scholarly journals Global transcriptome analysis reveals circadian control of splicing events in Arabidopsis thaliana

2019 ◽  
Author(s):  
Andrés Romanowski ◽  
Rubén G. Schlaen ◽  
Soledad Perez-Santangelo ◽  
Estefanía Mancini ◽  
Marcelo J. Yanovsky
Keyword(s):  
Arabidopsis Thaliana ◽  
Circadian Clock ◽  
Splice Site ◽  
Intron Retention ◽  
Exon Skipping ◽  
Global Scale ◽  
Analysis Tool ◽  
List Type ◽  
Mrna Levels ◽  
Splice Site Usage

SUMMARYThe circadian clock of Arabidopsis thaliana controls many physiological and molecular processes, allowing plants to anticipate daily changes in their environment. However, developing a detailed understanding of how oscillations in mRNA levels are connected to oscillations in post-transcriptional processes, such as splicing, has remained a challenge.Here we applied a combined approach using deep transcriptome sequencing and bioinformatics tools to identify novel circadian regulated genes and splicing events.Using a stringent approach, we identified 300 intron retention, 8 exon skipping, 79 alternative 3’ splice site usage, 48 alternative 5’ splice site usage, and 350 multiple (more than one event type) annotated events under circadian regulation. We also found 7 and 721 novel alternative exonic and intronic events. Depletion of the circadian regulated splicing factor AtSPF30 homolog, resulted in the disruption of a subset of clock controlled splicing events.Altogether, our global circadian RNA-seq coupled with an in silico, event centred, splicing analysis tool offers a new approach for studying the interplay between the circadian clock and the splicing machinery at a global scale. The identification of many circadian regulated splicing events broadens our current understanding of the level of control that the circadian clock has over this posttranscriptional regulatory layer.

scholarly journals Quantifying splice-site usage: a simple yet powerful approach to analyze splicing

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Craig I Dent ◽  
Shilpi Singh ◽  
Sourav Mukherjee ◽  
Shikhar Mishra ◽  
Rucha D Sarwade ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Splice Site ◽  
Rna Splicing ◽  
Intron Retention ◽  
Sequence Divergence ◽  
Exon Skipping ◽  
Splice Sites ◽  
Environmental Responses ◽  
Traditional Approaches ◽  
Splice Site Usage

Abstract RNA splicing, and variations in this process referred to as alternative splicing, are critical aspects of gene regulation in eukaryotes. From environmental responses in plants to being a primary link between genetic variation and disease in humans, splicing differences confer extensive phenotypic changes across diverse organisms (1–3). Regulation of splicing occurs through differential selection of splice sites in a splicing reaction, which results in variation in the abundance of isoforms and/or splicing events. However, genomic determinants that influence splice-site selection remain largely unknown. While traditional approaches for analyzing splicing rely on quantifying variant transcripts (i.e. isoforms) or splicing events (i.e. intron retention, exon skipping etc.) (4), recent approaches focus on analyzing complex/mutually exclusive splicing patterns (5–8). However, none of these approaches explicitly measure individual splice-site usage, which can provide valuable information about splice-site choice and its regulation. Here, we present a simple approach to quantify the empirical usage of individual splice sites reflecting their strength, which determines their selection in a splicing reaction. Splice-site strength/usage, as a quantitative phenotype, allows us to directly link genetic variation with usage of individual splice-sites. We demonstrate the power of this approach in defining the genomic determinants of splice-site choice through GWAS. Our pilot analysis with more than a thousand splice sites hints that sequence divergence in cis rather than trans is associated with variations in splicing among accessions of Arabidopsis thaliana. This approach allows deciphering principles of splicing and has broad implications from agriculture to medicine.

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 A comprehensive characterization of cis-acting splicing-associated variants in human cancer

2017 ◽  
Author(s):  
Yuichi Shiraishi ◽  
Keisuke Kataoka ◽  
Kenichi Chiba ◽  
Ai Okada ◽  
Yasunori Kogure ◽  
...  
Keyword(s):  
Splice Site ◽  
Human Cancer ◽  
Intron Retention ◽  
Exon Skipping ◽  
Driver Mutations ◽  
Splice Sites ◽  
Sequencing Data ◽  
Cis Acting ◽  
Statistical Framework ◽  
Cancer Types

AbstractAlthough many driver mutations are thought to promote carcinogenesis via abnormal splicing, the landscape of these splicing-associated variants (SAVs) remains unknown due to the complexity of splicing abnormalities. Here we developed a statistical framework to identify SAVs disrupting or newly creating splice site motifs and applied it to sequencing data from 8,976 samples across 31 cancer types. We constructed a catalog of 14,438 SAVs, approximately 50% of which consist of SAVs disrupting non-canonical splice sites (including the 3rd and 5th intronic bases of donor sites) or newly creating splice sites. Smoking-related signature substantially contributes to SAV generation. As many as 14.7% of samples harbor at least one SAVs in cancer-related genes, particularly in tumor suppressors. Importantly, in addition to previously reported intron retention, exon skipping or alternative splice site usage more frequently affected these genes. Our findings delineate a comprehensive portrait of SAVs, providing a basis for cancer precision medicine.

scholarly journals Splice-site Strength Estimation: A simple yet powerful approach to analyse RNA splicing

2020 ◽  
Author(s):  
Craig Dent ◽  
Shilpi Singh ◽  
Shikhar Mishra ◽  
Nawar Shamaya ◽  
Kok Ping Loo ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Intron Retention ◽  
Sequence Divergence ◽  
Exon Skipping ◽  
Splice Sites ◽  
Powerful Approach ◽  
Environmental Responses ◽  
Level Of Selection ◽  
Selection Of

RNA splicing, and variations in this process referred to as alternative splicing, are critical aspects of gene regulation in eukaryotes. From environmental responses in plants to being a primary link between genetic variation and disease in humans, splicing differences confer extensive phenotypic changes across diverse organisms1–3. Current approaches for analysing splicing rely on quantifying variant transcripts (i.e., isoforms) or splicing events (i.e., intron retention, exon skipping etc)4, 5. However, regulation of splicing occurs at the level of selection of individual splice sites, which results in variation in the abundance of isoforms and/or splicing events. Here, we present a simple approach to quantify the strength of individual splice sites, which determines their selection in a splicing reaction. Splice-site strength, as a quantitative phenotype, allows us to analyse splicing precisely in unprecedented ways. We demonstrate the power of this approach in defining the genomic determinants of the strength of individual splice-sites through GWAS. Our pilot-GWAS with more than thousand splice sites hints that cis-sequence divergence and competition between splice-sites and are among the primary determinants of variation in splicing among natural accessions of Arabidopsis thaliana. This approach allows deciphering the principles of splicing, which in turn has implications that range from agriculture to medicine.

scholarly journals Evidence for Splice Site Pairing via Intron Definition in Schizosaccharomyces pombe

2000 ◽  
Vol 20 (21) ◽  
pp. 7955-7970 ◽  
Author(s):  
Charles M. Romfo ◽  
Consuelo J. Alvarez ◽  
Willem J. van Heeckeren ◽  
Christopher J. Webb ◽  
Jo Ann Wise
Keyword(s):  
Drosophila Melanogaster ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Splice Site ◽  
Intron Retention ◽  
Exon Skipping ◽  
Splice Sites ◽  
Internal Exon ◽  
Intron Definition ◽  
Lines Of Evidence

ABSTRACT Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with pre-mRNAs fromDrosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition. Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition. First, mutating either or both splice sites flanking an internal exon in the S. pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates. Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals. Because nearly quantitative removal of the downstream intron incgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion. Third, several cryptic 5′ junctions in the second intron of fission yeastcdc2 are located within the intron, in contrast to their generally exonic locations in metazoa. The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5′ junctions can compete effectively with the standard 5′ splice site if they are closer to the 3′ splicing signals. Taken together, our data suggest that pairing of splice sites inS. pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.

scholarly journals Detection and Functional Verification of Noncanonical Splice Site Mutations in Hereditary Deafness

2021 ◽  
Vol 12 ◽  
Author(s):  
Penghui Chen ◽  
Longhao Wang ◽  
Yongchuan Chai ◽  
Hao Wu ◽  
Tao Yang
Keyword(s):  
Next Generation Sequencing ◽  
Splice Site ◽  
Negative Impact ◽  
Intron Retention ◽  
Exon Skipping ◽  
Next Generation Sequencing Data ◽  
Functional Verification ◽  
Next Generation ◽  
Hereditary Deafness ◽  
Generation Sequencing

Splice site mutations contribute to a significant portion of the genetic causes for mendelian disorders including deafness. By next-generation sequencing of 4 multiplex, autosomal dominant families and 2 simplex, autosomal recessive families with hereditary deafness, we identified a variety of candidate pathogenic variants in noncanonical splice sites of known deafness genes, which include c.1616+3A > T and c.580G > A in EYA4, c.322-57_322-8del in PAX3, c.991-15_991-13del in DFNA5, c.6087-3T > G in PTPRQ and c.164+5G > A in USH1G. All six variants were predicted to affect the RNA splicing by at least one of the computational tools Human Splicing Finder, NNSPLICE and NetGene2. Phenotypic segregation of the variants was confirmed in all families and is consistent with previously reported genotype-phenotype correlations of the corresponding genes. Minigene analysis showed that those splicing site variants likely have various negative impact including exon-skipping (c.1616+3A > T and c.580G > A in EYA4, c.991-15_991-13del in DFNA5), intron retention (c.322-57_322-8del in PAX3), exon skipping and intron retention (c.6087-3T > G in PTPRQ) and shortening of exon (c.164+5G > A in USH1G). Our study showed that the cryptic, noncanonical splice site mutations may play an important role in the molecular etiology of hereditary deafness, whose diagnosis can be facilitated by modified filtering criteria for the next-generation sequencing data, functional verification, as well as segregation, bioinformatics, and genotype-phenotype correlation analysis.

scholarly journals CBMT-38. THERAPEUTIC INHIBITION OF THE SPLICEOSOME IN GLIOBLASTOMA: CHANGES FORM MINOR TO MAJOR

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi41-vi41
Author(s):  
Tobias Bonifert ◽  
Sonali Arora ◽  
Matthew Fitzgibbon ◽  
Patrick Paddison
Keyword(s):  
Clinical Trials ◽  
Splice Site ◽  
Intron Retention ◽  
Exon Skipping ◽  
Clinical Development ◽  
Splice Sites ◽  
Depth Analysis ◽  
U2 Snrnp ◽  
Therapeutic Opportunity ◽  
Site Recognition

Abstract Chemical or genetic perturbation of the spliceosome function may represent a therapeutic opportunity for Glioblastoma multiforme (GBM) and other cancers. We and others have shown that oncogenic MYC activity causes spliceosome vulnerability and leads to deficits in 3’ splice site recognition upon spliceosome perturbation. Several spliceosome inhibitors in pre-clinical development and in clinical trials target the U2 snRNP subcomplex SF3B, which is essential for 3’ splice site recognition. We showed that knockdown of one of its subunits, PHF5A, causes GBM-specific changes in splicing, including increases in exon skipping, intron retention and activation of alternative 3’ and 5’ splice sites. However, in depth analysis of these events revealed they are disproportionately enriched for “minor” introns. This class of ~800 introns is specifically excised by an alternative splicing pathway which involves the U12-dependent spliceosome. Remarkably, we provide evidence that inhibition of PHF5A in GBM stem-like cells causes increased retention of most minor introns, as well as activation of cryptic and alternative U2 snRNP-dependent splicing sites. We further demonstrate that components of the U12 snRNP complex are differentially required for GSC-specific viability. Our results suggest that regulation of the splicing of U12-type introns may be critically important for GBM tumor cell viability.

scholarly journals Isoform transcriptome of developing human brain provides new insights into autism risk variants

2020 ◽  
Author(s):  
Kevin Chau ◽  
Pan Zhang ◽  
Jorge Urresti ◽  
Megha Amar ◽  
Akula Bala Pramod ◽  
...  
Keyword(s):  
Human Brain ◽  
Splice Site ◽  
Exon Skipping ◽  
List Type ◽  
Loss Of Function ◽  
Risk Genes ◽  
Neurodevelopmental Disease ◽  
Gene Level ◽  
Splicing Isoforms ◽  
The Impact

SummaryAlternative splicing plays important role in brain development, however its global contribution to human neurodevelopmental diseases (NDD) has not been fully investigated. Here, we examined the relationships between splicing isoforms expression in the brain and de novo loss-of-function mutations identified in the patients with NDDs. We constructed isoform transcriptome of the developing human brain, and observed differentially expressed isoforms and isoform co-expression modules undetectable by the gene-level analyses. These isoforms were enriched in loss-of-function mutations and microexons, co-expressed with a unique set of partners, and had higher prenatal expression. We experimentally tested the impact of splice site mutations in five NDD risk genes, including SCN2A, DYRK1A and BTRC, and demonstrated exon skipping. Furthermore, our results suggest that the splice site mutation in BTRC reduces translational efficiency, likely impacting Wnt signaling through impaired degradation of β-catenin. We propose that functional effect of mutations associated with human diseases should be investigated at isoform-rather than gene-level resolution.HighlightsDifferential isoform expression analysis of human brain transcriptome reveals neurodevelopmental processes and pathways undetectable by differential gene expression analyses.Splicing isoforms impacted by neurodevelopmental disease (NDD) risk mutations exhibit higher prenatal expression, are enriched in microexons and involved in neuronal-related functions.Isoform co-expression network analysis identifies modules with splicing and synaptic functions that are enriched in NDD mutations.Splice site mutations impacting NDD risk genes cause exon skipping and produce novel isoforms with altered biological properties.Functional impact of mutations should be investigated at isoform-rather than gene-level resolution

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