scholarly journals EPCO-10. CHANGES IN ALTERNATIVE SPLICING AND ASSOCIATED NEOANTIGENS DUE TO THERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii71-ii71
Author(s):  
Lin Wang ◽  
Karin Shamardani ◽  
Husam Babikir ◽  
Francisca Catalan ◽  
Takahide Nejo ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Recurrent Glioblastoma ◽  
Surface Proteins ◽  
Sequencing Data ◽  
Cell Therapies ◽  
Effect Of Therapy ◽  
Cell Type Specific ◽  
Human Gbm ◽  
Splicing Patterns

Abstract Recent studies have identified alternative splicing (AS) as a novel source of neoantigens for immunotherapy. Surprisingly little is known about the AS milieu in recurrent glioblastoma (GBM), despite this being the venue for most clinical trials. We profiled 29 primary-recurrent paired human GBM specimens via RNA sequencing and re-analyzed RNA-sequencing data from non-malignant human brain tissues. From these data, we reconstructed the landscape of AS in GBM through recurrence and contrasted that to isoforms observed in non-malignant brain. The AS events we identified were cross-referenced with single-cell GBM atlases to determine cell-type specific splicing patterns. From this we identified novel splicing events in cell-surface proteins that are suitable targets for engineered T-cell therapies. We found recurrent-specific isoforms of mitogen-activated kinase pathway genes which are expressed exclusively by GBM stem-like cells that enhance invasiveness. These studies shed light on the effect of therapy on AS and identify novel targets for emerging immunotherapies.

scholarly journals The evolution of alternative splicing in glioblastoma under therapy

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Lin Wang ◽  
Karin Shamardani ◽  
Husam Babikir ◽  
Francisca Catalan ◽  
Takahide Nejo ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Standard Of Care ◽  
Surface Proteins ◽  
Adult Brain ◽  
Primary Tumors ◽  
Cell Therapies ◽  
Therapeutic Development ◽  
Human Gbm ◽  
Tissue Specimens ◽  
Splicing Patterns

Abstract Background Alternative splicing is a rich source of tumor-specific neoantigen targets for immunotherapy. This holds promise for glioblastomas (GBMs), the most common primary tumors of the adult brain, which are resistant to standard-of-care therapy. Although most clinical trials enroll patients at recurrence, most preclinical studies have been done with specimens from primary disease. There are limited expression data from GBMs at recurrence and surprisingly little is known about the evolution of splicing patterns under therapy. Result We profile 37 primary-recurrent paired human GBM specimens via RNA sequencing. We describe the landscape of alternative splicing in GBM at recurrence and contrast that to primary and non-malignant brain-tissue specimens. By screening single-cell atlases, we identify cell-type-specific splicing patterns and novel splicing events in cell-surface proteins that are suitable targets for engineered T cell therapies. We identify recurrent-specific isoforms of mitogen-activated kinase pathway genes that enhance invasiveness and are preferentially expressed by stem-like cells. Conclusion These studies shed light on gene expression in recurrent GBM and identify novel targets for therapeutic development.

scholarly journals Quantifying alternative splicing from paired-end RNA-sequencing data

2014 ◽  
Vol 8 (1) ◽  
pp. 309-330 ◽  
Author(s):  
David Rossell ◽  
Camille Stephan-Otto Attolini ◽  
Manuel Kroiss ◽  
Almond Stöcker
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Sequencing Data

scholarly journals Identification of Genomewide Alternative Splicing Events in Sequential, Isogenic Clinical Isolates of Candida albicans Reveals a Novel Mechanism of Drug Resistance and Tolerance to Cellular Stresses

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Suraya Muzafar ◽  
Ravi Datta Sharma ◽  
Abdul Haseeb Shah ◽  
Naseem A. Gaur ◽  
Ujjaini Dasgupta ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Alternative Splicing ◽  
Rna Sequencing ◽  
Single Gene ◽  
Drug Susceptibility ◽  
Protein Isoforms ◽  
Sequencing Data ◽  
Splice Isoforms ◽  
Content Type

ABSTRACT Alternative splicing (AS)—a process by which a single gene gives rise to different protein isoforms in eukaryotes—has been implicated in many basic cellular processes, but little is known about its role in drug resistance and fungal pathogenesis. The most common human fungal pathogen, Candida albicans, has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Here, we report AS regulating drug resistance in C. albicans. Comparative RNA-sequencing of two different sets of sequential, isogenic azole-sensitive and -resistant isolates of C. albicans revealed differential expression of splice isoforms of 14 genes. One of these was the superoxide dismutase gene SOD3, which contains a single intron. The sod3Δ/Δ mutant was susceptible to the antifungals amphotericin B (AMB) and menadione (MND). While AMB susceptibility was rescued by overexpression of both the spliced and unspliced SOD3 isoforms, only the spliced isoform could overcome MND susceptibility, demonstrating the functional relevance of this splicing in developing drug resistance. Furthermore, unlike AMB, MND inhibits SOD3 splicing and acts as a splicing inhibitor. Consistent with these observations, MND exposure resulted in increased levels of unspliced SOD3 isoform that are unable to scavenge reactive oxygen species (ROS), resulting in increased drug susceptibility. Collectively, these observations suggest that AS is a novel mechanism for stress adaptation and overcoming drug susceptibility in C. albicans. IMPORTANCE The emergence of resistance in Candida albicans, an opportunistic pathogen, against the commonly used antifungals is becoming a major obstacle in its treatment. The necessity to identify new drug targets demands fundamental insights into the mechanisms used by this organism to develop drug resistance. C. albicans has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Using the RNA-sequencing data from isogenic pairs of azole-sensitive and -resistant isolates of C. albicans, here, we show how C. albicans uses modulations in mRNA splicing to overcome antifungal drug stress.

LncAS2Cancer: a comprehensive database for alternative splicing of lncRNAs across human cancers

2020 ◽  
Author(s):  
Yulan Deng ◽  
Hao Luo ◽  
Zhenyu Yang ◽  
Lunxu Liu
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Protein Coding ◽  
Functional Roles ◽  
Comprehensive Database ◽  
Human Cancers ◽  
Manual Curation ◽  
Annotation Information ◽  
Cancer Types ◽  
Splicing Patterns

Abstract Accumulating studies demonstrated that the roles of lncRNAs for tumorigenesis were isoform-dependent and their aberrant splicing patterns in cancers contributed to function specificity. However, there is no existing database focusing on cancer-related alternative splicing of lncRNAs. Here, we developed a comprehensive database called LncAS2Cancer, which collected 5335 bulk RNA sequencing and 1826 single-cell RNA sequencing samples, covering over 30 cancer types. By applying six state-of-the-art splicing algorithms, 50 859 alternative splicing events for 8 splicing types were identified and deposited in the database. In addition, the database contained the following information: (i) splicing patterns of lncRNAs under seven different conditions, such as gene interference, which facilitated to infer potential regulators; (ii) annotation information derived from eight sources and manual curation, to understand the functional impact of affected sequences; (iii) survival analysis to explore potential biomarkers; as well as (iv) a suite of tools to browse, search, visualize and download interesting information. LncAS2Cancer could not only confirm the known cancer-associated lncRNA isoforms but also indicate novel ones. Using the data deposited in LncAS2Cancer, we compared gene model and transcript overlap between lncRNAs and protein-coding genes and discusses how these factors, along with sequencing depth, affected the interpretation of splicing signals. Based on recurrent signals and potential confounders, we proposed a reliable score to prioritize splicing events for further elucidation. Together, with the broad collection of lncRNA splicing patterns and annotation, LncAS2Cancer will provide important new insights into the diverse functional roles of lncRNA isoforms in human cancers. LncAS2Cancer is freely available at https://lncrna2as.cd120.com/.

scholarly journals Splice junction centric approach to identify translated noncanonical isoforms in the human proteome

2018 ◽  
Author(s):  
Edward Lau ◽  
Yu Han ◽  
Maggie P. Y. Lam
Keyword(s):  
Alternative Splicing ◽  
Rna Sequencing ◽  
Splice Site ◽  
Splice Junction ◽  
Human Proteome ◽  
Protein Isoforms ◽  
Sequencing Data ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Disordered Regions

AbstractRNA sequencing has led to the discovery of many transcript isoforms created by alternative splicing, but the translational status and functional significance of most alternative splicing events remain unknown. Here we applied a splice junction-centric approach to survey the landscape of protein alternative isoform expression in the human proteome. We focused on alternative splice events where pairs of splice junctions corresponding to included and excluded exons with appreciable read counts are translated together into selective protein sequence databases. Using this approach, we constructed tissue-specific FASTA databases from ENCODE RNA sequencing data, then reanalyzed splice junction peptides in existing mass spectrometry datasets across 10 human tissues (heart, lung, liver, pancreas, ovary, testis, colon, prostate, adrenal gland, and esophagus). Our analysis reidentified 1,108 non-canonical isoforms annotated in SwissProt. We further found 253 novel splice junction peptides in 212 genes that are not documented in the comprehensive Uniprot TrEMBL or Ensembl RefSeq databases. On a proteome scale, non-canonical isoforms differ from canonical sequences preferentially at sequences with heightened protein disorder, suggesting a functional consequence of alternative splicing on the proteome is the regulation of intrinsically disordered regions. We further observed examples where isoform-specific regions intersect with important cardiac protein phosphorylation sites. Our results reveal previously unidentified protein isoforms and may avail efforts to elucidate the functions of splicing events and expand the pool of observable biomarkers in profiling studies.Acronyms and AbbreviationsA3SSalternative 3-prime splice site;A5SSalternative 5-prime splice site;FDRfalse discovery rate;IDRintrinsically disordered regions;MXEmutually exclusive exons;PSIpercent spliced in;PTCpremature termination codon;PTMpost-translational modifications;SEskipped exon;RIretained intron.

Random Forest Factorization Reveals Latent Structure in Single Cell RNA Sequencing Data

2021 ◽  
Author(s):  
Boris M. Brenerman ◽  
Benjamin D. Shapiro ◽  
Michael C. Schatz ◽  
Alexis Battle
Keyword(s):  
Random Forest ◽  
Single Cell ◽  
Rna Sequencing ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Sequencing Data ◽  
Random Forest Regression ◽  
Single Cell Rna Sequencing ◽  
Cell Type Specific ◽  
Gene Regulatory

AbstractSingle-cell RNA sequencing data contain patterns of correlation that are poorly captured by techniques that rely on linear estimation or assumptions of Gaussian behavior. We apply random forest regression to scRNAseq data from mouse brains, which identifies the co-regulation of genes within specific cellular contexts. By analyzing the estimators of the random forest, we identify several novel candidate gene regulatory networks and compare these networks in aged and young mice. We demonstrate that cell populations have cell-type specific phenotypes of aging that are not detected by other methods, including the collapse of differentiating oligodendrocytes but not precursors or mature oligodendrocytes.

scholarly journals Long non-coding RNA expression levels modulate cell-type specific splicing patterns by altering their interaction landscape with RNA-binding proteins

2019 ◽  
Author(s):  
Felipe Wendt Porto ◽  
Swapna Vidhur Daulatabad ◽  
Sarath Chandra Janga
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Cell Type ◽  
Specific Manner ◽  
Alternatively Spliced ◽  
A Cell ◽  
Specific Alternative ◽  
Cell Type Specific ◽  
Rna Interaction ◽  
Splicing Patterns

AbstractBackgroundRecent developments in our understanding of the interactions between long non-coding RNAs (lncRNAs) and cellular components have improved treatment approaches for various human diseases including cancer, vascular diseases, and neurological diseases. Although investigation of specific lncRNAs revealed their role in the metabolism of cellular RNA, our understanding of their contribution to post-transcriptional regulation is relatively limited. In this study, we explore the role of lncRNAs in modulating alternative splicing and their impact on downstream protein-RNA interaction networks.ResultsAnalysis of alternative splicing events across 39 lncRNA knockdown and wildtype RNA-sequencing datasets from three human cell lines: HeLa (Cervical Cancer), K562 (Myeloid Leukemia), and U87 (Glioblastoma), resulted in high confidence (fdr < 0.01) identification of 11630 skipped exon events and 5895 retained intron events, implicating 759 genes to be impacted at post-transcriptional level due to the loss of lncRNAs. We observed that a majority of the alternatively spliced genes in a lncRNA knockdown were specific to the cell type, in tandem, the functions annotated to the genes affected by alternative splicing across each lncRNA knockdown also displayed cell type specificity. To understand the mechanism behind this cell-type specific alternative splicing patterns, we analyzed RNA binding protein (RBP)-RNA interaction profiles across the spliced regions, to observe cell type specific alternative splice event RBP binding preference.ConclusionsDespite limited RBP binding data across cell lines, alternatively spliced events detected in lncRNA perturbation experiments were associated with RBPs binding in proximal intron-exon junctions, in a cell type specific manner. The cellular functions affected by alternative splicing were also affected in a cell type specific manner. Based on the RBP binding profiles in HeLa and K562 cells, we hypothesize that several lncRNAs are likely to exhibit a sponge effect in disease contexts, resulting in the functional disruption of RBPs, and their downstream functions. We propose that such lncRNA sponges can extensively rewire the post-transcriptional gene regulatory networks by altering the protein-RNA interaction landscape in a cell-type specific manner.

scholarly journals Cell type-specific weighting-factors for accurate virtual single-cell RNA-sequencing of diverse organs

2020 ◽  
Author(s):  
Kengo Tejima ◽  
Satoshi Kozawa ◽  
Thomas N. Sato
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Cell Type ◽  
Sequencing Data ◽  
Human Organ ◽  
Weighting Factors ◽  
Single Cell Rna Sequencing ◽  
Cell Type Specific ◽  
Bulk Tissue

AbstractComputational deconvolution of transcriptome data of organs/tissues uncovers their structural and functional complexities at cellular resolution without performing single-cell RNA-sequencing experiments. However, the deconvolution of highly heterogenous diverse organs/tissues remains a challenge. Herein, we report “cell type-specific weighting-factors” that are essential for accurate deconvolution, but critically lacking in the existing methods. We computed such weighting-factors for 97 cell-types across 10 mouse organs and demonstrate their effective usage in the Bayesian framework to generate their virtual single-cell RNA-sequencing data, hence accurately estimating both cell-type ratios and the complete transcriptome of each cell-type in these organs. The method also efficiently detects the temporal changes of such cell type-profiles during organ pathogenesis in disease models. Furthermore, we present its potential utility for human organ/bulk-tissue deconvolution. Taken together, the weighting-factors reported herein and their computation for new cell-types and/or new species such as human are essential tools/resources for studying high-resolution biology and disease.

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