scholarly journals Alternative Splicing Is a Frequent Event and Impacts Clinical Outcome in Myeloma: A Large RNA-Seq Data Analysis of Newly-Diagnosed Myeloma Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 638-638 ◽  
Author(s):  
Naim Rashid ◽  
Stephane Minvielle ◽  
Florence Magrangeas ◽  
Mehmet Kemal Samur ◽  
Alice Clynen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Exon Skipping ◽  
P Value ◽  
Newly Diagnosed ◽  
Rna Seq ◽  
Exon Level ◽  
Total Gene Expression ◽  
Isoform Switching ◽  
Alternative Splicing Events

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.

scholarly journals Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Anne-Laure Bougé ◽  
Eva Murauer ◽  
Emmanuelle Beyne ◽  
Julie Miro ◽  
Jessica Varilh ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Alternative Splicing ◽  
Cdna Sequence ◽  
454 Sequencing ◽  
Exon Skipping ◽  
Rna Seq ◽  
Splicing Pattern ◽  
Alternative Splicing Events ◽  
Diagnostic Research ◽  
Dmd Gene

Abstract We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3′ splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.

A Dynamic Alternative Splicing Program Regulates Gene Expression In A Differentiation Stage-Specific Manner During Terminal Erythropoiesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3413-3413 ◽  
Author(s):  
Harold Pimentel ◽  
Marilyn Parra ◽  
Jie Li ◽  
Sherry Gee ◽  
Dana Ghanem ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Structure And Function ◽  
Rna Seq ◽  
Arginine Residues ◽  
Alternative Splicing Events ◽  
And Function

Abstract Spatio-temporal regulation of switches in alternative pre-mRNA splicing modulate exon usage, critically remodeling the transcriptome during development and differentiation of many tissues, while aberrant regulation of alternative splicing disrupts these processes and plays a role in numerous human diseases. Recently, the discovery of splicing factor mutations in myelodysplasia has increased interest in splicing regulation in hematology. Previously, a functionally critical erythroid splicing switch in protein 4.1 pre-mRNA has been reported, in which activation of alternative exon 16 splicing in late erythroblasts is required for assembly of a mechanically stable red cell membrane. To explore globally the landscape of important alternative splicing events in the erythroid lineage, we applied RNA-seq analysis to five highly FACS-purified populations of human erythroblasts, cultured from CD34+ cord blood progenitors, representing proerythroblasts, early and late basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. Alternative splicing events predicted by computational analysis were filtered to remove low expression genes and low frequency splicing events, to derive a list of >3000 ‘major’ alternative splicing events of potential importance in erythroid biology. Many of these were validated by inspection of RNA-seq reads mapped on the human genome, and/or by RT-PCR analysis. In this unique differentiation system we found an extensive and dynamic alternative splicing program enriched in genes that function in cell cycle regulation, organelle organization, chromatin structure and function, and RNA processing. For example, we identified alternative splicing events in ∼25 genes encoding chromatin modifying enzymes that methylate, demethylate, or acetylate specific lysine or arginine residues in histones; in transcription modulators such as ATRX and BCL11A that regulate normal globin gene expression; and in ∼50 RNA binding proteins with various roles in post-transcriptional gene regulation. Comparison of PSI (percent spliced in) values across the differentiation series revealed that dozens of alternative exons exhibit substantial switches in splicing efficiency during terminal erythropoiesis. The majority of splicing switches occur in late-stage polychromatophilic and orthochromatophilic erythroblasts, temporally correlated with changes in transcript abundance for many splicing factors and with substantial cell remodeling prior to enucleation. One of the biggest switches in late erythroblasts involves inclusion of a 35nt exon in the NDEL1 (nuclear distribution factor E-homolog-like1) gene, which alters C-terminal structure of a protein that functions in nuclear migration and nucleokinesis in nonerythroid cells and may have a role in erythroblast enucleation. Most of the regulated splicing events insert or delete sequences predicted to modulate protein structure and function in late erythroblasts. However, a subset of altered splicing events have a different effect on gene expression by introducing premature translation termination codons (PTCs), leading us to hypothesize that alternative splicing-coupled nonsense-mediated-decay (AS-NMD) contributes to stage-specific down-regulation of numerous erythroid transcripts. Consistent with such a model, most genes that up-regulate PTC exons in late erythroblasts exhibit reduction in overall expression levels, and inhibition of NMD increases the apparent expression of PTC isoforms. In contrast, genes that up-regulate coding exons are not preferentially down-regulated in late erythroblasts. We conclude that a dynamically regulated alternative splicing program in terminally differentiating erythroblasts plays a major post-transcriptional role in shaping gene expression as the cells transition from proliferation to differentiation, ensuring synthesis of the appropriate constellation of proteins as the cells prepare for enucleation and production of mature red cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of kinase gene expression and splicing profile in prostate cancer with RNA-Seq data

2016 ◽  
Author(s):  
Huijuan Feng ◽  
Tingting Li ◽  
Xuegong Zhang
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Alternative Splicing ◽  
Differential Expression ◽  
Functional Enrichment ◽  
Cancer Development ◽  
Rna Seq ◽  
Differential Splicing ◽  
Kinase Gene ◽  
Isoform Switching

AbstractBackgroundAlternative splicing is a ubiquitous post-transcriptional process in most eukaryotic genes. Aberrant splicing isoforms and abnormal isoform ratios can contribute to cancer development. Kinase genes are key regulators of various cellular processes. Many kinases are found to be oncogenic and have been intensively investigated in the study of cancer and drugs. RNA-Seq provides a powerful technology for genome-wide study of alternative splicing in cancer besides the conventional gene expression profiling. But this potential has not been fully demonstrated yet.MethodsHere we characterized the transcriptome profile of prostate cancer using RNA-Seq data from viewpoints of both differential expression and differential splicing, with an emphasis on kinase genes and their splicing variations. We built up a pipeline to conduct differential expression and differential splicing analysis. Further functional enrichment analysis was performed to explore functional interpretation of the genes. With focus on kinase genes, we performed kinase domain analysis to identify the functionally important candidate kinase gene in prostate cancer. We further calculated the expression level of isoforms to explore the function of isoform switching of kinase genes in prostate cancer.ResultsWe identified distinct gene groups from differential expression and splicing analysis, which suggested that alternative splicing adds another level to gene expression regulation. Enriched GO terms of differentially expressed and spliced kinase genes were found to play different roles in regulation of cellular metabolism. Function analysis on differentially spliced kinase genes showed that differentially spliced exons of these genes are significantly enriched in protein kinase domains. Among them, we found that gene CDK5 has isoform switching between prostate cancer and benign tissues, which may affect cancer development by changing androgen receptor (AR) phosphorylation. The observation was validated in another RNA-Seq dataset of prostate cancer cell lines.ConclusionsOur work characterized the expression and splicing profile of kinase genes in prostate cancer and proposed a hypothetical model on isoform switching of CDK5 and AR phosphorylation in prostate cancer. These findings bring new understanding to the role of alternatively spliced kinases in prostate cancer and demonstrate the use of RNA-Seq data in studying alternative splicing in cancer.

scholarly journals Hotspot Mutations in SF3B1 Result in Increased Alternative Splicing in Multiple Myeloma and Activation of Key Cellular Pathways

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4454-4454
Author(s):  
Michael A Bauer ◽  
Cody Ashby ◽  
Christopher Wardell ◽  
Maria Ortiz ◽  
Erin Flynt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
P Value ◽  
Rna Seq ◽  
Splice Sites ◽  
Differential Splicing ◽  
Employment Equity ◽  
Differential Gene ◽  
Equity Ownership ◽  
Hotspot Mutations

Abstract Introduction: Mutations in the components of the spliceosome have been shown to occur at relatively high frequency in many cancers such as chronic lymphocytic leukemia, myelodysplastic syndromes and breast cancer. One component in particular, encoded by SF3B1, has hotspot missense mutations that result in a significant increase in alternatively spliced transcripts. RNA splicing in Multiple Myeloma (MM) has not been investigated and in particular the extent of mutations in SF3B1 and its effects on the transcriptome. Methods: Using the MMRF CoMMpass dataset (N=1273) of newly diagnosed MM patients, samples with whole exome sequencing (WES) were analyzed for mutations using Strelka and Mutect, and samples with SF3B1 mutations identified. A range of approaches were used to explore the effect of the SF3B1 mutations on the transcriptome and to determine possible downstream effects. Using RNA-seq with matched WES samples (n=615), the splice junction usage of SF3B1 mutants was compared against non-mutated samples which were matched for key MM molecular sub-types. The RNA-seq data was analyzed using a pipeline that included STAR and Salmon, aligning to human reference genome hg38, gene and transcript differential expression analysis tools DESeq2 and StringTie/Ballgown, differential splicing exon usage tools JunctionSeq/QoRTs, DEXSeq, and SUPPA and for Gene Set Enrichment Analysis (GSEA) the R package FGSEA was used. Results: From the WES data 1.7% (22/1273) of samples had mutations in SF3B1 of which 5 had mutations in the hotpot codons of K666 and K700. Differential isoform analysis of the 22 SF3B1 mutant samples compared to non-mutated samples did not identify any transcripts. However, when the analysis was restricted to the 5 samples with hotspot mutations differential gene expression identified 146 genes that were significantly differentially expressed at an adjusted p-value <0.05. Additionally, many genes that did not show an overall gene expression change between the control and the SF3B1 hotspot mutants did at transcriptional level where we observed isoform switching which included the protein coding genes BCL2L1, SNUR, ACKR3 and CRLF2. Results of differential gene analysis between the control and SF3B1 mutants were used in GSEA and significant normalized enrichment scores (NES) identifying increased protein secretion (p-value =0.009, NES= 1.9) and unfolded protein response (UPR) (p-value = 0.02, NES = 1.52) pathways. Conversely GSEA identified decreased apoptosis (p-value = 0.008, NES = -1.76), KRAS signaling (p-value = 0.008, NES = -1.92), TNFA signaling via NF-κB (p-value = 0.008, NES= 2.12) pathways in SF3B1 mutant samples. Investigation of splicing loci revealed that novel splice loci were significantly more abundant in the SF3B1 mutants versus control samples. Differential splicing analysis detected 474 genes to be significantly differentially spliced and of those 311 were not found to be differentially expressed at the gene level, indicating that alternative splicing is as important alternative mechanism to gene expression differences. 59 novel splice sites were identified, as well as 152 known splice sites and 218 exon significant differential usage with a p-value of < 0.05. The genes with most significant levels of alternative splicing and found by more than one approach were DYNLL1, TMEM14C, CRNDE, BRD4 and BCL2L1, several of which are also seen in other cancers with mutated SF3B1. Conclusions: Hotspot mutations in SF3B1 result in alternative splicing of genes as well as the introduction of novel splice sites. The confirmation that SF3B1 hotspot mutations in MM increases alternative splicing as well as the identification of the genes undergoing alternative splicing may present novel therapeutic targets. Gene expression analysis of these samples identifies key deregulated pathways, perhaps in response to alternative splicing, including the UPR and protein secretion pathways. These analyses indicate that disruption of these pathways are potential avenues of therapeutic intervention in patients with SF3B1 mutations. Disclosures Ortiz: Celgene Corporation: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.

Abundance of Alternative Splicing Events and Differentiation Stage-Specific Changes in Splicing Suggest A Major Role in Regulation of Gene Expression During Late Erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 978-978
Author(s):  
Harold Pimentel ◽  
Jie Li ◽  
Marilyn Parra ◽  
Dana Ghanem ◽  
Sherry Gee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Erythroid Differentiation ◽  
Splicing Factors ◽  
Rna Seq ◽  
Rt Pcr ◽  
Splicing Efficiency ◽  
Alternative Splicing Events ◽  
Differentiation Stage ◽  
Protein 4.1R

Abstract Abstract 978 Alternative pre-mRNA splicing plays a major role in development and differentiation by re-modeling the transcriptome to generate mRNAs that encode the biologically appropriate cell type-specific proteome. Earlier studies employing RT-PCR and exon microarrays demonstrated a small number of splicing changes during erythroid differentiation, one of which (in protein 4.1R) is critical for mechanical stability of the membrane skeleton. Here we report that the landscape of splicing changes executed during terminal erythropoiesis is far more extensive and highly dynamic, ultimately affecting the expression of many more proteins than previously recognized. Highly purified populations of FACS-sorted cells representing erythroblasts at distinct stages during terminal erythroid differentiation from proerythroblasts to orthochromatic erythroblasts of both human and mouse origin were used as the source of RNA for RNA-seq analysis. In total, hundreds of millions of sequence reads were obtained from three biological replicates for four (mouse) or five (human) cell populations, and reads were aligned to the Ensembl-annotated transcriptome using the Bowtie aligner. Transcript-level estimates were obtained using the streaming transcript abundance estimation tool, eXpress, expression of individual exons in “exon-inclusion” isoforms relative to total isoforms was represented as Ψ (psi), or percent spliced in, and statistical significance estimates adjusted for multiple comparisons by the Benjamini-Hochberg method. Thousands of alternative splicing events were predicted in genes with diverse functions in transcription, RNA processing, protein synthesis, membrane receptors, cytoskeletal structure, etc. Initial RT-PCR studies indicate that a high proportion of predicted alternative splicing events can be validated. Comparison of Ψ values across the differentiation series revealed that hundreds of alternative exons in erythroid transcripts exhibit substantial differences in splicing efficiency between proerythroblasts and orthrochromatic erythroblasts (ΔΨ>20%), suggesting that their splicing efficiency is regulated. Both increases and decreases in exon splicing efficiency were observed, indicating that multiple splicing regulatory pathways are active and that both splicing enhancer and splicing silencer factors are involved in the regulation. Interestingly, some of the splicing switches introduce premature translation termination codons, leading us to hypothesize that splicing-coupled nonsense mediated decay may down-regulate expression of a class of erythroid transcripts. To begin exploring mechanisms that regulate the late erythroid alternative splicing program, we used the RNA-seq data to derive differentiation stage-specific expression profiles of known splicing factors. Major changes in the expression profile of many splicing regulators were observed. hnRNPA1 was strongly down-regulated in late erythroblasts, in concert with up-regulation of the protein 4.1R splicing switch it has been shown to inhibit. Because many other exons are up-regulated with similar kinetics, hnRNPA1 may be a general inhibitor of alternative splicing in early erythroblasts. In contrast, RNA-seq data indicate that several other splicing factors including MBNL1, a known splicing factor in muscle and brain whose activity is disturbed in myotonic dystrophy, are substantially up-regulated in late erythropoiesis. We conclude that a highly dynamic alternative splicing program in terminally differentiating erythroblasts, in conjunction with the better studied transcriptional program, plays a major role in regulating gene expression to insure synthesis of the appropriate constellation of proteins both quantitatively and qualitatively as the cells are remodeled in preparation for production of mature red cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Alternative splicing landscapes in Arabidopsis thaliana across tissues and stress conditions highlight major functional differences with animals

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Guiomar Martín ◽  
Yamile Márquez ◽  
Federica Mantica ◽  
Paula Duque ◽  
Manuel Irimia
Keyword(s):  
Gene Expression ◽  
Arabidopsis Thaliana ◽  
Alternative Splicing ◽  
Stress Responses ◽  
Target Genes ◽  
Intron Retention ◽  
Single Gene ◽  
Exon Skipping ◽  
Environmental Response ◽  
Biotic Stresses

Abstract Background Alternative splicing (AS) is a widespread regulatory mechanism in multicellular organisms. Numerous transcriptomic and single-gene studies in plants have investigated AS in response to specific conditions, especially environmental stress, unveiling substantial amounts of intron retention that modulate gene expression. However, a comprehensive study contrasting stress-response and tissue-specific AS patterns and directly comparing them with those of animal models is still missing. Results We generate a massive resource for Arabidopsis thaliana, PastDB, comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets reveals that A. thaliana shows high levels of AS, similar to fruitflies, and that, compared to animals, disproportionately uses AS for stress responses. We identify core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that is tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, are overrepresented across regulated AS sets in A. thaliana, being also largely involved in modulating gene expression through NMD and uORF inclusion. Conclusions Non-intron retention events have likely been functionally underrated in plants. AS constitutes a distinct regulatory layer controlling gene expression upon internal and external stimuli whose target genes and master regulators are hardwired at the genomic level to specifically undergo post-transcriptional regulation. Given the higher relevance of AS in the response to different stresses when compared to animals, this molecular hardwiring is likely required for a proper environmental response in A. thaliana.

scholarly journals Comparative RNA-Seq Analyses of Solenopsis japonica (Hymenoptera: Formicidae) Reveal Gene in Response to Cold Stress

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1610
Author(s):  
Mohammad Vatanparast ◽  
Youngjin Park
Keyword(s):  
Gene Expression ◽  
Cold Stress ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Predatory Behavior ◽  
P Value ◽  
Molecular Response ◽  
Rna Seq ◽  
Protein Database ◽  
Brood Chamber

Solenopsis japonica, as a fire ant species, shows some predatory behavior towards earthworms and woodlice, and preys on the larvae of other ant species by tunneling into a neighboring colony’s brood chamber. This study focused on the molecular response process and gene expression profiles of S. japonica to low (9 °C)-temperature stress in comparison with normal temperature (25 °C) conditions. A total of 89,657 unigenes (the clustered non-redundant transcripts that are filtered from the longest assembled contigs) were obtained, of which 32,782 were annotated in the NR (nonredundant protein) database with gene ontology (GO) terms, gene descriptions, and metabolic pathways. The results were 81 GO subgroups and 18 EggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) keywords. Differentially expressed genes (DEGs) with log2fold change (FC) > 1 and log2FC < −1 with p-value ≤ 0.05 were screened for cold stress temperature. We found 215 unigenes up-regulated and 115 unigenes down-regulated. Comparing transcriptome profiles for differential gene expression resulted in various DE proteins and genes, including fatty acid synthases and lipid metabolism, which have previously been reported to be involved in cold resistance. We verified the RNA-seq data by qPCR on 20 up- and down-regulated DEGs. These findings facilitate the basis for the future understanding of the adaptation mechanisms of S. japonica and the molecular mechanisms underlying the response to low temperatures.

scholarly journals Exploration of Alternative Splicing (AS) Events in MDV-Infected Chicken Spleens

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1857
Author(s):  
Lulu Wang ◽  
Gang Zheng ◽  
Yiming Yuan ◽  
Ziyi Wang ◽  
Changjun Liu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Infection Process ◽  
Marek's Disease ◽  
Individual Development ◽  
Control Group ◽  
P Value ◽  
Marek’S Disease ◽  
Rna Seq ◽  
Infected Chicken ◽  
Structural Variations

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.

scholarly journals Hypomethylating Agents Do Not Alter Novel Splicing Events in Myeloid Neoplasms

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 37-38
Author(s):  
Hussein A Abbas ◽  
Feng Wang ◽  
Yue Wei ◽  
Hui Yang ◽  
Guillermo Montalban Bravo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alternative Splicing ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Exon Skipping ◽  
Splice Junction ◽  
The Novel ◽  
Hypomethylating Agents ◽  
Myeloid Malignancies ◽  
Alternative Splicing Events

Background: Aberrant mRNA splicing occurs in myeloid malignancies and affects genes involved in tumor suppression, heme biosynthesis and mitochondrial iron metabolism. Functional studies demonstrated impaired cellular differentiation upon targeting of aberrant splice variants. Hypomethylating agents (HMA) constitute the backbone of therapy of myeloid malignancies. Whether HMA treatment in myeloid malignancies alters the novel splicing transcriptional landscape and whether it correlates with responses remain largely unexplored. Methods: Total RNA sequencing was done on CD34+ cells from 79 patients bone marrow samples involved by acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), TF1 cell lines and CD34+ murine bone marrow cells. Novel alternatively spliced transcripts were detected using SplAdder and included the following splicing events: alterative 3' splice junction, alternative 5' splice junction, exon skipping, intron retention, multiple exon skipping and mutually exclusive exons. All alternatively splicing events were normalized to total transcript count in order to correct for total transcript levels. A false discovery rate of &lt;0.1 was used to identify significant events. Results: A total of 79 myeloid disease patients (27.8% females, 72.1% males) with a median age of 70 years (range, 31-87 years) were included in this study. In aggregate analysis of all 79 myeloid malignancies (39.2% (n=31) pre-treatment and 60.8% (n=48) post-treatment), there were 160 versus 37 (4.3 folds), 112 versus 40 (2.8 folds), 292 versus 51 (5.7 folds), 172 versus 80 (2.1 folds) and 29 versus 9 (3.2 folds) and 2 versus 0 novel splicing events occurring in pre- versus post- HMA treatment, respectively, in alterative 3' splice junction, alternative 5' splice junction, exon skipping, intron retention, multiple exon skipping and mutually exclusive exons, respectively. This suggested that treatment with HMA led to downregulation of novel alternative splicing events after normalization to total transcripts. However, upon excluding AML patients from the analysis, there were no significant events associated with treatment suggesting that the findings could be due to random events. To further explore whether HMA therapy influenced novel splicing events, we examined the novel splicing pattern in 7 MDS patients with paired BM samples at pre- and post-HMA and found no significant differences in alternative splicing events before and after the treatment. We then examined TF1 (human erythroleukemia) cell lines at pre- and post- HMA time points, but did not identify notable differences in the novel alternative splicing events with respect to HMA treatment. To assess whether CD34+ bone marrow cells from mice treated with hypomethylating agents have differential novel alternatively spliced events, we conducted similar analysis and did not find any discernible differences pre- and post- HMA treatment. These findings suggest that HMA does not influence novel alternative splicing events. Conclusions: Aberrant splicing has been linked to myeloid neoplasms especially myelodysplastic syndrome with mutations in splice variant genes. Our findings suggest that HMA does not influence novel alternative splicing events in myeloid malignancies. Therefore, the alternative splicing in myeloid disease is inherent to the disease and not affected by treatment. Disclosures Garcia-Manero: H3 Biomedicine: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Onconova: Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Amphivena Therapeutics: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Research Funding.

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