of heat-stress-related genes in multiple biological pathways and provides a different regulation mechanism from DEGs.

Polyadenylate-binding protein cytoplasmic 1 mediates alternative splicing events of immune-related genes in gastric cancer cells.

Polyadenylate-binding protein cytoplasmic 1 (PABPC1) is dysregulated in malignancies, which is considered as a potential therapeutic target for many cancer types. By alternative splicing (AS) for gastric cancer (GC), we described PABPC1-modulated AS events in this study. PABPC1 expression was analyzed in 408 GC tissues from The Cancer Genome Altas (TCGA) database. Human gastric adenocarcinoma (AGS) cells were transfected with PABPC1-specific small interfering RNA (siPABPC1) with siCtrl as a negative control. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was done for the determination of transcripts. To detect the differentially expressed genes (DEGs) and 10 different types of AS events, RNA sequencing (RNA-seq) was performed. DEGs were analyzed for functional categories including gene ontology, and the Kyoto encyclopedia of genes and genomes pathway were analyzed for DEGs. GC displayed an elevated expression of PABPC1. PABPC1 was efficiently knocked down in AGS cells. Here, we excavated 1234 PABPC1-regulated DEGs, among which 502 were down-regulated and 732 were up-regulated compared to the siCtrl group. A total of 94 DEGs were involved in inflammation and immune response. Results from qRT-PCR validated the up-regulation of 10 immune and inflammation-related DEGs in the siPABPC1 group. PABPC1 deficiency causes 1304 AS events differentially occurred in AGS cells. The most common type of AS events regulated by PABPC2 is alternative 5' splice sites. qRT-PCR confirmed the transcription level of five immune-related genes, in which AS events were detected in the siPABPC1 group. PABPC1 knockdown mediates AS events and thus the transcript level of immune and inflammation-related genes in AGS cells. PABPC1-regulated oncogenic AS events display potential as targets for therapeutic development.

Decoding the concealed transcriptional signature of the apoptosis-related BCL2 antagonist/killer 1 (BAK1) gene in human malignancies.

BCL2 antagonist/killer (BAK) is a multidomain pro-apoptotic effector protein, encoded by the human BAK1 gene, which has emerged as a key checkpoint in the apoptotic machinery. Disassembly of BAK's tertiary structure, such as the truncation of the α1 helix, leads to deregulation of the pro-apoptotic functions and reduction of the protein's stability, thus being implicated in human malignancies. Although many studies have already clarified the vital role of BAK in cellular mechanisms, its pre-mRNA maturation process under cancerous and physiological human cells is neglected. In the present work, we developed and employed a custom multiplexed nanopore sequencing approach that enabled the identification and structural characterization of previously undescribed BAK1 mRNA transcripts (BAK1 v.2-v.11). The described novel mRNAs are derived from multiple types of alternative splicing events, including exon skipping and intron retentions. The implemented multiplexed long-read sequencing approach provided the detailed expression profile of the novel mRNAs in a wide panel of human malignancies and at the same time allowed their relative quantification as compared to the annotated BAK1 v.1. The validation of each novel transcript was carried out with qPCR-based assays. Our results strongly support that most of the novel BAK1 mRNAs harbor open reading frames with conserved BH domains that provide new insights into the correlated mechanisms of apoptosis suppression and cancer. The current study highlights for the first time the hidden aspects of BAK1's transcriptional landscape in both physiological and cancerous human cells and distinguishes the amino acid sequence of the putative BAK isoforms that may possess key apoptosis-related functions not only in diseases, but also under normal cellular conditions.

Identification of prognostic alternative splicing signature in gastric cancer

BackgroundAberrant alternative splicing (AS) events could be viewed as prognostic indicators in a large number of malignancies. This study aims to identify prognostic AS events, illuminate the function of the splicing variants biomarkers and provide reliable evidence for formulating public health strategies for gastric cancer (GC) surveillance.MethodsRNA-Seq data, clinical information and percent spliced in (PSI) values were available in The cancer genome atlas (TCGA) and TCGA SpliceSeq data portal. A three-step regression method was conducted to identify prognostic AS events and construct multi-AS-based signatures. The associations between prognostic AS events and splicing factors were also investigated.ResultsWe identified a total of 1,318 survival-related AS events in GC, parent genes of which were implicated in numerous oncogenic pathways. The final prognostic signatures stratified by seven types of AS events or not stratified performed well in risk prediction for GC patients. Moreover, five signatures based on AA, AD, AT, ES and RI events function as independent prognostic indicators after multivariate adjustment of other clinical variables. Splicing network also showed marked correlation between the expression of splicing factors and PSI value of AS events in GC patients.ConclusionOur findings provide a landscape of AS events and regulatory network in GC, indicating that AS events might serve as prognostic biomarkers and therapeutic targets for GC.

Delineating genome-wide alternative splicing landscapes and their functional significance in orchids

Orchids are one of the most attractive groups of flowers with exceptional morphological diversity and high commercial value. Orchid plants are evolved to thrive in diverse habitats under different climatic conditions. Alternative splicing (AS) is an important post-transcriptional regulatory mechanism to modulate transcript diversity for necessary physiological functions in plants. Present study deals with the genome-wide AS landscapes and their functional relevance in six orchid species, information on which was inadequate so far. A total of 180 transcriptome datasets representing different tissues and treatments were chosen from the orchid taxa with well-annotated, publicly accessible genome asssemblies. Cleaned RNA-seq reads were aligned against corresponding reference genomes followed by de-novo transcriptome assembly and detection of novel AS events with significant read support. Alternative acceptor sites (A3S) and intron retentions (IR) were the most prevalent type of AS events. Under cold acclimation, exon skipping events were observed to be elevated in D. officinale leaves. Incidences of intron retentions were elevated in response to pathogen stress, that further amplified offering jasmonate mediated immunity in D. catenatum. GO enrichment of AS-affected genes suggested that most of them are functionally involved in cellular metabolism and RNA processing. A major fraction of genes could be detected that are not differentially expressed in general, however, specific splice variants of them are significantly up/down-regulated with the biotic or abiotic stress mediated responses. Functional information on the AS-affected genes and expressional dynamics of the resultant isoforms would serve as potential genomic resources for orchid research.

Analysis of Genomic Alternative Splicing Patterns in Rat under Heat Stress Based on RNA-Seq Data.

Heat stress is one of the most severe challenges faced in livestock production in summer. Alternative splicing as an important post-transcriptional regulation is rarely studied in heat-stressed animals. Here, we performed and analyzed RNA-sequencing assays on the liver of Sprague-Dawley rats in control (22 °C, n = 5) and heat stress (4 °C for 120 min, H120; n = 5) groups, resulting in the identification of 636 differentially expressed genes. Identification analysis of the alternative splicing events revealed that heat stress-induced alternative splicing events increased by 20.18%. Compared with other types of alternative splicing events, the alternative start increased the most (43.40%) after heat stress. Twenty-eight genes were differentially alternatively spliced (DAS) between the control and H120 groups, among which Acly, Hnrnpd and mir3064 were also differentially expressed. For DAS genes, Srebf1, Shc1, Srsf5 and Ensa were associated with insulin, while Cast, Srebf1, Tmem33, Tor1aip2, Slc39a7 and Sqstm1 were enriched in the composition of the endoplasmic reticulum. In summary, our study conducts a comprehensive profile of alternative splicing in heat-stressed rats, indicating that alternative splicing is one of the molecular mechanisms of heat stress response in mammals and providing reference data for research on heat tolerance in mammalian livestock.

Systemic characterization of alternative splicing related to prognosis, immune infiltration, and drug sensitivity analysis in ovarian cancer.

BackgroundAlternative splicing (AS) plays an essential role in tumorigenesis and progression. This study intended to construct an innovative prognostic model based on AS events to gain more precise survival prediction and search for potential therapeutic targets in ovarian cancer.MethodsSeven types of AS events in ovarian serous cystadenocarcinoma (OV) patients with RNA-seq were obtained using The Cancer Genome Atlas (TCGA) SpliceSeq tool and database. Cox and Kaplan-Meier curve analyses were employed to establish the prognostic models. Relying on drug sensitivity data from the CellMiner database, Genomics of Drug Sensitivity (GDS) was adopted to estimate the platinum-sensitive analysis. Furthermore, a prognostic splicing factor (SF)-AS network was constructed using Cytoscape. Finally, in order to explore the influence of the tumor microenvironment on the prognosis of OV patients, we first combined a similar network fusion and consensus clustering (SNF-CC) algorithm to identify three OV subtypes based on survival-related AS events and then utilized single-sample Gene Set Enrichment Analysis (ssGSEA) method to perform immune cell infiltration analysis.ResultsA total of 48,049 AS events and 21,841 related genes were selected from 318 OV samples, and 2,206 AS events associated with disease-free survival (DFS) were identified. Multivariate Cox and Kaplan-Meier curve analyses were then employed to establish the prognostic models. Receiver operating characteristic (ROC) analysis from 0.59 to 0.75 showed that these models were highly efficient in distinguishing patient survival. GDS was adopted with the CellMiner database to provide some insights for platinum-sensitive analysis of OV. Furthermore, a prognostic SF-AS network, which discovered a significant connection between SFs and prognostic AS genes, was constructed using Cytoscape. The combined SNF-CC algorithm revealed three distinct OV subtypes based on the prognostic AS events, and the associations between this novel molecular classification and immune cell infiltration were further explored.ConclusionsWe developed a powerful prognostic AS signature for OV and provided a deeper understanding of SF-AS network regulatory mechanisms, as well as platinum-sensitive and cancer immune microenvironments. These results revealed various candidate biomarkers and potential targets for OV treatment strategies.

Genome-wide transcriptome analysis of the early developmental stages of Echinococcus granulosus protoscoleces reveals extensive alternative splicing events in the spliceosome pathway

BackgroundThe complex life cycle of Echinococcus granulosus involves numerous environmental conditions within different intermediate and definitive hosts. This requires adaptation at different levels of transcript regulation. Alternative splicing (AS) and the related cellular functions as one of the major fields of post-genomics has been poorly studied in tapeworms. In the present study, we investigated AS events and their potential biological effects in E. granulosus.MethodsWhole transcriptome sequencing data of four groups of protoscoleces were prepared for RNA-seq library construction. Fresh protoscoleces were either used as non-induced controls (NT group) or incubated for 15 min with pepsin (PEP group) and cultivated in a biphasic medium for 12 and 24 h (12 and 24 h groups). The frequency and different types of AS events were identified using rMATS software. Functional annotations and gene ontology of differential AS (DAS) genes were performed using Blast2GO software. AS events were experimentally validated by PCR on the protoscolex cDNAs using specific primers for each gene.ResultsAt least one AS event was found in 38.1% of the genes (3904 out of 10,245) in the protoscoleces during early strobilar development. The genes were associated primarily with cellular and metabolic processes and binding and catalytic activity. KEGG pathway analysis of DAS events revealed a number of genes belonging to different components of the spliceosome complex. These genes tended to belong to common SR proteins, U1-related factors, U2-related factors, complex A-specific factors and other splicing-related proteins.ConclusionsThe high number of AS events in the transcriptome regulatory mechanisms indicates the essential rapid molecular processes required by the parasite for adaptation in different environments.Graphical

Splicing Factor SRSF1 Promotes Tumorigenesis Via Oncogenic Splice-Switching and Predicts Poor Prognosis in Multiple Myeloma

ObjectiveDysregulation of alternative splicing (AS) triggers many tumors, yet its contribution to multiple myeloma (MM) has not been elucidated. Our study aims to reveal the role of AS in MM progression, and to explore the molecular mechanisms of the candidate splicing factor (SF), thus valuates its feasibility as a treatment target and prognostic factor.Design and results 1.Gene set enrichment analysis (GSEA) revealed a significant difference in the expression pattern of the alternative splicing pathway genes, notably enriched in MM patients (Figure 1A). And, the splicing factor SRFS1 expression showed concordant differential expression in MM versus normal donors in two datasets (GSE6477, GSE13591). Kaplan-Meier analysis showed that high SRSF1 expression in CD138 + MM cells was associated with decreased PFS and poor OS (GSE9782) (Figure 1B). Moreover, the SRSF1 levels appeared a progressive increase in the progression of plasma cell dyscrasias (MGUS, SMM, MM and PCL) (Figure 1C). A cohort of 57 newly diagnosed MM patients was uniformly treated and followed. Univariate and multivariate analyses indicated that high SRSF1 expression was correlated with decreased PFS and OS. The c-indices of the Cox model fit with the combination of RISS and SRSF1 were higher than those of models fit without SRSF1, suggesting that SRSF1 improved the prognostic stratification of patients with MM.2.In vitro, SRSF1 downregulation in MM cells resulted in decreased proliferation, increased apoptosis and cell cycle arrest in G1 phase. In orthotopic MM xenograft models, bioluminescent imaging monitoring revealed a decreased burden of disease and a prolongation in overall survival in mice injected with SRSF1-shRNA cells versus the control (Figure 1D-F).3.Pearson correlation analysis suggested that the expression of TF YY1 was positively correlated with SRSF1 in five sets of GEO (Figure 1G). CHIP-seq BigWig files were downloaded from GSE31477 and Integrative Genomics Viewer (IGV) tracks displayed the binding site of YY1 at SRSF1 promoter locus from chr17: 56084421 to 56084751 (Figure 1H). CHIP-qPCR assay confirmed that endogenous YY1 directly interacted with the core element of the SRSF1 promoter in myeloma cells. Silencing of YY1 expression decreased the luciferase activity of the reporter construct carrying wild-type SRSF1 promoter region but not mutant in the YY1 binding site. Consistent with the effects of SRSF1, downregulation YY1 displayed anticancer activity in myeloma cells. Kaplan-Meier analysis showed that high YY1 expression in CD138 + myeloma cells was associated with decreased PFS and poor OS.4.RNA-seq analysis revealed a total of 7678 SRSF1-regulated AS events in LP-1 cell with an FDR cut-off of <0.05. Among them, the index of percent spliced in percentage-spliced-in (PSI) was upregulated in 4098 AS events and downregulated in 3580 AS via the downregulation of SRSF1. Changes in alternative splicing events were analyzed by replicate multivariate analysis of transcript splicing (rMATS) to identify downstream key genes regulated by SRSF1. Various types of AS events, including skipped exons (SEs), alternative 5‘ ss exons (A5SSs), alternative 3‘ ss exons (A3SSs), retained introns (RIs), and mutually exclusive exons (MXEs), can be regulated by SRSF1, and of these, SE is the most common type of SRSF1-regulated AS events (Figure 1I-O). The KEGG Pathway and GO analysis revealed that SRSF1-regulated AS events were involved in spliceosome, cell cycle and cellular process. Among the most significant AS events, we noted RBBP6, which isoforms have been reported to exhibit opposite effect on oncogenic activity.5.RNA-seq and subsequent validation indicated that SRSF1 knockdown caused a significantly decreased iso1 and increased iso3 of RBBP6. An opposite effect on myeloma proliferation between iso1 and iso3 was observed upon overexpression of individual proteins. Moreover, the level of RBBP6 iso1 is associated with poor prognosis, whereas iso3 expression is associated with favorable prognosis in MM patientsConclusionOur study uncovered that SRSF1-regulated aberrant AS facilitated myeloma progression through creating reducing normal isoforms and increasing oncogenic isoforms (Figure 1P). Moreover, SRSF1 and its critical splicing target could act as an independent prognostic factor for MM patients. Thus, SRSF1 might be considered as a valuable therapeutic target and a potential prognostic biomarker for MM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of Genome-Wide Alternative Splicing Profiling and Development of Potential Drugs in Lung Adenocarcinoma.

Alternative splicing (AS) is significantly related to tumor development as well as a patient’s clinical characteristics. This study was designed to systematically analyze the survival-associated AS signatures in Lung adenocarcinoma (LUAD). Among 30,735 AS events in 9,635 genes, we found that there were 1,429 AS in 1,125 genes which were conspicuously related to the overall survival of LUAD patients. Then, according to the seven types of AS events, we established AS signatures and constructed a new combined prognostic model. The Kaplan-Meier curve results showed that seven types of AS signatures and the combined prognostic model could divide patients into distinct prognoses. The ROC curve shows that all eight AS signatures had powerful predictive properties with different AUCs ranging from 0.708 to 0.849. Additionally, the elevated risk scores were positively related to higher TNM stage and metastasis. Interestingly, AS events and splicing factors (SFs) network shed light on a meaningful connection between prognostic AS genes and corresponding SFs. Moreover, we found that the combined prognostic model signature has a higher predictive ability than the mRNA signature. Furthermore, tumors at high risk might evade immune recognition by decreasing the expression of antigen presentation genes. Finally, we predicted the three most significant small molecule drugs to inhibit LUAD. Among them, NVP-AUY922 had the lowest IC50 value and might become a potential drug to prolong a patient’s survival. In conclusion, our study established a potential prognostic signature for LUAD patients, revealed a splicing network between AS and SFs and possible immune escape mechanism, and provided several small-molecule drugs to inhibit tumorigenesis.

Role of alternative splicing events in endometrial cancer prognosis.

Alternative splicing (AS), as a potent and pervasive mechanism of transcriptional regulation, can expand the genome's coding capacity. Growing evidence suggests that the AS events may be associated with various types of cancer. This study aims to explore the prognostic value of AS in endometrial cancer (EC). Differently expressed AS (DEAS) events were screened by pairing the percent spliced in (PSI) value of tumor and paracancerous tissues in The Cancer Genome Atlas (TCGA) database, and gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed on their parental gene analysis of organisms. Subsequently, univariate Cox analysis was used to identify the prognostic AS events and a stepwise multi-factor Cox regression analysis was performed to further construct prognostic models. Furthermore, the diagnostic value of the prognostic model was evaluated by receiver operating characteristic (ROC) curve and Kaplan-Meier analysis. Finally, the regulatory network of AS events and splicing factory in the model was also constructed. A total of 28 281 AS events were detected in EC. Of them, 42 DEAS were identified, and their parental genes were involved in tumor-related processes such as meiotic nuclear division, alpha-amino acid biosynthetic process, nuclear division, and so on. Univariate Cox analysis identified 2 289 prognostic-related AS events and constructed Cox prognostic models based on 7 different types and all types of AS events, in which the area under the curve of ROC of all types was as high as 0.882 and was better than that of 7 different splicing types. Finally, 12 splicing factors and AS events showed an obvious regulatory relationship. We use the whole genome analysis of AS events to establish a scientific prognostic prediction model for EC patients, which provides a reliable theoretical basis for the evaluation of EC clinical prognosis.

Identification of survival-related alternative splicing signatures in acute myeloid leukemia.

Aberrant RNA alternative splicing (AS) variants play critical roles in tumorigenesis and prognosis in human cancers. Here, we conducted a comprehensive profiling of aberrant AS events in acute myeloid leukemia (AML). RNA AS profile, including seven AS types, and the percent spliced in (PSI) value for each patient were generated by SpliceSeq using RNA-seq data from TCGA. Univariate followed by multivariate Cox regression analysis were used to identify survival-related AS events and develop the AS signatures. A nomogram was developed, and its predictive efficacy was assessed. About 27,892 AS events and 3,178 events were associated with overall survival (OS) after strict filtering. Parent genes of survival-associated AS events were mainly enriched in leukemia-associated processes including chromatin modification, autophagy, and T-cell receptor signaling pathway. The 10 AS signature based on seven types of AS events showed better efficacy in predicting OS of patients than those built on a single AS event type. The area under curve (AUC) value of the 10 AS signature for 3-year OS was 0.91. Gene set enrichment analysis (GSEA) confirmed that these survival-related AS events contribute to AML progression. Moreover, the nomogram showed good predictive performance for patient's prognosis. Finally, the correlation network of AS variants with splicing factor genes found potential important regulatory genes in AML. The present study presented a systematic analysis of survival-related AS events and developed AS signatures for predicting the patient’s survival. Further studies are needed to validate the signatures in independent AML cohorts and might provide a promising perspective for developing therapeutic targets.