Quantifying alternative splicing from paired-end RNA-sequencing data

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.

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

10.1101/372995 ◽

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.

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

Neuro-Oncology ◽

10.1093/neuonc/noaa215.289 ◽

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.

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Leveraging transcript quantification for fast computation of alternative splicing profiles

10.1101/008763 ◽

2014 ◽

Cited By ~ 4

Author(s):

Gael P Alamancos ◽

Amadís Pagès ◽

Juan L Trincado ◽

Nicolás Bellora ◽

Eduardo Eyras

Keyword(s):

Alternative Splicing ◽

Rna Sequencing ◽

De Novo ◽

Computation Time ◽

Sequencing Data ◽

Standard Methods ◽

Transcript Quantification ◽

Cellular Processes ◽

Reconstruction Methods ◽

Transcript Reconstruction

Alternative splicing plays an essential role in many cellular processes and bears major relevance in the understanding of multiple diseases, including cancer. High-throughput RNA sequencing allows genome-wide analyses of splicing across multiple conditions. However, the increasing number of available datasets represents a major challenge in terms of computation time and storage requirements. We describe SUPPA, a computational tool to calculate relative inclusion values of alternative splicing events, exploiting fast transcript quantification. SUPPA accuracy is comparable and sometimes superior to standard methods using simulated as well as real RNA sequencing data compared to experimentally validated events. We assess the variability in terms of the choice of annotation and provide evidence that using complete transcripts rather than more transcripts per gene provides better estimates. Moreover, SUPPA coupled with de novo transcript reconstruction methods does not achieve accuracies as high as using quantification of known transcripts, but remains comparable to existing methods. Finally, we show that SUPPA is more than 1000 times faster than standard methods. Coupled with fast transcript quantification, SUPPA provides inclusion values at a much higher speed than existing methods without compromising accuracy, thereby facilitating the systematic splicing analysis of large datasets with limited computational resources. The software is implemented in Python 2.7 and is available under the MIT license at https://bitbucket.org/regulatorygenomicsupf/suppa

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HEDGEHOG/GLI Modulates the PRR11-SKA2 Bidirectional Transcription Unit in Lung Squamous Cell Carcinomas

Genes ◽

10.3390/genes12010120 ◽

2021 ◽

Vol 12 (1) ◽

pp. 120

Author(s):

Yiyun Sun ◽

Dandan Xu ◽

Chundong Zhang ◽

Yitao Wang ◽

Lian Zhang ◽

...

Keyword(s):

Rna Sequencing ◽

Squamous Cell ◽

Gene Pair ◽

Gene List ◽

Ectopic Expression ◽

Lung Squamous Cell Carcinoma ◽

Gene Set Enrichment Analysis ◽

The Cancer Genome Atlas ◽

Sequencing Data ◽

Gene Set

We previously demonstrated that proline-rich protein 11 (PRR11) and spindle and kinetochore associated 2 (SKA2) constituted a head-to-head gene pair driven by a prototypical bidirectional promoter. This gene pair synergistically promoted the development of non-small cell lung cancer. However, the signaling pathways leading to the ectopic expression of this gene pair remains obscure. In the present study, we first analyzed the lung squamous cell carcinoma (LSCC) relevant RNA sequencing data from The Cancer Genome Atlas (TCGA) database using the correlation analysis of gene expression and gene set enrichment analysis (GSEA), which revealed that the PRR11-SKA2 correlated gene list highly resembled the Hedgehog (Hh) pathway activation-related gene set. Subsequently, GLI1/2 inhibitor GANT-61 or GLI1/2-siRNA inhibited the Hh pathway of LSCC cells, concomitantly decreasing the expression levels of PRR11 and SKA2. Furthermore, the mRNA expression profile of LSCC cells treated with GANT-61 was detected using RNA sequencing, displaying 397 differentially expressed genes (203 upregulated genes and 194 downregulated genes). Out of them, one gene set, including BIRC5, NCAPG, CCNB2, and BUB1, was involved in cell division and interacted with both PRR11 and SKA2. These genes were verified as the downregulated genes via RT-PCR and their high expression significantly correlated with the shorter overall survival of LSCC patients. Taken together, our results indicate that GLI1/2 mediates the expression of the PRR11-SKA2-centric gene set that serves as an unfavorable prognostic indicator for LSCC patients, potentializing new combinatorial diagnostic and therapeutic strategies in LSCC.

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RNA Sequencing Data from Human Intracranial Aneurysm Tissue Reveals a Complex Inflammatory Environment Associated with Rupture

Molecular Diagnosis & Therapy ◽

10.1007/s40291-021-00552-4 ◽

2021 ◽

Author(s):

Vincent M. Tutino ◽

Haley R. Zebraski ◽

Hamidreza Rajabzadeh-Oghaz ◽

Lee Chaves ◽

Adam A. Dmytriw ◽

...

Keyword(s):

Intracranial Aneurysm ◽

Rna Sequencing ◽

Sequencing Data

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Mixed Distribution Models Based on Single-Cell RNA Sequencing Data

Interdisciplinary Sciences Computational Life Sciences ◽

10.1007/s12539-021-00427-6 ◽

2021 ◽

Author(s):

Min Wu ◽

Junhua Xu ◽

Tao Ding ◽

Jie Gao

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Sequencing Data ◽

Distribution Models ◽

Mixed Distribution ◽

Single Cell Rna Sequencing

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In-depth transcriptomic analysis of human retina reveals molecular mechanisms underlying diabetic retinopathy

Scientific Reports ◽

10.1038/s41598-021-88698-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kolja Becker ◽

Holger Klein ◽

Eric Simon ◽

Coralie Viollet ◽

Christian Haslinger ◽

...

Keyword(s):

Diabetic Retinopathy ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Vision Loss ◽

Ganglion Cells ◽

Expression Profiles ◽

Cell Types ◽

Sequencing Data ◽

Disease Stages ◽

Post Transcriptional Regulation

AbstractDiabetic Retinopathy (DR) is among the major global causes for vision loss. With the rise in diabetes prevalence, an increase in DR incidence is expected. Current understanding of both the molecular etiology and pathways involved in the initiation and progression of DR is limited. Via RNA-Sequencing, we analyzed mRNA and miRNA expression profiles of 80 human post-mortem retinal samples from 43 patients diagnosed with various stages of DR. We found differentially expressed transcripts to be predominantly associated with late stage DR and pathways such as hippo and gap junction signaling. A multivariate regression model identified transcripts with progressive changes throughout disease stages, which in turn displayed significant overlap with sphingolipid and cGMP–PKG signaling. Combined analysis of miRNA and mRNA expression further uncovered disease-relevant miRNA/mRNA associations as potential mechanisms of post-transcriptional regulation. Finally, integrating human retinal single cell RNA-Sequencing data revealed a continuous loss of retinal ganglion cells, and Müller cell mediated changes in histidine and β-alanine signaling. While previously considered primarily a vascular disease, attention in DR has shifted to additional mechanisms and cell-types. Our findings offer an unprecedented and unbiased insight into molecular pathways and cell-specific changes in the development of DR, and provide potential avenues for future therapeutic intervention.

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