Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation.

Complexity of the Alternative Splicing Landscape in Plants

High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely.

/pdf/alternative-splicing-at-the-intersection-of-biological-7tohgrm2d3.pdf

Alternative Splicing at the Intersection of Biological Timing, Development, and Stress Responses

Summary
Arabidopsis thaliana is a long established model species for plant molecular biology, genetics and genomics, and studies of A.thaliana gene function provide the basis for formulating hypotheses and designing experiments involving other plants, including economically important species. A comprehensive understanding of the A.thaliana genome and a detailed and accurate understanding of the expression of its associated genes is therefore of great importance for both fundamental research and practical applications. Such goal is reliant on the development of new genetic and genomic resources, involving new methods of data acquisition and analysis. We present here the genome-wide analysis of A.thaliana gene expression profiles across different organs and developmental stages using high-throughput transcriptome sequencing. The expression of 25,706 protein-coding genes, as well as their stability and their spatiotemporal specificity, was assessed in 79 organs and developmental stages. A search for alternative splicing events identified 37,873 previously unreported splice junctions, approximately 30% of occurred in intergenic regions. These potentially represent novel spliced genes that are not included in the TAIR10 database. These data are housed in an open-access web-based database, TraVA (Transcriptome Variation Analysis, http://travadb.org/), which allows visualization and analysis of gene expression profiles and differential gene expression between organs and developmental stages.

This article is protected by copyright. All rights reserved.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/tpj.13312

A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling.

Photosystem II (PSII) is an integral-membrane, multisubunit complex that initiates electron flow in oxygenic photosynthesis. The biogenesis of this complex machine involves the concerted assembly of at least 20 different polypeptides as well as the incorporation of a variety of inorganic and organic cofactors. Many factors have recently been identified that constitute an integrative network mediating the stepwise assembly of PSII components. One recurring theme is the subcellular organization of the assembly process in specialized membranes that form distinct biogenesis centers. Here, we review our current knowledge of the molecular components and events involved in PSII assembly and their high degree of evolutionary conservation.

Photosystem II assembly: from cyanobacteria to plants.

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5′ and 3′ untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser.

/pdf/rna-seq-of-arabidopsis-pollen-uncovers-novel-transcription-3t9m8iz4yj.pdf

RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing

 Premise of the study: High-throughput sequencing of cDNA libraries prepared from diverse samples (RNA-seq) can reveal genome-wide changes in alternative splicing. Using RNA-seq data to assess splicing at the level of individual genes requires the ability to visualize read alignments alongside genomic annotations. To meet this need, we added RNA-seq visualization capability to Integrated Genome Browser (IGB), a free desktop genome visualization tool. To illustrate this capability, we present an in-depth analysis of abiotic stresses and their effects on alternative splicing of SR45a (AT1G07350), a putative splicing regulator from Arabidopsis thaliana .  Methods: cDNA libraries prepared from Arabidopsis plants that were subjected to heat and dehydration stresses were sequenced on an Illumina GAIIx sequencer, yielding more than 511 million high-quality 75-base, single-end sequence reads. Reads were aligned onto the reference genome and visualized in IGB.  Key results: Using IGB, we confi rmed exon-skipping alternative splicing in SR45a. Exon-skipped variant AT1G07350.1 encodes full-length SR45a protein with intact RS and RNA recognition motifs, while nonskipped variant AT1G07350.2 lacks the C-terminal RS region due to a frameshift in the alternative exon. Heat and drought stresses increased both transcript abundance and the proportion of exon-skipped transcripts encoding the full-length protein. We identifi ed new splice sites and observed frequent intron retention fl anking the alternative exon.  Conclusions: This study underlines the importance of visual inspection of RNA-seq alignments when investigating alternatively spliced genes. We showed that heat and dehydration stresses increase overall abundance of SR45a mRNA while also increasing production of transcripts encoding the full-length SR45a protein relative to other splice variants.

https://bsapubs.onlinelibrary.wiley.com/doi/pdf/10.3732/ajb.1100355

Mining Arabidopsis thaliana RNA-seq data with Integrated Genome Browser reveals stress-induced alternative splicing of the putative splicing regulator SR45a

Around 14% of protein-coding genes of Arabidopsis thaliana genes from the TAIR9 genome release are annotated as producing multiple transcript variants through alternative splicing. However, for most alternatively spliced genes in Arabidopsis, the relative expression level of individual splicing variants is unknown. We investigated prevalence of alternative splicing (AS) events in Arabidopsis thaliana using ESTs. We found that for most AS events with ample EST coverage, the majority of overlapping ESTs strongly supported one major splicing choice, with less than 10% of ESTs supporting the minor form. Analysis of ESTs also revealed a small but noteworthy subset of genes for which alternative choices appeared with about equal prevalence, suggesting that for these genes the variant splicing forms co-occur in the same cell types. Of the AS events in which both forms were about equally prevalent, more than 80% affected untranslated regions or involved small changes to the encoded protein sequence. Currently available evidence from ESTs indicates that alternative splicing in Arabidopsis occurs and affects many genes, but for most genes with documented alternative splicing, one AS choice predominates. To aid investigation of the role AS may play in modulating function of Arabidopsis genes, we provide an on-line resource (ArabiTag) that supports searching AS events by gene, by EST library keyword search, and by relative prevalence of minor and major forms.

/pdf/prevalence-of-alternative-splicing-choices-in-arabidopsis-23ax9dlx3w.pdf

Prevalence of alternative splicing choices in Arabidopsis thaliana

Ultrahigh-throughput sequencing of cDNA (RNA-Seq) is an invaluable resource for investigating alternative splicing in an organism. Alternative splicing is a form of posttranscriptional regulation in which primary RNA transcripts from a single gene can be spliced in multiple ways leading to different RNA and protein products. In plants and other species, it has been shown that many genes involved in circadian regulation are alternatively spliced. As new RNA-Seq data sets become available, these data will lead to new insights into links between regulation RNA splicing and the circadian system. Analyzing RNA-Seq data sets requires software tools that can display RNA-Seq read alignments alongside gene models, enabling assessment of how treatments or developmental stages affect splicing patterns and production of novel variants. The Integrated Genome Browser (IGB) software program is a free and flexible desktop tool that enables discovery and quantification of alternative splicing. In this protocol, we use IGB and a cold-stress RNA-Seq data set to examine alternative splicing of Arabidopsis thaliana LHY, a circadian clock regulator. IGB is freely available from http://www.bioviz.org .

A protocol for visual analysis of alternative splicing in RNA-Seq data using integrated genome browser.

1 Background: Blueberries are a rich source of antioxidants and other beneficial compounds that 2

https://biorxiv.org/content/biorxiv/early/2014/10/08/010116.full.pdf

Ketan Patel

Papers

RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing

Mining Arabidopsis thaliana RNA-seq data with Integrated Genome Browser reveals stress-induced alternative splicing of the putative splicing regulator SR45a

Prevalence of alternative splicing choices in Arabidopsis thaliana

A protocol for visual analysis of alternative splicing in RNA-Seq data using integrated genome browser.

candidate genes involved in fruit ripening, biosynthesis of bioactive compounds, and stage- 2 specific alternative splicing 3