Characterization of Alternative Splicing (AS) Events during Chicken (Gallus gallus) Male Germ-Line Stem Cell Differentiation with Single-Cell RNA-seq.
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ABSTRACT:
SUBMITTER: Sun C
PROVIDER: S-EPMC8160964 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Accumulating evidence supports the significance of aberrant alternative splicing (AS) events in cancer; however, genome-wide profiling of progression-free survival (PFS)-related AS events in testicular germ cell tumors (TGCT) has not been reported. Here, we analyzed high-throughput RNA-sequencing data and percent-spliced-in values for 150 patients with TGCT. Using univariate and multivariate Cox regression analysis and a least absolute shrinkage and selection operator method, we identified the top 15 AS events most closely associated with disease progression. A risk-associated AS score (ASS) for the 15 AS events was calculated for each patient. ASS, pathological stage, and T stage were significantly associated with disease progression by univariate analysis, but only ASS and pathological stage remained significant by multivariate analysis. The ability of these variables to predict 5-year progression was assessed using receiver operating characteristic curve analysis. ASS had stronger predictive value than a combination of age, pathological stage, and T stage (area under the curve = 0.899 and 0.715, respectively). Furthermore, Kaplan-Meier analysis of patients with low and high ASS demonstrated that high ASS was associated with significantly worse PFS than low ASS (P = 1.46 × 10-7). We also analyzed the biological functions of the PFS-related AS-related genes and found enrichment in pathways associated with DNA repair and modification. Finally, we identified a regulatory network of splicing factors with expression levels that correlated significantly with AS events in TGCT. Collectively, this study identifies a novel method for risk stratification of patients and provides insight into the molecular events underlying TGCT.
| S-EPMC8152425 | biostudies-literature
Project description:Marek's disease (MD) was an immunosuppression disease induced by Marek's disease virus (MDV). MD caused huge economic loss to the global poultry industry, but it also provided an ideal model for studying diseases induced by the oncogenic virus. Alternative splicing (AS) simultaneously produced different isoform transcripts, which are involved in various diseases and individual development. To investigate AS events in MD, RNA-Seq was performed in tumorous spleens (TS), spleens from the survivors (SS) without any lesion after MDV infection, and non-infected chicken spleens (NS). In this study, 32,703 and 25,217 AS events were identified in TS and SS groups with NS group as the control group, and 1198, 1204, and 348 differently expressed (DE) AS events (p-value < 0.05 and FDR < 0.05) were identified in TS vs. NS, TS vs. SS, SS vs. NS, respectively. Additionally, Function enrichment analysis showed that ubiquitin-mediated proteolysis, p53 signaling pathway, and phosphatidylinositol signaling system were significantly enriched (p-value < 0.05). Small structural variations including SNP and indel were analyzed based on RNA-Seq data, and it showed that the TS group possessed more variants on the splice site region than those in SS and NS groups, which might cause more AS events in the TS group. Combined with previous circRNA data, we found that 287 genes could produce both circular and linear RNAs, which suggested these genes were more active in MD lymphoma transformation. This study has expanded the understanding of the MDV infection process and provided new insights for further analysis of resistance/susceptibility mechanisms.
| S-EPMC8701255 | biostudies-literature
Project description:BACKGROUND: In this paper, we address the problem of identifying and quantifying polymorphisms in RNA-seq data when no reference genome is available, without assembling the full transcripts. Based on the fundamental idea that each polymorphism corresponds to a recognisable pattern in a De Bruijn graph constructed from the RNA-seq reads, we propose a general model for all polymorphisms in such graphs. We then introduce an exact algorithm, called KISSPLICE, to extract alternative splicing events. RESULTS: We show that KISSPLICE enables to identify more correct events than general purpose transcriptome assemblers. Additionally, on a 71 M reads dataset from human brain and liver tissues, KISSPLICE identified 3497 alternative splicing events, out of which 56% are not present in the annotations, which confirms recent estimates showing that the complexity of alternative splicing has been largely underestimated so far. CONCLUSIONS: We propose new models and algorithms for the detection of polymorphism in RNA-seq data. This opens the way to a new kind of studies on large HTS RNA-seq datasets, where the focus is not the global reconstruction of full-length transcripts, but local assembly of polymorphic regions. KISSPLICE is available for download at http://alcovna.genouest.org/kissplice/.
| S-EPMC3358658 | biostudies-literature
Project description:As a member of the large Ran-binding protein family, Ran-binding protein 9 (RANBP9) has been suggested to play a critical role in diverse cellular functions in somatic cell lineages in vitro, and this is further supported by the neonatal lethality phenotype in Ranbp9 global knockout mice. However, the exact molecular actions of RANBP9 remain largely unknown. By inactivation of Ranbp9 specifically in testicular somatic and spermatogenic cells, we discovered that Ranbp9 was dispensable for Sertoli cell development and functions, but critical for male germ cell development and male fertility. RIP-Seq and proteomic analyses revealed that RANBP9 was associated with multiple key splicing factors and directly targeted >2,300 mRNAs in spermatocytes and round spermatids. Many of the RANBP9 target and non-target mRNAs either displayed aberrant splicing patterns or were dysregulated in the absence of Ranbp9. Our data uncovered a novel role of Ranbp9 in regulating alternative splicing in spermatogenic cells, which is critical for normal spermatogenesis and male fertility.
| S-EPMC4256260 | biostudies-literature
Project description:Transposable elements can drive genome evolution, but their enhanced activity is detrimental to the host and therefore must be tightly regulated. The Piwi-interacting small RNA (piRNA) pathway is vital for the regulation of transposable elements, by inducing transcriptional silencing or post-transcriptional decay of mRNAs. Here we show that piRNAs and piRNA biogenesis components regulate precursor mRNA splicing of P-transposable element transcripts in vivo, leading to the production of the non-transposase-encoding mature mRNA isoform in Drosophila germ cells. Unexpectedly, we show that the piRNA pathway components do not act to reduce transcript levels of the P-element transposon during P-M hybrid dysgenesis, a syndrome that affects germline development in Drosophila. Instead, splicing regulation is mechanistically achieved together with piRNA-mediated changes to repressive chromatin states, and relies on the function of the Piwi-piRNA complex proteins Asterix (also known as Gtsf1) and Panoramix (Silencio), as well as Heterochromatin protein 1a (HP1a; encoded by Su(var)205). Furthermore, we show that this machinery, together with the piRNA Flamenco cluster, not only controls the accumulation of Gypsy retrotransposon transcripts but also regulates the splicing of Gypsy mRNAs in cultured ovarian somatic cells, a process required for the production of infectious particles that can lead to heritable transposition events. Our findings identify splicing regulation as a new role and essential function for the Piwi pathway in protecting the genome against transposon mobility, and provide a model system for studying the role of chromatin structure in modulating alternative splicing during development.
| S-EPMC5933846 | biostudies-literature
Project description:BACKGROUND:While the reconstruction of transcripts from a sample of RNA-Seq data is a computationally expensive and complicated task, the detection of splicing events from RNA-Seq data and a gene annotation is computationally feasible. This latter task, which is adequate for many transcriptome analyses, is usually achieved by aligning the reads to a reference genome, followed by comparing the alignments with a gene annotation, often implicitly represented by a graph: the splicing graph. RESULTS:We present ASGAL (Alternative Splicing Graph ALigner): a tool for mapping RNA-Seq data to the splicing graph, with the specific goal of detecting novel splicing events, involving either annotated or unannotated splice sites. ASGAL takes as input the annotated transcripts of a gene and a RNA-Seq sample, and computes (1) the spliced alignments of each read in input, and (2) a list of novel events with respect to the gene annotation. CONCLUSIONS:An experimental analysis shows that ASGAL allows to enrich the annotation with novel alternative splicing events even when genes in an experiment express at most one isoform. Compared with other tools which use the spliced alignment of reads against a reference genome for differential analysis, ASGAL better predicts events that use splice sites which are novel with respect to a splicing graph, showing a higher accuracy. To the best of our knowledge, ASGAL is the first tool that detects novel alternative splicing events by directly aligning reads to a splicing graph. AVAILABILITY:Source code, documentation, and data are available for download at http://asgal.algolab.eu .
| S-EPMC6247705 | biostudies-literature
Project description:SYMPK is a scaffold protein that supports polyadenylation machinery assembly on nascent transcripts and is also involved in alternative splicing in some mammalian somatic cells. However, the role of SYMPK in germ cells remains unknown. Here, we report that SYMPK is highly expressed in male germ cells, and germ cell-specific knockout (cKO) of Sympk in mouse leads to male infertility. Sympk cKODdx4-cre mice showed reduced spermatogonia at P4 and almost no germ cells at P18. Sympk cKOStra8-Cre spermatocytes exhibit defects in homologous chromosome synapsis, DNA double-strand break (DSB) repair, and meiotic recombination. RNA-Seq analyses reveal that SYMPK is associated with alternative splicing, besides regulating the expressions of many genes in spermatogenic cells. Importantly, Sympk deletion results in abnormal alternative splicing and a decreased expression of Sun1. Taken together, our results demonstrate that SYMPK is pivotal for meiotic progression by regulating pre-mRNA alternative splicing in male germ cells.
| S-EPMC8380814 | biostudies-literature
Project description:RNA-Seq is an efficient way to comprehensively identify single nucleotide polymorphisms (SNPs) and alternative splicing (AS) events from the expressed genes. In this study, we conducted transcriptome sequencing of four Asian lotus (Nelumbo nucifera) cultivars using Illumina HiSeq2000 platform to identify SNPs and AS events in lotus. A total of 505 million pair-end RNA-Seq reads were generated from four cultivars, of which 86% were mapped to the lotus reference genome. Using the four sets of data together, a total of 357,689 putative SNPs were identified with an average density of one SNP per 2.2 kb. These SNPs were located in 1,253 scaffolds and 15,016 expressed genes. A/G and C/T were the two major types of SNPs in the Asian lotus transcriptome. In parallel, a total of 177,540 AS events were detected in the four cultivars and were distributed in 64% of the expressed genes of lotus. The predominant type of AS events was alternative 5' first exon, which accounted for 41.2% of all the observed AS events, and exon skipping only accounted for 4.3% of all AS. Gene Ontology analysis was conducted to analyze the function of the genes containing SNPs and AS events. Validation of selected SNPs and AS events revealed that 74% of SNPs and 80% of AS events were reliable, which indicates that RNA-Seq is an efficient approach to uncover gene-associated SNPs and AS events. A large number of SNPs and AS events identified in our study will facilitate further genetic and functional genomics research in lotus.
| S-EPMC4416007 | biostudies-literature
Project description:(1) Background: A simplistic understanding of the central dogma falls short in correlating the number of genes in the genome to the number of proteins in the proteome. Post-transcriptional alternative splicing contributes to the complexity of the proteome and is critical in understanding gene expression. mRNA-sequencing (RNA-seq) has been widely used to study the transcriptome and provides opportunity to detect alternative splicing events among different biological conditions. Despite the popularity of studying transcriptome variants with RNA-seq, few efficient and user-friendly bioinformatics tools have been developed for the genome-wide detection and visualization of alternative splicing events. (2) Results: We propose AS-Quant, (Alternative Splicing Quantitation), a robust program to identify alternative splicing events from RNA-seq data. We then extended AS-Quant to visualize the splicing events with short-read coverage plots along with complete gene annotation. The tool works in three major steps: (i) calculate the read coverage of the potential spliced exons and the corresponding gene; (ii) categorize the events into five different categories according to the annotation, and assess the significance of the events between two biological conditions; (iii) generate the short reads coverage plot for user specified splicing events. Our extensive experiments on simulated and real datasets demonstrate that AS-Quant outperforms the other three widely used baselines, SUPPA2, rMATS, and diffSplice for detecting alternative splicing events. Moreover, the significant alternative splicing events identified by AS-Quant between two biological contexts were validated by RT-PCR experiment. (3) Availability: AS-Quant is implemented in Python 3.0. Source code and a comprehensive user's manual are freely available online.
| S-EPMC8123109 | biostudies-literature
Project description:MotivationUnderstanding the occurrence and regulation of alternative splicing (AS) is a key task towards explaining the regulatory processes that shape the complex transcriptomes of higher eukaryotes. With the advent of high-throughput sequencing of RNA (RNA-Seq), the diversity of AS transcripts could be measured at an unprecedented depth. Although the catalog of known AS events has grown ever since, novel transcripts are commonly observed when working with less well annotated organisms, in the context of disease, or within large populations. Whereas an identification of complete transcripts is technically challenging and computationally expensive, focusing on single splicing events as a proxy for transcriptome characteristics is fruitful and sufficient for a wide range of analyses.ResultsWe present SplAdder, an alternative splicing toolbox, that takes RNA-Seq alignments and an annotation file as input to (i) augment the annotation based on RNA-Seq evidence, (ii) identify alternative splicing events present in the augmented annotation graph, (iii) quantify and confirm these events based on the RNA-Seq data and (iv) test for significant quantitative differences between samples. Thereby, our main focus lies on performance, accuracy and usability.AvailabilitySource code and documentation are available for download at http://github.com/ratschlab/spladder Example data, introductory information and a small tutorial are accessible via http://bioweb.me/spladderContacts: andre.kahles@ratschlab.org or gunnar.ratsch@ratschlab.orgSupplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC4908322 | biostudies-literature