Analysis Of Alternative Splicing Events Research Articles

Transcriptomic Analysis of Alternative Splicing Events during Different Fruit Ripening Stages of Coffea arabica L.

To date, genomic and transcriptomic data on Coffea arabica L. in public databases are very limited, and there has been no comprehensive integrated investigation conducted on alternative splicing (AS). Previously, we have constructed and sequenced eighteen RNA-seq libraries of C. arabica at different ripening stages of fruit development. From this dataset, a total of 3824, 2445, 2564, 2990, and 3162 DSGs were identified in a comparison of different fruit ripening stages. The largest proportion of DSGs, approximately 65%, were of the skipped exon (SE) type. Biologically, 9 and 29 differentially expressed DSGs in the spliceosome pathway and carbon metabolism pathway, respectively, were identified. These DSGs exhibited significant variations, primarily in S1 vs. S2 and S5 vs. S6, and they involve many aspects of organ development, hormone transduction, and the synthesis of flavor components. Through the examination of research findings regarding the biological functions and biochemical pathways associated with DSGs and DEGs, it was observed that six DSGs significantly enriched in ABC transporters, namely, LOC113712394, LOC113726618, LOC113739972, LOC113725240, LOC113730214, and LOC113707447, were continually down-regulated at the fruit ripening stage. In contrast, a total of four genes, which were LOC113732777, LOC113727880, LOC113690566, and LOC113711936, including those enriched in the cysteine and methionine metabolism, were continually up-regulated. Collectively, our findings may contribute to the exploration of alternative splicing mechanisms for focused investigations of potential genes associated with the ripening of fruits in C. arabica.

Profiling of the gene expression and alternative splicing landscapes of Eucalyptus grandis.

Eucalyptus is a widely planted hardwood tree speciesdue to its fast growth, superior wood properties and adaptability. However, the post-transcriptional regulatory mechanisms controlling tissue development and stress responses in Eucalyptus remain poorly understood. In this study, we performed a comprehensive analysis of the gene expression profile and the alternative splicing (AS) landscape of E. grandis using strand-specific RNA-Seq, which encompassed 201 libraries including different organs, developmental stages, and environmental stresses. We identified 10 416 genes (33.49%) that underwent AS, and numerous differentially expressed and/or differential AS genes involved in critical biological processes, such as primary-to-secondary growth transition of stems, adventitious root formation, aging and responses to phosphorus- or boron-deficiency. Co-expression analysis of AS events and gene expression patterns highlighted the potential upstream regulatory role of AS events in multiple processes. Additionally, we highlighted the lignin biosynthetic pathway to showcase the potential regulatory functions of AS events in the KNAT3 and IRL3 genes within this pathway. Our high-quality expression atlas and AS landscape serve as valuable resources for unravelling the genetic control of woody plant development, long-term adaptation, and understanding transcriptional diversity in Eucalyptus. Researchers can conveniently access these resources through the interactive ePlant browser (https://bar.utoronto.ca/eplant_eucalyptus).

Regulation of Alternative Splicing in B-Cell ALL By DYRK1A

DYRK1A, located in the Down syndrome critical region of chromosome 21, is a serine and threonine kinase that controls multiple cellular processes including apoptosis, cell cycle, transcription and signal transduction. We previously demonstrated that DYRK1A is required for maturation of B cells and is a therapeutic target in B-cell ALL, including both DS and non-DS subtypes. Key substrates within the B-cell lineage include D-type cyclins, FOXO1, and STAT3. Given that several studies have reported that DYRK1A phosphorylates various splicing factors and participates in splicing of Tau in neuronal cells, we investigated the contribution of DYRK1A to splicing in malignant B cells. We first treated B-ALL cell lines with two DYRK1A inhibitors, EHT 1610 and GNF 2133 and performed RNA-sequencing to detect changes in alternative splicing (AS). GO analysis of AS events showed RNA binding proteins among top differentially spliced genes. Of note, mis-splicing of RBM39 was one of the top differentially AS events associated with DYRK1A inhibition; this event involves inclusion of a “poison” exon that contains a premature termination codon and leads to a transcript predicted to be degraded by nonsense-mediated mRNA decay (NMD). We confirmed NMD by knocking down an essential NMD factor, UPF1, which stabilized the NMD isoform. The NMD isoform was also detected in B-ALL patient samples treated with DYRK1A inhibitors and in NALM6 cells in which DYRK1A was inducibly degraded. RBM39 has been reported to be a therapeutic target in AML, where cells with splicing factor mutations are more sensitive to loss of RBM39 than cells lacking splicing factor mutations. To determine whether RBM39 is a therapeutic target in B-ALL, we silenced RMB39 with shRNAs in vitro and in vivo. Genetic knockdown of RBM39 impeded B-ALL cell growth both in vitro and in xenograft models. Furthermore, inducible silencing of RBM39 hampered B-ALL progression in vivo evidenced by a significant increase in survival of mice engrafted with human B-ALL cells. We also found that indisulam and its analogs, which lead to DCAF15-mediated degradation of RBM39, suppressed the growth of B-ALL cells in vitro and in vivo. Importantly, the anti-tumor effect of RBM39 disruption extended beyond DS-ALL to Ph-like B-ALL, which continues to be an area of unmet medical need. We next analyzed the contributions of splicing factors that are known DYRK1A substrates to AS of RBM39. Knockdown of SRSF1 and SRSF2 led to increased inclusion of the poison exon even in the absence of DYRK1A inhibition, indicating that they normally function to exclude the exon. By contrast, knockdown of SF3B1 led to a reduction of the NMD isoform in the presence of EHT1610. Surprisingly, phosphorylation of SF3B1 by DYRK1A was not essential for the change in RBM39 splicing, as cells engineered to harbor two SF3B1 T434A phospho-deficient mutant alleles, did not show aberrant splicing. These results indicate that SF3B1, SRSF1, and SRSF2 are not the key substrates that mediate the effect of DYRK1A on alternative splicing. Next, given that DYRK1A is known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (Pol II) on Ser2 and Ser5, we investigated the contribution of Pol II phosphorylation to RBM39 splicing. IP-MS revealed an interaction between RNA Pol II and SF3B1, consistent with previous reports of co-transcriptional splicing. We found that inhibition of DYRK1A disrupted the colocalization of SF3B1 and RNA Pol II as well as with RBM39 RNA. However, DYRK1A inhibition had a transient effect on Ser2 and Ser5 phosphorylation, suggesting that cells have alternative pathways for Ser2/5 phosphorylation. Combining DYRK1A inhibition with inhibition or loss of CDK9, which also phosphorylates the CTD of RNA Pol II, led to a persistent decrease in RNA Pol II phosphorylation, marked increased NMD isoform and a profound reduction in RBM39 protein. Finally, we found that combining the CDK9 inhibitor dinaciclib with EHT 1610 synergistically inhibited growth of B-ALL patient cells in vitro and in vivo shown by significantly improved survival. Ectopic expression of RBM39 partially rescued the growth inhibition upon dinaciclib and EHT 1610 treatment suggesting downregulation of RBM39 is the key event in drug targeting. Together, our results reveal that DYRK1A controls RNA splicing by regulating the SF3B1-RNA Pol II interaction and that RBM39 is a therapeutic target in B-ALL.

Transcriptomic analysis of alternative splicing events for different stages of growth and development in Sistan Yaghooti grape clusters

Sistan Yaghooti grape variety, despite characteristics such as early ripening, is vulnerable to cluster rot due to small berries and dense clusters. In this regard, AS may serve as a regulatory mechanism during developmental processes and in response to environmental signals. RNA-Seq analysis was performed to measure gene expression and the extent of AS events in the cluster growth and development stages of Sistan Yaghooti grape. The number of AS events increased during stages, suggesting that it contributes to the grapevine's adaptability to various stresses. In addition, DEG and DAS genes showed little overlap in cluster growth stages. Functional analysis of 19,194 DAS -gene sets showed that VIT_06s0004g06670 gene is involved in the activation of calcium channels (Ca2+) through the activation of 5 PLC biosynthetic pathways. Among the 27,229 DEG -sets, VIT_07s0005g05320 gene showed higher expression. Interestingly, this gene is involved in the synthesis of an EF -hand domain-containing protein capable of binding to Ca2+ by activating 4 biochemical pathways. These genes increase cytosolic Ca2+ concentration, enhancing plant stress tolerance and resistance to cracking. These results show that AS can respond independently to different types of stress. Among the other DAS genes, the GA2ox gene (VvGA2ox) showed an increase in AS events during cluster development. This gene is critical for initiating the degradation process of GA and plays a crucial role in different stages of seed development. Therefore, it is very likely that this gene is one of the main factors responsible for the density and seedlessness of Sistan Yaghooti grape.

Comparative analysis of alternative splicing events in skeletal muscle of different sheep

This paper aims to investigate the relationship between genes with alternative splicing (AS) events and breed-specific differences in muscle development in two breeds of sheep. RNA-seq was utilized to identify genes with AS between Small-tailed Han sheep and Dorset sheep. The gene lists of differentially spliced genes were identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted on these genes. In this study, 299 genes with 356 AS indicated significant differences between two diffrerent breeds. There are differences in 31 genes with 35 AS. Cassette, alt5′ and alt3’ exhibited the highest levels of enrichment across various significant levels. GO and KEGG enrichment analysis demonstrated a significant correlation between Wnt, TGF-beta, Notch and MAPK signaling pathways and the development of muscle in sheep. These findings indicate that genes with AS are linked to variations in muscle development in sheep. These results offer significant scientific and practical implications for improving the quality of sheep meat.

Systematic Collection and Analysis of Alternative Splicing Events in Potato Plants

Systematic Collection and Analysis of Alternative Splicing Events in Potato Plants

Comparative Analysis of Alternative Splicing Events in Foliar Transcriptomes of Potato Plants Inoculated with <i>Phytophthora Infestans</i>

Alternative splicing (AS) is a common process during gene expression of plants in coping various biotic or abiotic stresses. The work reports identification and analysis of AS events in foliar samples of two potato lines, including a wild type line and a pathogen resistant transgenic line (+RB), inoculated with Phytophthora infestans . After combining all RNA-seq data collected from 36 samples, a total of 10,246 AS events were identified, including 1,563 exon skipping, 1,368 alternative donor sites, 3,091 alternative acceptor sites, 884 intron retention, and 3,340 complex events, which consisted of more than one basic event. These AS events were generated from 45,874 isoform transcripts expressed from 13,704 genes. It was estimated 30.2% of genes undergoing AS in this analysis. Furthermore, we identified 406 specific AS events, which were generated from 281 genes, and 766 differentially expressed transcripts (DETs) in the sample collected 24 hours after inoculation of P. infestans  in +RB lines. These DETs were expressed from 763 genes, and among them, 338 genes were alternatively spliced. These results indicate that both AS and differential gene expression may contribute to the resistance against P. infestans  in +RB line of potato plants.

Transcriptome Analysis Reveals the Involvement of Alternative Splicing in the Nitrogen Starvation Response of Chlamydomonas reinhardtii

Alternative splicing (AS) is a regulatory mechanism of post-transcriptional regulation that plays an important role in plant response to abiotic stresses. However, corresponding research involving the mechanism of AS in the nitrogen starvation response of C. reinhardtii is rare. This study performed a comprehensive and systematic analysis of AS events in C. reinhardtii at nine time points (0 h, 10 m, 30 m, 1 h, 6 h, 8 h, 24 h, and 48 h) under nitrogen starvation. It used STAR and rMATS tools to identify and quantify the probability of the AS event happening through the transcriptome high-throughput sequencing data. A total of 5806 AS events in 3500 genes were identified, and the retained intron and skipped exon were considered the main AS types. The genes related to the AS event in nitrogen starvation were mainly involved in spliceosome and transporter and enriched in the citrate cycle and fatty acid degradation pathways. These results suggested that AS may play an important role in the nitrogen starvation response in C. reinhardtii, and provided insights into post-transcriptional regulation under nitrogen starvation.

Global analysis of alternative splicing events based on long- and short-read RNA sequencing during grape berry development

• This study highlights that alternative splicing in grape fruit development is a major event. • Differentially spliced genes potentially associated with early ripening traits were identified. • Gene annotation in the grape reference genome will benefit from this information.

Splicing Analysis Unveils Convergent Transcriptomic Pathways in U2AF1 S34 and Q157 Mutant Myeloid Neoplasia

Splicing factor mutations (SFMT) are frequent in MDS and their pathogenic consequences may include missplicing and changes in alternative splicing (AS). However, divergent sequence-sequence effects on misspliced genes were recorded in U2AF1MT at the two canonical hotspots (S34, Q157). Most of the current studies have been focused on the identification of distinctive features of splicing dysfunction between the two hotspots, unveiling various unique hotspot specific splicing events. Classical instances of missplicing events include those in ATG7,H2AFY and STRAP specific to U2AF1S34 MDS. However, whereas we and others have demonstrated certain unique clinical features of MDS patients carrying S34 vs Q1571, these two alterations produce a generally similar disease phenotype and likely rely on a common transformation mechanism. This conclusion suggests that missplicing events might converge in pathways relevant to MDS pathogenesis, which may be much more widespread than what is believed. While defining a topology of AS events (ASE), our analysis of the global landscape of RNA splicing in myeloid neoplasia (MN)2 also opened the possibility to search for subsets of patients sharing RNA splicing features and gene expression (GE). We now curated RNA-Seq data from diagnostic samples of 1258 MN patients by comparing RNA splicing profiles of clonal primary cells with S34 (n=19) and Q157 (n=26) (VAFs >10%, both) in search for overlapping ASE representing new cooperative mechanisms germane to leukemogenesis. ASE were filtered based on percent spliced in (PSI) (variation threshold > 0.01). Limma analysis for differential GE and PSI was applied. All data were analyzed on the genome assembly GRCh37/hg19. Patients without SFMT plus a cohort of 63 age-matched controls were used to detect exclusive ASE common in U2AF1 S34 and Q157. Pathway analysis showed an enrichment of chromatin remodeling, histone demethylases and signal transduction genes. A total of 17564 ASE were called with 30% (n=5106) of them found to be significant (qvalues ≤ .05 and inclusion level difference ≥ 5%). In-depth analysis of ASE showed that 70% of significant PSI changes resulted from exon skipping (ES) followed by intron retention (17%) and alternative 3′ and 5′ splice site events (14%). No significant mutually exclusive ASE was found. Considering that ES might result in truncated proteins and consequent lack of functional domains, we mapped ASE to protein domains. Top PSI changes which were common between S34 and Q157 were found in chromatin remodeling (EZH2,JMJD1C, ARID2/4B, CREBBP), O-linked β-N-acetylglucosamine transferase (OGT), a partner of TET2, GTPase activating (GNAS, ARHGAP9, HBS1L), RNA processing/ binding (RBM5, DDX21, DHX38,HNRNPM, PRPF3), unfolded protein response (XBP1), and ubiquitination (CUL3, NAE1, UBQLN1) genes. Lesions affecting chromosome 7 either structurally (cytogenetic alterations) or by mutations and CNV (EZH2 among others) are associated with adverse prognosis in MN. In total, 8% of the patients had deletion 7 and 4% carried EZH2MT. EZH2 mRNA expression levels were significantly lower in U2AF1MTvs controls (LFC=-0.88; P= 1e-11). Of note is that only 6 cases carried EZH2MT, of whom none harbored deletion 7. This suggests that EZH2 low expression levels might be due to other mechanisms. Looking at most relevant findings, we identified a significant and single ES (ex12) in EZH2 flanking the pre-SET domain (cysteine rich, CXC: aa503-605) crucial to maintain histone methyltransferase activity. Beside somatic mutations, EZH2 function can be altered by other mechanisms, e.g., cooperative mutations. The presence of an ES possibly suggests that an alternative mechanism of gene inactivation beside mutations in the CXC/SET domain and chromosome loss might play a role. Similarly, a CREBBP ES (ex11) was detected upstream the kinase inducible interacting (KIX) domain, a docking site for transcriptional activators (e.g., MLL, c-Myb sites). Finally, U2AF1MT have been also associated with deletion 20q or with a hyperactive long GNAS isoform. We found indeed an ES in GNAS and other GTPases, further emphasizing the broad dysfunctions in U2AF1 cooperative genes. In conclusion, our study describes convergent splicing features common to both U2AF1 S34 and Q157 to conceptualize the essential mechanisms contributing to clonal expansion due to lesions in U2AF1.

In silico analysis of alternative splicing events implicated in intracellular trafficking during B-lymphocyte differentiation.

There are multiple regulatory layers that control intracellular trafficking and protein secretion, ranging from transcriptional to posttranslational mechanisms. Finely regulated trafficking and secretion is especially important for lymphocytes during activation and differentiation, as the quantity of secretory cargo increases once the activated cells start to produce and secrete large amounts of cytokines, cytotoxins, or antibodies. However, how the secretory machinery dynamically adapts its efficiency and specificity in general and specifically in lymphocytes remains incompletely understood. Here we present a systematic bioinformatics analysis to address RNA-based mechanisms that control intracellular trafficking and protein secretion during B-lymphocyte activation, and differentiation, with a focus on alternative splicing. Our in silico analyses suggest that alternative splicing has a substantial impact on the dynamic adaptation of intracellular traffic and protein secretion in different B cell subtypes, pointing to another regulatory layer to the control of lymphocyte function during activation and differentiation. Furthermore, we suggest that NERF/ELF2 controls the expression of some COPII-related genes in a cell type-specific manner. In addition, T cells and B cells appear to use different adaptive strategies to adjust their secretory machineries during the generation of effector and memory cells, with antibody secreting B cell specifically increasing the expression of components of the early secretory pathway. Together, our data provide hypotheses how cell type-specific regulation of the trafficking machinery during immune cell activation and differentiation is controlled that can now be tested in wet lab experiments.

Post-transcriptional regulation of 2-acetyl-1-pyrroline (2-AP) biosynthesis pathway, silicon, and heavy metal transporters in response to Zn in fragrant rice.

Fragrant rice (Oryza sativa L.) has a high economic and nutritional value, and the application of micronutrients regulates 2-acetyl-1-pyrroline (2-AP) production, which is responsible for aroma in fragrant rice. Alternative splicing (AS) is an important post-transcriptional regulatory mechanism to generate transcript variability and proteome diversity in plants. However, no systematic investigation of AS events in response to micronutrients (Zn) has been performed in fragrant rice. Furthermore, the post-transcriptional regulation of genes involved in 2-AP biosynthesis is also not known. In this study, a comprehensive analysis of AS events under two gradients of Zn treatment in two different fragrant rice cultivars (Meixiangzhan-2 and Xiangyaxiangzhan) was performed based on RNA-seq analysis. A total of 386 and 598 significant AS events were found in Meixiangzhan-2 treated with low and high doses of Zn, respectively. In Xiangyaxiangzhan, a total of 449 and 598 significant AS events were found in low and high doses of Zn, respectively. Go analysis indicated that these genes were highly enriched in physiological processes, metabolism, and cellular processes in both cultivars. However, genotype and dose-dependent AS events were also detected in both cultivars. By comparing differential AS (DAS) events with differentially expressed genes (DEGs), we found a weak overlap among DAS and DEGs in both fragrant rice cultivars indicating that only a few genes are post-transcriptionally regulated in response to Zn treatment. We further report that Zn differentially regulates the expression of 2-AP biosynthesis-related genes in both cultivars and Zn treatment altered the editing frequency of single nucleotide polymorphism (SNPs) in the genes involved in 2-AP biosynthesis. Finally, we showed that epigenetic modifications associated with active gene transcription are generally enriched over 2-AP biosynthesis-related genes. Similar to the 2-AP pathway, we found that heavy metal transporters (genes related to silicon, iron, Zn and other metal transport) are also regulated at transcriptional and post-transcriptional levels in response to Zn in fragrant rice. Taken together, our results provide evidence of the post-transcriptional gene regulation in fragrant rice in response to Zn treatment and highlight that the 2-AP biosynthesis pathway and heavy metal transporters may also be regulated through epigenetic modifications. These findings will serve as a cornerstone for further investigation to understand the molecular mechanisms of 2-AP biosynthesis and regulation of heavy metal transporters in fragrant rice.

A Regulatory Axis between Epithelial Splicing Regulatory Proteins and Estrogen Receptor α Modulates the Alternative Transcriptome of Luminal Breast Cancer

Epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the splicing pattern during epithelial to mesenchymal transition (EMT) in a physiological context and in cancer, including breast cancer (BC). Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs of patients with poor prognosis and correlates with estrogen receptor α (ERα) levels. Analysis of ERα genome-binding profiles in cell lines and primary breast tumors showed its binding in the proximity of ESRP1 and ESRP2 genes, whose expression is strongly decreased by ERα silencing in hormone-deprived conditions. The combined knock-down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs correlated with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein–protein interactions altered by ESRP1/2 silencing. The comparison of ERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected in primary BCs and endocrine-resistant cell lines. Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.

Tissue-Specific Analysis of Alternative Splicing Events and Differential Isoform Expression in Large Yellow Croaker (Larimichthys crocea) After Cryptocaryon irritans Infection.

The large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. Recently, cryptocaryonosis caused by Cryptocryon irritans infection has brought huge economic losses and threatens the healthy and sustainable development of the L. crocea industry. However, the molecular mechanism and regulation process for L. crocea resistance to C. irritans infection has not been fully researched. Alternative splicing (AS) is an important post-transcriptional regulatory mechanism that allows cells to produce transcriptional and proteomic diversity. The results of AS are tissue dependent, and the expression of tissue-specific transcription subtype genes is determined by AS and transcriptional regulation. However, studies on the tissue specificity of AS events in L. crocea following infection with C. irritans have not been performed. In this study, the L. crocea were artificially infected with C. irritans; their skin and gill were collected at 0h, 24h, 48h, 72h, and 96h post infection. After sequencing and differential expression analysis, a set of 452, 692, 934, 711, 534, and 297 differential alternative splicing(DAS) eventswere identified in 0h, 12h, 24h, 48h, 72h, and 96h post infection respectively. Furthermore, 4160differentially expressed isoforms (DEIs) and 4209 DEI genes were identified from all time point groups. GO enrichment and pathway analysis indicated that many genes of DAS and DEIs were rich in immune-related GO terms and KEGG pathways, such as the Toll and Imd signaling pathway, NOD-like receptor signaling pathway, TNF signaling pathway, and TNF signaling pathway. Among hub DEI genes, alternative splicing-related genes (cwc25, prpf8, and sf3a3), skin function-related gene (fa2h), and oxygen deprivation-related gene (hyo1) were found in DEI genes. This study provided insight into the temporal change of DAS and DEIs between skin and gill of L. crocea against C. irritans infection and revealed that these differences mightplay immune-related roles in the infection process.

Identification and Validation of Novel Immune-Related Alternative Splicing Signatures as a Prognostic Model for Colon Cancer.

BackgroundIndividual immune-related alternative splicing (AS) events have been found to be significant in immune regulation and cancer prognosis. However, a comprehensive analysis of AS events in cancer cells based on immune-related genes (IRGs) has not been performed, and its clinical value is unknown.MethodsColon cancer cases with AS data were obtained from TCGA, and then, we identified overall survival-related AS events (OS-ASEs) based on IRGs by univariate analyses. Using Lasso regression, multivariate Cox regression, Kaplan–Meier analysis and nomograms, we constructed an AS risk model based on the calculated risk score. Furthermore, associations of the risk score with clinical and immune features were confirmed through the Wilcoxon rank sum test, association analysis, etc. Finally, by qRT–PCR, cell coculture and CCK-8 analyses, we validated the significance of OS-ASEs in colon cancer cell lines and clinical samples.ResultsA total of 3,119 immune-related AS events and 183 OS-ASEs were identified, and 9 OS-ASEs were ultimately used to construct a comprehensive risk model for colon cancer patients. Low-risk patients had better OS and DFS rates than high risk patients. Furthermore, a high risk score corresponded to high numbers of multiple tumour-infiltrating immune cells and high expression of HLA-D region genes and immune checkpoint genes. Notably, we identified for the first time that anti-PD-L1 or anti-CTLA-4 antibodies may decrease the OS of specific colon cancer patients in the low-risk group. Additionally, the in vitro experiment validated that CD46-9652-ES and PSMC5-43011-ES are positively correlated with the infiltration of immune cells and promote the growth of colon cancer cells. CD46-9652-ES can contribute to T cell-mediated tumour cell killing. PSMC5-43011-ES was observed to induce M2 polarization of macrophages.ConclusionsThis study identified and validated immune-related prognostic AS signatures that can be used as a novel AS prognostic model and provide a novel understanding of the relationship between the immune microenvironment and clinical outcomes.

Analysis of Whole-Transcriptome RNA-Seq Data Reveals the Involvement of Alternative Splicing in the Drought Response of Glycyrrhiza uralensis.

Alternative splicing (AS) is a post-transcriptional regulatory mechanism that increases protein diversity. There is growing evidence that AS plays an important role in regulating plant stress responses. However, the mechanism by which AS coordinates with transcriptional regulation to regulate the drought response in Glycyrrhiza uralensis remains unclear. In this study, we performed a genome-wide analysis of AS events in G. uralensis at different time points under drought stress using a high-throughput RNA sequencing approach. We detected 2,479 and 2,764 AS events in the aerial parts (AP) and underground parts (UP), respectively, of drought-stressed G. uralensis. Of these, last exon AS and exon skipping were the main types of AS. Overall, 2,653 genes undergoing significant AS regulation were identified from the AP and UP of G. uralensis exposed to drought for 2, 6, 12, and 24 h. Gene Ontology analyses indicated that AS plays an important role in the regulation of nitrogen and protein metabolism in the drought response of G. uralensis. Notably, the spliceosomal pathway and basal transcription factor pathway were significantly enriched with differentially spliced genes under drought stress. Genes related to splicing regulators in the AP and UP of G. uralensis responded to drought stress and underwent AS under drought conditions. In summary, our data suggest that drought-responsive AS directly and indirectly regulates the drought response of G. uralensis. Further in-depth studies on the functions and mechanisms of AS during abiotic stresses will provide new strategies for improving plant stress resistance.

Genome-Wide Analysis for the Regulation of Gene Alternative Splicing by DNA Methylation Level in Glioma and its Prognostic Implications

Glioma is a primary high malignant intracranial tumor with poorly understood molecular mechanisms. Previous studies found that both DNA methylation modification and gene alternative splicing (AS) play a key role in tumorigenesis of glioma, and there is an obvious regulatory relationship between them. However, to date, no comprehensive study has been performed to analyze the influence of DNA methylation level on gene AS in glioma on a genome-wide scale. Here, we performed this study by integrating DNA methylation, gene expression, AS, disease risk methylation at position, and clinical data from 537 low-grade glioma (LGG) and glioblastoma (GBM) individuals. We first conducted a differential analysis of AS events and DNA methylation positions between LGG and GBM subjects, respectively. Then, we evaluated the influence of differential methylation positions on differential AS events. Further, Fisher’s exact test was used to verify our findings and identify potential key genes in glioma. Finally, we performed a series of analyses to investigate influence of these genes on the clinical prognosis of glioma. In total, we identified 130 glioma-related genes whose AS significantly affected by DNA methylation level. Eleven of them play an important role in glioma prognosis. In short, these results will help to better understand the pathogenesis of glioma.

ASpediaFI: Functional Interaction Analysis of Alternative Splicing Events

Alternative splicing (AS) regulates biological processes governing phenotypes and diseases. Differential AS (DAS) gene test methods have been developed to investigate important exonic expression from high-throughput datasets. However, the DAS events extracted using statistical tests are insufficient to delineate relevant biological processes. In this study, we developed a novel application, Alternative Splicing Encyclopedia: Functional Interaction (ASpediaFI), to systemically identify DAS events and co-regulated genes and pathways. ASpediaFI establishes a heterogeneous interaction network of genes and their feature nodes (i.e., AS events and pathways) connected by co-expression or pathway gene set knowledge. Next, ASpediaFI explores the interaction network using the random walk with restart algorithm and interrogates the proximity from a query gene set. Finally, ASpediaFI extracts significant AS events, genes, and pathways. To evaluate the performance of our method, we simulated RNA sequencing (RNA-seq) datasets to consider various conditions of sequencing depth and sample size. The performance was compared with that of other methods. Additionally, we analyzed three public datasets of cancer patients or cell lines to evaluate how well ASpediaFI detects biologically relevant candidates. ASpediaFI exhibits strong performance in both simulated and public datasets. Our integrative approach reveals that DAS events that recognize a global co-expression network and relevant pathways determine the functional importance of spliced genes in the subnetwork. ASpediaFI is publicly available at https://bioconductor.org/packages/ASpediaFI.

RNA-seq reveal RNA binding protein GNL3 as a key mediator in the development of psoriasis vulgaris by regulating the IL23/IL17 axis

BackgroundPsoriasis is a systemic chronic inflammatory skin disorder that was prone to recurrence. The RNA binding protein GNL3 has an important function in maintaining the proliferative ability of stem cells, and its overexpression leads to apoptosis. GNL3 is expressed in the epidermis, however, its regulatory mechanism in psoriasis vulgaris is still poorly understood. ObjectiveTo identify the role of GNL3 in the pathogenesis of psoriasis vulgaris. Materials and methodsRNA-seq was performed to obtain the data of genes' expression and splicing events in Hela cells after shGNL3 and shCtrl was transferred. High quality results of differentially expressed genes (DEGs) and alternative splicing events (ASEs) were further attained by quality control and analysis. Through the functional enrichment analysis of DEGs and ASEs, the regulating effect of GNL3 was discussed, and the hypothesis was further confirmed in HaCat cells and psoriasis lesions. ResultsThe mRNA expression of IL23A in Hela cells was upregulated in GNL3 knockdown, and the ratio of ASE occurred in TNFAIP3 was increased. However, in HaCaT cells, the mRNA expression level of IL23A was downregulated in GNL3 knockdown, and the ratio of ASE of TNFAIP3 was decreased. Additionally, the results obtained in HaCaT cells was further validated in the lesional psoriatic skin. ConclusionGNL3 takes an important part in the development of psoriasis vulgaris by regulating the IL23/IL17 axis, which may serve as the basis of effective targeted treatment in future.

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.