Investigation for a multi-silique trait in Brassica napus by alternative splicing analysis

Background Flower and fruit development are vital stages of the angiosperm lifecycle. We previously investigated the multi-silique trait in the rapeseed (Brassica napus) line zws-ms on a genomic and transcriptomic level, leading to the identification of two genomic regions and several candidate genes associated with this trait. However, some events on the transcriptome level, like alternative splicing, were poorly understood. Methods Plants from zws-ms and its near-isogenic line (NIL) zws-217 were both grown in Xindu with normal conditions and a colder area Ma’erkang. Buds from the two lines were sampled and RNA was isolated to perform the transcriptomic sequencing. The numbers and types of alternative splicing (AS) events from the two lines were counted and classified. Genes with AS events and expressed differentially between the two lines, as well as genes with AS events which occurred in only one line were emphasized. Their annotations were further studied. Results From the plants in Xindu District, an average of 205,496 AS events, which could be sorted into five AS types, were identified. zws-ms and zws-217 shared highly similar ratios of each AS type: The alternative 5′ and 3′ splice site types were the most common, while the exon skipping type was observed least often. Eleven differentially expressed AS genes were identified, of which four were upregulated and seven were downregulated in zws-ms. Their annotations implied that five of these genes were directly associated with the multi-silique trait. While samples from colder area Ma’erkang generated generally reduced number of each type of AS events except for Intron Retention; but the number of differentially expressed AS genes increased significantly. Further analysis found that among the 11 differentially expressed AS genes from Xindu, three of them maintained the same expression models, while the other eight genes did not show significant difference between the two lines in expression level. Additionally, the 205 line-specific expressed AS genes were analyzed, of which 187 could be annotated, and two were considered to be important. Discussion This study provides new insights into the molecular mechanism of the agronomically important multi-silique trait in rapeseed on the transcriptome level and screens out some environment-responding candidate genes.

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 During the Chlamydomonasreinhardtii Cell Cycle

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401622 ◽

2020 ◽

Vol 10 (10) ◽

pp. 3797-3810

Author(s):

Manishi Pandey ◽

Gary D. Stormo ◽

Susan K. Dutcher

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Alternative Splicing ◽

Chlamydomonas Reinhardtii ◽

Intron Retention ◽

Exon Skipping ◽

Dark Phase ◽

Periodic Patterns ◽

Genome Wide ◽

Splicing Pattern

Genome-wide analysis of transcriptome data in Chlamydomonas reinhardtii shows periodic patterns in gene expression levels when cultures are grown under alternating light and dark cycles so that G1 of the cell cycle occurs in the light phase and S/M/G0 occurs during the dark phase. However, alternative splicing, a process that enables a greater protein diversity from a limited set of genes, remains largely unexplored by previous transcriptome based studies in C. reinhardtii. In this study, we used existing longitudinal RNA-seq data obtained during the light-dark cycle to investigate the changes in the alternative splicing pattern and found that 3277 genes (19.75% of 17,746 genes) undergo alternative splicing. These splicing events include Alternative 5′ (Alt 5′), Alternative 3′ (Alt 3′) and Exon skipping (ES) events that are referred as alternative site selection (ASS) events and Intron retention (IR) events. By clustering analysis, we identified a subset of events (26 ASS events and 10 IR events) that show periodic changes in the splicing pattern during the cell cycle. About two-thirds of these 36 genes either introduce a pre-termination codon (PTC) or introduce insertions or deletions into functional domains of the proteins, which implicate splicing in altering gene function. These findings suggest that alternative splicing is also regulated during the Chlamydomonas cell cycle, although not as extensively as changes in gene expression. The longitudinal changes in the alternative splicing pattern during the cell cycle captured by this study provides an important resource to investigate alternative splicing in genes of interest during the cell cycle in Chlamydomonas reinhardtii and other eukaryotes.

Download Full-text

Swan: a library for the analysis and visualization of long-read transcriptomes

Bioinformatics ◽

10.1093/bioinformatics/btaa836 ◽

2020 ◽

Author(s):

Fairlie Reese ◽

Ali Mortazavi

Keyword(s):

Rna Sequencing ◽

Cell Lines ◽

Intron Retention ◽

Exon Skipping ◽

Differentially Expressed ◽

Transcript Isoforms ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Long Read

Abstract Motivation Long-read RNA-sequencing technologies such as PacBio and Oxford Nanopore have discovered an explosion of new transcript isoforms that are difficult to visually analyze using currently available tools. We introduce the Swan Python library, which is designed to analyze and visualize transcript models. Results Swan finds 4909 differentially expressed transcripts between cell lines HepG2 and HFFc6, including 279 that are differentially expressed even though the parent gene is not. Additionally, Swan discovers 285 reproducible exon skipping and 47 intron retention events not recorded in the GENCODE v29 annotation. Availability and implementation The Swan library for Python 3 is available on PyPi at https://pypi.org/project/swan-vis/ and on GitHub at https://github.com/mortazavilab/swan_vis.

Download Full-text

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

10.1101/2020.11.10.374751 ◽

2020 ◽

Author(s):

Guiomar Martín ◽

Yamile Márquez ◽

Federica Mantica ◽

Paula Duque ◽

Manuel Irimia

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Stress Responses ◽

Target Genes ◽

Intron Retention ◽

Single Gene ◽

Exon Skipping ◽

Environmental Response ◽

Biotic Stresses ◽

External Stimuli

AbstractBackgroundAlternative 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.ResultsWe generated a massive resource for A. thaliana (PastDB; pastdb.crg.eu), comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets revealed that A. thaliana shows high levels of AS (similar to fruitflies) and that, compared to animals, disproportionately uses AS for stress responses. We identified core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that was tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, were overrepresented across regulated AS sets in A. thaliana, being also largely involved in modulating gene expression through NMD and uORF inclusion.ConclusionsNon-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

Novel Form of Alternative Splicing of NFKB1. Its Role in Polycythemia and Adaptation to High Altitude in Andean Aymara

Blood ◽

10.1182/blood-2018-99-117463 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2316-2316

Author(s):

Jihyun Song ◽

Seonggyun Han ◽

Ricardo Amaru ◽

Teddy Quispe ◽

Dongwook Kim ◽

...

Keyword(s):

Alternative Splicing ◽

High Altitude ◽

Cell Activation ◽

Conflicts Of Interest ◽

Intron Retention ◽

Exon Skipping ◽

Cardiovascular Development ◽

Splice Sites ◽

Inflammatory Pathways ◽

Exon 4

Abstract Evolutionary adaptations to high altitude in Tibetans, Ethiopians, and Andean populations of South America have shown that Tibetans and Ethiopians have normal hemoglobin %, while most of Aymara and Quechua of the Andean highlands are polycythemic. Whole genome sequencing (WGS) in Quechua identified enriched SENP1 and ANP32D genes correlating with polycythemia (Zhou et al, Am J Hum Genet. 2013 Sep 5; 93(3): 452-462) but these genes were neither enriched nor segregated with polycythemia in Aymara. Instead, we identified that genes enriched in Aymara are related to regulation of cardiovascular development in high-altitude adapted Andeans, BRINP3, NOS2, and TBX5 (Crawford et al, Am J Hum Genet. 2017 Nov 2;101(5):752-767). To further search for Aymara propensity to polycythemia, we analyzed transcriptomes from Aymara and Europeans living in La Paz, Bolivia (3,639-4,150m) from limited amount of peripheral blood reticulocytes, platelets and granulocytes, but only granulocyte RNA was adequate for unbiased whole transcriptome analyses. In Aymaras, 2,585 genes were upregulated and 365 genes were downregulated (Adjp<0.05, fold difference <-2.0, and >2.0). Many of these modulated genes are involved in inflammatory pathways including B-cell activation (FDR=0.005) and NF-κB signaling pathway (FDR=0.011). We then analyzed differential exon usage in the transcriptome and identified 2,475 genes with alternative splicing events, comprising 1,568 exon skipping, 485 intron retention, 175 alternative 3' splice sites, 144 alternative 5' splice sites, and 902 mutually exclusive exons. These alternative spliced genes were also overrepresented in inflammatory pathways (TNF receptor, IL-1 and IL-23 mediated signaling, and NF-κB signaling). Notably we detected the previously unreported NFKB1 alternate transcripts skipping exon 4 or 5, which lead to the out-of-framed NFKB1 mRNA, generating the truncated nonfunctional NF-κB protein (Figure). Inflammation is a potent suppressor of erythropoiesis and the NF-κB is transcriptional regulator of plethora of inflammatory genes. Further, NF-κB also interacts with erythropoiesis-regulators, hypoxia-inducible factors (HIFs). By the integrative analysis of the Aymara transcriptome and WGS, we identified 46 NFKB1 splicing quantitative trait loci (sQTLs). Among these 46 sQTLs, five single nucleotide polymorphisms (SNP) were in high linkage disequilibrium, and two (rs230511 and rs230504) were more enriched in Aymara (allele frequency: 0.878) (Figure) and within a genomic region where Andeans are genetically differentiated from lowland Native Americans (peak FST = 0.37, peak PBSn1 = 0.31). These sQTLs rs230511 and rs230504 were corelated with two functionally important exon skipping (exon 4 and 5) in NFKB1 as described above. Furthermore, these two SNPs were correlated with higher hemoglobin levels and lower leukocytes; the wild-type NFKB1 transcript inversely correlated with hemoglobin%. We report Aymara have differentially expressed and alternatively spliced transcripts of genes modulating inflammation, particularly NFKB1. This Aymara enriched NFKB1 haplotype variant stands out as a major cause of Aymara adaptation to high altitude, as this truncated nonfunctional NF-κB variant peptide correlates with higher hemoglobin, lower leukocytes and suppresses inflammation. These data indicate that NFKB1 SNPs enriched in Aymara are associated with alternative spliced NFKB1 transcripts which contribute to polycythemia in Aymara. Further evaluation of NF-κB and HIFs' transcriptional activity and their correlation with inflammatory makers, hepcidin and erythroferrone in Aymara and Europeans living at the same high altitude is under way. JS and SH contributed equally to this work. YL and JTP act as equivalent co-senior authors. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Selective Targeting of Alternative Splicing Deregulation in Pediatric Acute Myeloid Stem and Progenitor Cells

Blood ◽

10.1182/blood-2020-136596 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-8

Author(s):

Inge Van Der Werf ◽

Phoebe Mondala ◽

Raymond Diep ◽

Larissa Balaian ◽

Cayla Mason ◽

...

Keyword(s):

Stem Cells ◽

Alternative Splicing ◽

Intron Retention ◽

Leukemia Cell ◽

Exon Skipping ◽

Pediatric Leukemia ◽

The Past ◽

Pediatric Aml ◽

Student’S T ◽

Dose Dependent

INTRODUCTION Because acute myeloid leukemia (AML) is the leading cause of pediatric leukemia relapse-related mortality, there is a desperate need for developing new therapeutics and providing mechanistic insights into the cell type and molecular context specific causes of relapse. Myeloid malignancies, including AML, have a propensity to disrupt the spliceosome either through acquisition of splicing factor gene mutations or epigenetic spliceosome disruption in leukemia stem cells (reviewed in Chua, Van Der Werf, Jamieson, Signer. Cancer Cell 2020;26:138-159). While leukemia stem cells (LSCs) promote relapse in adult AML as a result of their inherent capacity to become dormant and resist therapies that target dividing cells, the role of alternative splicing (AS) deregulation in relapse and therapeutic vulnerability of LSCs has not been thoroughly studied in pediatric AML. METHODS Recently, we completed pre-IND enabling studies with a selective splicing modulator, 17S-FD-895. Previously, we showed that 17S-FD-895 selectively eliminated adult AML LSC (Crews...Burkart, Jamieson. Cell Stem Cell 2016;19:599-612). As a stable pladienolide-derived small molecule splicing modulator, 17S-FD-895 targets a key component of the spliceosome, SF3B1, thereby modulating mRNA splicing. To investigate the role of splicing deregulation in pediatric AML LSC maintenance, we developed a sensitive in vitro and in vivo lentiviral splicing reporter assay, whole transcriptome RNA sequencing (RNA-seq) stem and progenitor cell splicing analysis pipelines, qRT-PCR splice isoform specific biomarkers of response, as well as stromal co-culture, hematopoietic progenitor colony survival and replating assays to assay LSC eradication. RESULTS Because splicing regulation is cell type and context dependent, we generated a comprehensive transcriptome expression map of FACS-purified hematopoietic stem cells (HSCs; CD34+CD38-Lin-) and hematopoietic progenitor cells (HPCs;CD34+CD38+Lin). By utilizing a splice variant-specific alignment algorithm, we evaluated genome wide alternative splicing events and uncovered widespread exon skipping in pediatric AML compared to non-leukemic donors. More than 2000 exon skipping events were identified in pediatric AML HSCs and HPCs. In addition, both pediatric AML HSC and HPC demonstrated a downregulation of the splicing regulator RNA-Binding fox 2 (RBFOX2), which has been linked to embryonic stem cell splice variant signatures that are vital for leukemia cell survival (Holm...Jamieson. PNAS 2015;112:15444-15449; Denichenko et al Nat Commun 2019;10:1590). Treatment with 17S-FD-895 induced a dose-dependent reduction in clonogenicity (p=0.001; Student's t-test) and self-renewal of CD34+ cells (p=0.001; Student's t-test) isolated from pediatric AML samples. Pediatric AML samples were significantly more sensitive to splicing modulation than adult de novo or adult secondary AML. Moreover, normal cord blood HSC and HPC samples were unaffected by splicing modulator treatment. Our lentiviral splicing reporter assays demonstrated a dose dependent increase in MAPT intron retention in in pediatric leukemia cell lines as measured by a switch from GFP to RFP. Finally, splice isoform specific RT-PCR demonstrated a dose-dependent increase in SF3B1 intron retention following treatment as well as MCL1 exon 2 skipping, producing pro-apoptotic MCL1-S transcripts. CONCLUSIONS Cumulatively, our data indicate that spliceosome modulation via 17S-FD-895-mediated targeting of SF3B1 constitutes a novel potential therapeutic strategy for pediatric patients with AML. Disclosures Kaspers: Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; AbbVie: Ended employment in the past 24 months; Janssen R&D: Ended employment in the past 24 months; Helsinn Healthcare: Ended employment in the past 24 months. Crews:Ionis Pharmaceuticals: Research Funding. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.

Download Full-text

spliceR: An R package for classification of alternative splicing and prediction of coding potential from RNA-seq data

10.7287/peerj.preprints.80 ◽

2013 ◽

Author(s):

Kristoffer Vitting-Seerup ◽

Bo T Porse ◽

Albin Sandelin ◽

Johannes E Waage

Keyword(s):

Alternative Splicing ◽

Intron Retention ◽

Exon Skipping ◽

R Package ◽

Full Length ◽

Alternative Transcript ◽

Rna Seq ◽

Donor And Acceptor ◽

Coding Potential

Background: With the increasing depth and decreasing costs of RNA-sequencing researchers are now able to profile the transcriptome with unprecedented detail. These advances not only allow for precise approximation of gene expression levels, but also for the characterization of alternative transcript usage/switching between conditions. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing and prediction of coding potential. Results: spliceR uses the full-length transcripts output from RNA-seq assemblers, to detect single- and multiple exon skipping, alternative donor and acceptor sites, intron retention, alternative first or last exon usage, and mutually exclusive exon events. For each of these events spliceR also annotates the genomic coordinates of the differentially spliced elements facilitating downstream sequence analysis. Furthermore, isoform fraction values are calculated for effective post-filtering, i.e. identification of transcript switching between conditions. Lastly spliceR predicts the coding potential, as well as the potential nonsense mediated decay (NMD) sensitivity of each transcript. Conclusions: spliceR is a easy-to-use tool that allows detection of alternative splicing, transcript switching and NMD sensitivity from RNA-seq data, extending the usability of RNA-seq and assembly technologies. spliceR is implemented as an R package and is freely available from the Bioconductor repository (http://www.bioconductor.org/packages/2.13/bioc/html/spliceR.html).

Download Full-text

Changes in Ovary Transcriptome and Alternative splicing at estrus from Xiang pigs with Large and Small Litter Size

10.1101/547810 ◽

2019 ◽

Author(s):

Fuping Zhang ◽

Liangting Tang ◽

Xueqin Ran ◽

Ning Mao ◽

Yiqi Ruan ◽

...

Keyword(s):

Alternative Splicing ◽

Candidate Genes ◽

Differentially Expressed Genes ◽

Litter Size ◽

Molecular Mechanisms ◽

Reproductive Traits ◽

Differentially Expressed ◽

Rna Seq ◽

Small Litter ◽

Small Litter Size

AbstractBackground/AimsLitter size is one of the most important reproductive traits in pig breeding, which is affected by multiple genes and the environment. Ovaries are the most important reproductive organs and have a profound impact on the reproduction efficiency. Therefore, genetic differences in the ovaries may contribute to the observed differences in litter size. Although QTLs and candidate genes have been reported to affect the litter size in many pig breeds, however, the findings cannot elucidate the marked differences of the reproductive traits between breeds. The aim of present work is to elucidate the mechanisms of the differences for the reproductive traits and identify candidate genes associated with litter size in Xiang pig breed.MethodsThe changes in ovary transcriptome and alternative splicing were investigated at estrus between Xiang pigs with large and small litter size by RNA-seq technology. The RNA-seq results were confirmed by RT-qPCR method.ResultsWe detected 16,219 - 16,285 expressed genes and 12 types of alternative splicing (AS) events in Xiang pig samples. A total of 762 differentially expressed genes were identified by XL (Xiang pig group with larger litter size) vs XS (Xiang pig group with small litter size) sample comparisons. A total of 34 genes were upregulated and 728 genes were downregulated in XL ovary samples compared with the XS samples. Alternative splicing (AS) rates in XL samples were slightly lower than that observed in XS samples. Most of differentially expressed genes were differentially regulated on AS level. Eleven candidate genes were potentially identified to be related to Xiang pig fecundity and litter size, which may be closely related to the gonad development, oocyte maturation or embryo quality.ConclusionThe significant changes in the expression of the protein-coding genes and the level of alternative splicing in estrus ovarian transcriptome between XL and XS groups probably are the molecular mechanisms of phenotypic variation in litter size.

Download Full-text

spliceR: An R package for classification of alternative splicing and prediction of coding potential from RNA-seq data

10.7287/peerj.preprints.80v2 ◽

2013 ◽

Author(s):

Kristoffer Vitting-Seerup ◽

Bo T Porse ◽

Albin Sandelin ◽

Johannes E Waage

Keyword(s):

Alternative Splicing ◽

Intron Retention ◽

Exon Skipping ◽

R Package ◽

Full Length ◽

Alternative Transcript ◽

Rna Seq ◽

Donor And Acceptor ◽

Coding Potential

Download Full-text

Fine Mapping of a New Major QTL-qGLS8 for Gray Leaf Spot Resistance in Maize

Frontiers in Plant Science ◽

10.3389/fpls.2021.743869 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hongbo Qiu ◽

Chunhong Li ◽

Wenzhu Yang ◽

Kang Tan ◽

Qiang Yi ◽

...

Keyword(s):

Transcription Factor ◽

Candidate Genes ◽

Fine Mapping ◽

Phenotypic Variation ◽

Leaf Spot ◽

Gray Leaf Spot ◽

Chromosome 8 ◽

Genetic Maps ◽

Isogenic Line ◽

Significant Difference

Gray leaf spot (GLS), caused by different species of Cercospora, is a fungal, non-soil-borne disease that causes serious reductions in maize yield worldwide. The identification of major quantitative trait loci (QTLs) for GLS resistance in maize is essential for developing marker-assisted selection strategies in maize breeding. Previous research found a significant difference (P < 0.01) in GLS resistance between T32 (highly resistant) and J51 (highly susceptible) genotypes of maize. Initial QTL analysis was conducted in an F2 : 3 population of 189 individuals utilizing genetic maps that were constructed using 181 simple sequence repeat (SSR) markers. One QTL (qGLS8) was detected, defined by the markers umc1130 and umc2354 in three environments. The qGLS8 QTL detected in the initial analysis was located in a 51.96-Mb genomic region of chromosome 8 and explained 7.89–14.71% of the phenotypic variation in GLS resistance in different environments. We also developed a near isogenic line (NIL) BC3F2 population with 1,468 individuals and a BC3F2-Micro population with 180 individuals for fine mapping. High-resolution genetic and physical maps were constructed using six newly developed SSRs. The QTL-qGLS8 was narrowed down to a 124-kb region flanked by the markers ym20 and ym51 and explained up to 17.46% of the phenotypic variation in GLS resistance. The QTL-qGLS8 contained seven candidate genes, such as an MYB-related transcription factor 24 and a C3H transcription factor 347), and long intergenic non-coding RNAs (lincRNAs). The present study aimed to provide a foundation for the identification of candidate genes for GLS resistance in maize.

Download Full-text