A survey of computational methods in transcriptome-wide alternative splicing analysis

Alternative splicing is widely recognized for its roles in regulating genes and creating gene diversity. However, despite many efforts, the repertoire of gene splicing variation is still incompletely characterized, even in humans. Here we describe a new computational system, ASprofile, and its application to RNA-seq data from Illumina’s Human Body Map project (>2.5 billion reads). Using the system, we identified putative alternative splicing events in 16 different human tissues, which provide a dynamic picture of splicing variation across the tissues. We detected 26,989 potential exon skipping events representing differences in splicing patterns among the tissues. A large proportion of the events (>60%) were novel, involving new exons (~3000), new introns (~16000), or both. When tracing these events across the sixteen tissues, only a small number (4-7%) appeared to be differentially expressed (‘switched’) between two tissues, while 30-45% showed little variation, and the remaining 50-65% were not present in one or both tissues compared. Novel exon skipping events appeared to be slightly less variable than known events, but were more tissue-specific. Our study represents the first effort to build a comprehensive catalog of alternative splicing in normal human tissues from RNA-seq data, while providing insights into the role of alternative splicing in shaping tissue transcriptome differences. The catalog of events and the ASprofile software are freely available from the Zenodo repository(http://zenodo.org/record/7068; doi:10.5281/zenodo.7068) and from our web site http://ccb.jhu.edu/software/ASprofile.

Download Full-text

Alternative Splicing Increases Sirtuin Gene Family Diversity and Modulates Their Subcellular Localization and Function

International Journal of Molecular Sciences ◽

10.3390/ijms22020473 ◽

2021 ◽

Vol 22 (2) ◽

pp. 473

Author(s):

Xiaomin Zhang ◽

Fathima S. Ameer ◽

Gohar Azhar ◽

Jeanne Y. Wei

Keyword(s):

Alternative Splicing ◽

Subcellular Localization ◽

Structural Changes ◽

Catalytic Domain ◽

Gene Diversity ◽

Exon Skipping ◽

Age Related ◽

C Terminus ◽

Domain Loss ◽

And Function

Alternative splicing generates multiple distinct isoforms that increase transcriptome and proteome diversity. There are seven sirtuin genes in humans, each consists of multiple exons that are likely to undergo alternative splicing. Our aim was to characterize the effect of alternative splicing on the sirtuin genes. Here, we report the identification of 23 human sirtuin isoforms, most of which were not previously reported. Five of the sirtuin genes had more than one isoform, whereas sirtuin-6 had nine isoforms. Exon skipping was the main event. Most of the sirtuin isoforms were deficient in parts of the protein domains, including the catalytic domain, the N- or C-terminus, nuclear localization signal or mitochondrial targeting signal. The domain loss caused potential structural changes. Three SIRT1 isoforms had a differential effect on the mitochondrial oxygen consumption rate. Age-related changes in the expression of SIRT1 isoforms were observed in the human heart in fetus, adults, and very old individuals. We also identified 15 sirtuin isoforms in mice. Our data indicate that alternative splicing increases sirtuin gene diversity and may modulate subcellular localization and function, thereby adding complexity to the gene regulation of mitochondrial respiration, metabolism, and cardiac function during maturation and aging.

Download Full-text

Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues

F1000Research ◽

10.12688/f1000research.2-188.v2 ◽

2013 ◽

Vol 2 ◽

pp. 188 ◽

Cited By ~ 24

Author(s):

Liliana Florea ◽

Li Song ◽

Steven L Salzberg

Keyword(s):

Alternative Splicing ◽

Gene Diversity ◽

Exon Skipping ◽

Human Tissues ◽

Rna Seq ◽

Gene Splicing ◽

Link Type ◽

Normal Human ◽

Splicing Patterns

Alternative splicing is widely recognized for its roles in regulating genes and creating gene diversity. However, despite many efforts, the repertoire of gene splicing variation is still incompletely characterized, even in humans. Here we describe a new computational system, ASprofile, and its application to RNA-seq data from Illumina’s Human Body Map project (>2.5 billion reads). Using the system, we identified putative alternative splicing events in 16 different human tissues, which provide a dynamic picture of splicing variation across the tissues. We detected 26,989 potential exon skipping events representing differences in splicing patterns among the tissues. A large proportion of the events (>60%) were novel, involving new exons (~3000), new introns (~16000), or both. When tracing these events across the sixteen tissues, only a small number (4-7%) appeared to be differentially expressed (‘switched’) between two tissues, while 30-45% showed little variation, and the remaining 50-65% were not present in one or both tissues compared. Novel exon skipping events appeared to be slightly less variable than known events, but were more tissue-specific. Our study represents the first effort to build a comprehensive catalog of alternative splicing in normal human tissues from RNA-seq data, while providing insights into the role of alternative splicing in shaping tissue transcriptome differences. The catalog of events and the ASprofile software are freely available from the Zenodo repository(http://zenodo.org/record/7068; doi:10.5281/zenodo.7068) and from our web site http://ccb.jhu.edu/software/ASprofile.

Download Full-text

Computational comparison of common event-based differential splicing tools: practical considerations for laboratory researchers

BMC Bioinformatics ◽

10.1186/s12859-021-04263-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Ittai B. Muller ◽

Stijn Meijers ◽

Peter Kampstra ◽

Steven van Dijk ◽

Michel van Elswijk ◽

...

Keyword(s):

Alternative Splicing ◽

Correlation Coefficients ◽

Differential Splicing ◽

Sequencing Data ◽

Computational Performance ◽

Input Size ◽

Cpu Utilization ◽

Dataset Size ◽

Log Linear ◽

Event Based

Abstract Background Computational tools analyzing RNA-sequencing data have boosted alternative splicing research by identifying and assessing differentially spliced genes. However, common alternative splicing analysis tools differ substantially in their statistical analyses and general performance. This report compares the computational performance (CPU utilization and RAM usage) of three event-level splicing tools; rMATS, MISO, and SUPPA2. Additionally, concordance between tool outputs was investigated. Results Log-linear relations were found between job times and dataset size in all splicing tools and all virtual machine (VM) configurations. MISO had the highest job times for all analyses, irrespective of VM size, while MISO analyses also exceeded maximum CPU utilization on all VM sizes. rMATS and SUPPA2 load averages were relatively low in both size and replicate comparisons, not nearing maximum CPU utilization in the VM simulating the lowest computational power (D2 VM). RAM usage in rMATS and SUPPA2 did not exceed 20% of maximum RAM in both size and replicate comparisons while MISO reached maximum RAM usage in D2 VM analyses for input size. Correlation coefficients of differential splicing analyses showed high correlation (β > 80%) between different tool outputs with the exception of comparisons of retained intron (RI) events between rMATS/MISO and rMATS/SUPPA2 (β < 60%). Conclusions Prior to RNA-seq analyses, users should consider job time, amount of replicates and splice event type of interest to determine the optimal alternative splicing tool. In general, rMATS is superior to both MISO and SUPPA2 in computational performance. Analysis outputs show high concordance between tools, with the exception of RI events.

Download Full-text

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

Genome Biology ◽

10.1186/s13059-020-02258-y ◽

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.

Download Full-text

Alternative splicing of synaptotagmins involving transmembrane exon skipping

FEBS Letters ◽

10.1016/s0014-5793(99)01382-4 ◽

1999 ◽

Vol 460 (3) ◽

pp. 417-422 ◽

Cited By ~ 28

Author(s):

Molly Craxton ◽

Michel Goedert

Keyword(s):

Alternative Splicing ◽

Exon Skipping

Download Full-text

Abstract 344: Finding the Achilles’ heel of Muscle Giant---TALEN-mediated Gene-editing in Zebrafish Titin

Circulation Research ◽

10.1161/res.117.suppl_1.344 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Yu-Huan Shih ◽

Xiaolei Xu

Keyword(s):

Alternative Splicing ◽

Gene Editing ◽

Muscle Development ◽

Exon Skipping ◽

Transcription Activator ◽

Detailed Characterization ◽

A Domains ◽

Underlying Mechanisms ◽

Sarcomere Assembly

Background: TITIN (TTN) has more than 300 exons and encodes a gigantic protein that is crucial for heart and muscle development. Mutations in TTN caused a variety of human diseases including cardiomyopathy and muscular dystrophy. Recently, dilated cardiomyopathy-associated mutations on TTN have been found more frequently in exons encoding A-band domains but less in exons encoding the N-terminal Z-disc domains, suggesting that mutations in different exons of TTN cause distinct consequences. To elucidate the underlying mechanisms, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to introduce truncating mutations in different exons of ttn, and then study their effects on heart and somites. Results: We generated truncational mutations in different exons of zebrafish titins encoding Z-disc, N2B, Novex-3, and A domains, respectively. Because zebrafish contains two titin homologues, ttna and ttnb, we introduced mutations in both genes at the corresponding loci. While both Z-disc and A band mutations on ttna disrupted sarcomere assembly in heart and somites, Z-disc or A band mutations on ttnb only affect somites without affecting the heart. Interestingly, a Z-disc mutation on ttna resulted in milder phenotypes than an A-band mutation, while a Z-disc mutation on ttnb generated severer phenotypes than an A-band mutation. No phenotype was observed in the homozygous fish in either ttna-novex-3 or ttnb-N2B mutant fish. Conclusions: A spectrum of truncational mutations in ttna and ttnb has been generated in zebrafish using the TALEN technology. Mutations in different exons result in different phenotypes. Detailed characterization of these mutants and double mutants will be presented, which shall elicit distinct contribution of alternative splicing and exon skipping as two candidate mechanisms during pathogenesis of Titinopathies.

Download Full-text

AStrap: identification of alternative splicing from transcript sequences without a reference genome

Bioinformatics ◽

10.1093/bioinformatics/bty1008 ◽

2018 ◽

Vol 35 (15) ◽

pp. 2654-2656 ◽

Cited By ~ 5

Author(s):

Guoli Ji ◽

Wenbin Ye ◽

Yaru Su ◽

Moliang Chen ◽

Guangzao Huang ◽

...

Keyword(s):

Machine Learning ◽

Alternative Splicing ◽

Single Molecule ◽

Reference Genome ◽

De Novo ◽

Supplementary Information ◽

Model Organisms ◽

Sequencing Data ◽

Extensive Evaluation ◽

Reference Genomes

Abstract Summary Alternative splicing (AS) is a well-established mechanism for increasing transcriptome and proteome diversity, however, detecting AS events and distinguishing among AS types in organisms without available reference genomes remains challenging. We developed a de novo approach called AStrap for AS analysis without using a reference genome. AStrap identifies AS events by extensive pair-wise alignments of transcript sequences and predicts AS types by a machine-learning model integrating more than 500 assembled features. We evaluated AStrap using collected AS events from reference genomes of rice and human as well as single-molecule real-time sequencing data from Amborella trichopoda. Results show that AStrap can identify much more AS events with comparable or higher accuracy than the competing method. AStrap also possesses a unique feature of predicting AS types, which achieves an overall accuracy of ∼0.87 for different species. Extensive evaluation of AStrap using different parameters, sample sizes and machine-learning models on different species also demonstrates the robustness and flexibility of AStrap. AStrap could be a valuable addition to the community for the study of AS in non-model organisms with limited genetic resources. Availability and implementation AStrap is available for download at https://github.com/BMILAB/AStrap. Supplementary information Supplementary data are available at Bioinformatics online.

Download Full-text

The Effect of 6-Thioguanine on Alternative Splicing and Antisense-Mediated Exon Skipping Treatment for Duchenne Muscular Dystrophy

PLoS Currents ◽

10.1371/currents.md.597d700f92eaa70de261ea0d91821377 ◽

2012 ◽

Cited By ~ 4

Author(s):

Ingrid E. C. Verhaart ◽

Annemieke Aartsma-Rus

Keyword(s):

Alternative Splicing ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Exon Skipping

Download Full-text

ASPP2κ Is Expressed In Human Colorectal Carcinoma And Promotes Chemotherapy Resistance And Tumorigenesis

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.727203 ◽

2021 ◽

Vol 8 ◽

Author(s):

Ingmar Rieger ◽

Vasileia Tsintari ◽

Mathis Overkamp ◽

Falko Fend ◽

Charles D. Lopez ◽

...

Keyword(s):

Alternative Splicing ◽

Chemotherapy Resistance ◽

Human Tumor ◽

Exon Skipping ◽

Predictive Marker ◽

Hematologic Malignancies ◽

Response To Therapy ◽

Dominant Negative ◽

Gene Products ◽

Resistance To Therapy

Alternative splicing is a common physiologic mechanism to generate numerous distinct gene products from one gene locus, which can result in unique gene products with differing important functional outcomes depending on cell context. Aberrant alternative splicing is a hallmark of cancer that can contribute to oncogenesis and aggressiveness of the disease as well as resistance to therapy. However, aberrant splicing might also result in novel targets for cancer therapy. ASPP2 is a haplo-insufficient tumor suppressor, that functions through both p53-dependent as well as p53-independent mechanisms to enhance cell death after stress. Interestingly, the common human tumor TP53 mutations result in a loss of the binding sites to ASPP2, leading to impaired induction of apoptosis. Vice versa, attenuation of ASPP2 has been described to be associated with high-risk disease, therapy failure and poor clinical outcome especially in tumors harboring the TP53 wildtype (WT) isoform. We have recently identified a novel, dominant-negative splicing variant of ASPP2, named ASPP2κ, with oncogenic potential. Exon-skipping results in a reading-frame shift with a premature translation stop, omitting most of the ASPP2 C-terminus - which harbors the p53-binding domain. Consequently, the ASPP2-p53 interaction is abrogated, which in part impacts on oncogenesis, aggressiveness of disease and response to therapy. Since ASPP2κ has been shown in hematologic malignancies to promote tumorigenesis, we further wished to determine if aberrant ASPP2κ expression plays a role in human solid tumors. In this report, we find that ASPP2κ is frequently expressed in human colorectal tumors (CRC). Using ASPP2κ overexpressing and interference CRC models, we demonstrate a functional role of ASPP2κ in contributing to oncogenesis and resistance to therapy in CRC by 1) enhancing proliferation, 2) promoting cell migration and, 3) conferring resistance to chemotherapy induced apoptosis. Our findings have far-reaching consequences for future diagnostic and therapeutic strategies for ASPP2κ expressing colorectal cancer patients and provide proof-of-principle to further explore ASPP2κ as potential predictive marker and target for therapy in clinical trials.

Download Full-text