scholarly journals The landscape of the alternatively spliced transcriptome remains stable during aging across different species and tissues

2019 ◽  
Author(s):  
Patricia Sieber ◽  
Emanuel Barth ◽  
Manja Marz

ABSTRACTAging is characterized by a decline of cellular homeostasis over time, leading to various severe disorders and death. Alternative splicing is an important regulatory level of gene expression and thus takes on a key role in the maintenance of accurate cell and tissue function. Missplicing of certain genes has already been linked to several age-associated diseases, such as progeria, Alzheimer’s disease, Parkinson’s disease and cancer. Nevertheless, many studies focus only on transcriptional variations of single genes or the expression changes of spliceosomal genes, coding for the proteins that aggregate to the spliceosomal machinery. Little is known on the general change of present and switching isoforms in different tissues over time. In this descriptive RNA-Seq study, we report differences and commonalities of isoform usage during aging among different tissues within one species and compare changes of alternative splicing among different, evolutionarily distinct species. Although we identified a multitude of differntially spliced genes among different time points, we observed little to no general changes in the transcriptomic landscape of the investigated samples. Although there is undoubtedly considerable influence of specifically spliced genes on certain age-associated processes, this work shows that alternative splicing remains stable for the majority of genes with aging.

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yanlei Yue ◽  
Ze Jiang ◽  
Enoch Sapey ◽  
Tingting Wu ◽  
Shi Sun ◽  
...  

Abstract Background In soybean, some circadian clock genes have been identified as loci for maturity traits. However, the effects of these genes on soybean circadian rhythmicity and their impacts on maturity are unclear. Results We used two geographically, phenotypically and genetically distinct cultivars, conventional juvenile Zhonghuang 24 (with functional J/GmELF3a, a homolog of the circadian clock indispensable component EARLY FLOWERING 3) and long juvenile Huaxia 3 (with dysfunctional j/Gmelf3a) to dissect the soybean circadian clock with time-series transcriptomal RNA-Seq analysis of unifoliate leaves on a day scale. The results showed that several known circadian clock components, including RVE1, GI, LUX and TOC1, phase differently in soybean than in Arabidopsis, demonstrating that the soybean circadian clock is obviously different from the canonical model in Arabidopsis. In contrast to the observation that ELF3 dysfunction results in clock arrhythmia in Arabidopsis, the circadian clock is conserved in soybean regardless of the functional status of J/GmELF3a. Soybean exhibits a circadian rhythmicity in both gene expression and alternative splicing. Genes can be grouped into six clusters, C1-C6, with different expression profiles. Many more genes are grouped into the night clusters (C4-C6) than in the day cluster (C2), showing that night is essential for gene expression and regulation. Moreover, soybean chromosomes are activated with a circadian rhythmicity, indicating that high-order chromosome structure might impact circadian rhythmicity. Interestingly, night time points were clustered in one group, while day time points were separated into two groups, morning and afternoon, demonstrating that morning and afternoon are representative of different environments for soybean growth and development. However, no genes were consistently differentially expressed over different time-points, indicating that it is necessary to perform a circadian rhythmicity analysis to more thoroughly dissect the function of a gene. Moreover, the analysis of the circadian rhythmicity of the GmFT family showed that GmELF3a might phase- and amplitude-modulate the GmFT family to regulate the juvenility and maturity traits of soybean. Conclusions These results and the resultant RNA-seq data should be helpful in understanding the soybean circadian clock and elucidating the connection between the circadian clock and soybean maturity.


BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Clemens Falker-Gieske ◽  
Andrea Mott ◽  
Sören Franzenburg ◽  
Jens Tetens

Abstract Background Retinol (RO) and its active metabolite retinoic acid (RA) are major regulators of gene expression in vertebrates and influence various processes like organ development, cell differentiation, and immune response. To characterize a general transcriptomic response to RA-exposure in vertebrates, independent of species- and tissue-specific effects, four publicly available RNA-Seq datasets from Homo sapiens, Mus musculus, and Xenopus laevis were analyzed. To increase species and cell-type diversity we generated RNA-seq data with chicken hepatocellular carcinoma (LMH) cells. Additionally, we compared the response of LMH cells to RA and RO at different time points. Results By conducting a transcriptome meta-analysis, we identified three retinoic acid response core clusters (RARCCs) consisting of 27 interacting proteins, seven of which have not been associated with retinoids yet. Comparison of the transcriptional response of LMH cells to RO and RA exposure at different time points led to the identification of non-coding RNAs (ncRNAs) that are only differentially expressed (DE) during the early response. Conclusions We propose that these RARCCs stand on top of a common regulatory RA hierarchy among vertebrates. Based on the protein sets included in these clusters we were able to identify an RA-response cluster, a control center type cluster, and a cluster that directs cell proliferation. Concerning the comparison of the cellular response to RA and RO we conclude that ncRNAs play an underestimated role in retinoid-mediated gene regulation.


2020 ◽  
Author(s):  
Zixiao Zhao ◽  
Christine G. Elsik ◽  
Bruce E. Hibbard ◽  
Kent S. Shelby

AbstractBackgroundAlternative splicing is one of the major mechanisms that increases transcriptome diversity in eukaryotes, including insect species that have gained resistance to pesticides and Bt toxins. In western corn rootworm (Diabrotica virgifera virgifera LeConte), neither alternative splicing nor its role in resistance to Bt toxins has been studied.ResultsTo investigate the mechanisms of Bt resistance we carried out single-molecule real-time (SMRT) transcript sequencing and Iso-seq analysis on resistant, eCry3.1Ab-selected and susceptible, unselected, western corn rootworm neonate midguts which fed on seedling maize with and without eCry3.1Ab for 12 and 24 hours. We present transcriptome-wide alternative splicing patterns of western corn rootworm midgut in response to feeding on eCry3.1Ab-expressing corn using a comprehensive approach that combines both RNA-seq and SMRT transcript sequencing techniques. We found that 67.73% of multi-exon genes are alternatively spliced, which is consistent with the high transposable element content of the genome. One of the alternative splicing events we identified was a novel peritrophic matrix protein with two alternative splicing isoforms. Analysis of differential exon usage between resistant and susceptible colonies showed that in eCry3.1Ab-resistant western corn rootworm, expression of one isoform was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the other isoform.ConclusionOur results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced isoforms of peritrophic matrix protein may be associated with eCry3.1Ab resistance in western corn rootworm.


BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Paulo Rapazote-Flores ◽  
Micha Bayer ◽  
Linda Milne ◽  
Claus-Dieter Mayer ◽  
John Fuller ◽  
...  

Abstract Background The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. Results A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts – BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427–433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20–28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5′ and 3′ UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. Conclusion A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.


2015 ◽  
Vol 28 (3) ◽  
pp. 298-309 ◽  
Author(s):  
Alyssa Burkhardt ◽  
Alex Buchanan ◽  
Jason S. Cumbie ◽  
Elizabeth A. Savory ◽  
Jeff H. Chang ◽  
...  

Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of P. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations were improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome and nearly 55% of the intron-containing genes from P. cubensis had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in P. cubensis, with alternative 5′- and alternative 3′-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations and, through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant-pathogenic oomycetes.


2020 ◽  
Author(s):  
Gergely Csaba ◽  
Evi Berchtold ◽  
Armin Hadziahmetovic ◽  
Markus Gruber ◽  
Constantin Ammar ◽  
...  

ABSTRACTWhile absolute quantification is challenging in high-throughput measurements, changes of features between conditions can often be determined with high precision. Therefore, analysis of fold changes is the standard method, but often, a doubly differential analysis of changes of changes is required. Differential alternative splicing is an example of a doubly differential analysis, i.e. fold changes between conditions for different isoforms of a gene. EmpiRe is a quantitative approach for various kinds of omics data based on fold changes for appropriate features of biological objects. Empirical error distributions for these fold changes are estimated from Replicate measurements and used to quantify feature fold changes and their directions. We assess the performance of EmpiRe to detect differentially expressed genes applied to RNA-Seq using simulated data. It achieved higher precision than established tools at nearly the same recall level. Furthermore, we assess the detection of alternatively Spliced genes via changes of isoform fold changes (EmpiReS) on distribution-free simulations and experimentally validated splicing events. EmpiReS achieves the best precision-recall values for simulations based on different biological datasets. We propose EmpiRe(S) as a general, quantitative and fast approach with high reliability and an excellent trade-off between sensitivity and precision in (doubly) differential analyses.


Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4500-4500
Author(s):  
Mariateresa Fulciniti ◽  
Michael A Lopez ◽  
Anil Aktas Samur ◽  
Eugenio Morelli ◽  
Hervé Avet-Loiseau ◽  
...  

Abstract Gene expression profile has provided interesting insights into the disease biology, helped develop new risk stratification, and identify novel druggable targets in multiple myeloma (MM). However, there is significant impact of alternative pre-mRNA splicing (AS) as one of the key transcriptome modifier. These spliced variants increases the transcriptomic complexity and its misregulation affect disease behavior impacting therapeutic consideration in various disease processes including cancer. Our large well annotated deep RNA sequencing data from purified MM cells data from 420 newly-diagnosed patients treated homogeneously have identified 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). These observed global alternative splicing events in MM involves aberrant splicing of critical growth and survival genes affects the disease biology as well as overall survival. Moreover, the decrease of cell viability observed in a large panel of MM cell lines after inhibition of splicing at the pre-mRNA complex and stalling at the A complex confirmed that MM cells are exquisitely sensitive to pharmacological inhibition of splicing. Based on these data, we further focused on understanding the molecular mechanisms driving aberrant alternative splicing in MM. An increasing body of evidence indicates that altered expression of regulatory splicing factors (SF) can have oncogenic properties by impacting AS of cancer-associated genes. We used our large RNA-seq dataset to create a genome wide global alterations map of SF and identified several splicing factors significantly dysregulated in MM compared to normal plasma cells with impact on clinical outcome. The splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1), regulating initiation of spliceosome assembly, was selected for further evaluation, as its impact on clinical outcome was confirmed in two additional independent myeloma datasets. In gain-of (GOF) studies enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells; and conversely, in loss-of function (LOF) studies, downregulation of SRSF1, using stable or doxy-inducible shRNA systems significantly inhibited MM cell proliferation and survival over time. We utilized SRSF1 mutants to dissect the mechanisms involved in the SRSF1-mediated MM growth induction, and observed that the growth promoting effect of SRSF1 in MM cells was mainly due to its splicing activity. We next investigated the impact of SRSF1 on allelic isoforms of specific gene targets by RNA-seq in LOF and confirmed in GOF studies. Splicing profiles showed widespread changes in AS induced by SRSF1 knock down. The most recurrent splicing events were skipped exon (SE) and alternative first (AF) exon splicing as compared to control cells. SE splice events were primarily upregulated and AF splice events were evenly upregulated and downregulated. Genes in which splicing events in these categories occurred mostly did not show significant difference in overall gene expression level when compared to control, following SRSF1 depletion. When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affects genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells. We have observed increased levels of SRSF phosphorylation, which regulates it's subcellular localization and activity, in MM cell lines and primary patient MM cells compared to normal donor PBMCs. Moreover, we evaluated the chemical compound TG003, an inhibitor of Cdc2-like kinase (CLK) 1 and 4 that regulate splicing by fine-tuning the phosphorylation of SR proteins. Treatment with TG003 decreased SRSF1 phosphorylation preventing the spliceosome assembly and inducing a dose dependent inhibition of MM cell viability. In conclusions, here we provide mechanistic insights into myeloma-related splicing dysregulation and establish SRSF1 as a tumor promoting gene with therapeutic potential. Disclosures Avet-Loiseau: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:OncoPep: Other: Board of director.


2017 ◽  
Author(s):  
Alexander Platzer ◽  
Julia Polzin ◽  
Ping Penny Han ◽  
Klaus Rembart ◽  
Thomas Nussbaumer

AbstractMetagenomics, RNA-seq, WGS (Whole Genome Sequencing) and other types of next-generation sequencing techniques provide quantitative measurements for single strains and genes over time. To obtain a global overview of the experiment and to explore the full potential of a given dataset, intuitive and interactive visualization tools are needed. Therefore, we established BioSankey, which allows to visualize microbial species in microbiome studies and gene expression over time as a Sankey diagram. These diagrams are embedded into a project-specific HTML page, that contains all information as provided during the installation process. BioSankey can be easily applied to analyse bacterial communities in time-series datasets. Furthermore, it can be used to analyse the fluctuations of differentially expressed genes (DEG). The output of BioSankey is a project-specific HTML page, which depends only on JavaScript to enable searches of interesting species or genes of interest without requiring a web server or connection to a database to exchange results among collaboration partners. BioSankey is a tool to visualize different data elements from single and dual RNA-seq datasets as well as from metagenomes studies.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 638-638 ◽  
Author(s):  
Naim Rashid ◽  
Stephane Minvielle ◽  
Florence Magrangeas ◽  
Mehmet Kemal Samur ◽  
Alice Clynen ◽  
...  

Abstract Alternative splicing is an important post-translational change that alters gene function. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. Here we have analyzed alternative splicing in myeloma using high throughput RNA-seq. Our analytic pipeline for RNA-seq data used in this investigation not only provides information on expression levels for genes, but also provides information on the expression of known splice variants of genes (isoforms), and can identify novel exon level events across individuals (i.e. exon skipping events). We conducted a study of 328 newly-diagnosed patients with multiple myeloma treated homogeneously with novel agent combination containting lenalidomide, bortezomib and dexamethsone with or without high-dose melphalan followed by lenalidomide maintenance in the IFM/DFCI study. RNA isolated from purified CD138+ MM cells collected at the time of diagnosis and from 18 normal donor plasma cells were processed by RNA-seq (100 million paired end reads on Illumina HiSeq) and analyzed using a custom computational and statistical pipeline. Following read alignment to hg19, we utilized RSEM to quantify both gene-level and isoform-level expression of known ENSEMBL transcripts. We then implemented a novel testing approach based on compositional regression to discover genes that show significant isoform switching between the 328 MM samples and 18 Normal Plasma Cell (NPC) samples from healthy donors. Using various programs and their modifications, we also identified novel alternative splicing events, such as exon skipping and mutually exclusive exon usage, among others. Patient data for MM characteristics, cytogenetic and FISH as well as clinical survival outcomes were also analyzed and correlated with genomic data. We observed over 600 genes showing significant changes in relative isoform abundances (isoform switching) between MM and normal samples. A number of previously characterized genes including MYCL1 (adj. p = 0.0014) and CCND3 (adj. p = 0.0013), and MAP kinase-related genes (MAP3K8, MAPKAPK2, MAPKAPK3, MAP4K4) exhibited significant isoform switching compared to normal, in addition to some not well characterized genes. Genes showing the greatest magnitude of isoform switching include MEFV (adj. p = 2.7 x 10-5), showing a two fold change in the relative major isoform abundance compared to normal, and has been previously shown to have a role in lymphoid neoplasms. We applied hierarchical clustering to the isoforms showing significant changes in isoform-switching and identified 4 distinct clusters, which are currently being investigated for correlation with clinical subtypes of MM. Exon level analyses of alternative splicing events, such as exon skipping, are currently underway. Clinical data including MM characteristics, cytogenetics, FISH and survival outcomes was available for a subset of 265 patients. We found that 109 genes showed significant isoform switching between t(4;14) and non-t(4;14) patients, such as CD44 (adj. p =1.8 x 10-6) and WHSC1 (adj. p =5.1 x 10-28). Comparing del17p (28 in total) and non del17p patients, we found no significant splicing changes after multiple testing adjustment. Of these genes, only a subset (40%) were shown to be differentially expressed in terms of total gene expression, suggesting the importance of examining alternative splicing events in addition to total gene expression. With respect to treatment response, we compared the expression of gene isoforms between patients achieving complete response (CR) versus others and identified 38 isoforms associated with response to treatment (adj. p value < 0.05), with SEPT9, SLC2A5, and UBX6 having the strongest associations (adj. p-value < 3 x 10-4). Using a univariate cox regression model, 4 spliced isoforms relating to 3 genes were identified as having significant correlation with event-free survival (EFS) (FDR-adjusted cox p value < 0.05). We are in the process of now integrating the gene expression data with altered splicing data to develop an integrated survival model. In summary, this study highlights the significant frequency, biological and clinical importance of alternative splicing in MM and points to the need for evaluation of not only the expression level of genes but also post-translational modifications. The genes identified here are important targets for therapy as well as possible immune modulation. Disclosures Moreau: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees.


2021 ◽  
Vol 12 ◽  
Author(s):  
Ting-Lin Pang ◽  
Zhan Ding ◽  
Shao-Bo Liang ◽  
Liang Li ◽  
Bei Zhang ◽  
...  

Interrupted exons in the pre-mRNA transcripts are ligated together through RNA splicing, which plays a critical role in the regulation of gene expression. Exons with a length ≤ 30 nt are defined as microexons that are unique in identification. However, microexons, especially those shorter than 8 nt, have not been well studied in many organisms due to difficulties in mapping short segments from sequencing reads. Here, we analyzed mRNA-seq data from a variety of Drosophila samples with a newly developed bioinformatic tool, ce-TopHat. In addition to the Flybase annotated, 465 new microexons were identified. Differentially alternatively spliced (AS) microexons were investigated between the Drosophila tissues (head, body, and gonad) and genders. Most of the AS microexons were found in the head and two AS microexons were identified in the sex-determination pathway gene fruitless.


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