A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype

BackgroundAlternative splicing (AS) significantly enhances transcriptome complexity. It is differentially regulated in a wide variety of cell types and plays a role in several cellular processes. Here we describe a detailed survey of alternative splicing in grape based on 124 SOLiD RNAseq analyses from different tissues, stress conditions and genotypes.ResultsWe used the RNAseq data to update the existing grape gene prediction with 2,258 new coding genes and 3,336 putative long non-coding RNAs. Several gene structures have been improved and alternative splicing was described for about 30% of the genes. A link between AS and miRNAs was shown in 139 genes where we found that AS affects the miRNA target site. A quantitative analysis of the isoforms indicated that most of the spliced genes have one major isoform and tend to simultaneously co-express a low number of isoforms, typically two, with intron retention being the most frequent alternative splicing event.ConclusionsAs described in Arabidopsis, also grape displays a marked AS tissue-specificity, while stress conditions produce splicing changes to a minor extent. Surprisingly, some distinctive splicing features were also observed between genotypes. This was further supported by the observation that the panel of Serine/Arginine-rich splicing factors show a few, but very marked differences between genotypes. The finding that a part the splicing machinery can change in closely related organisms can lead to some interesting hypotheses for evolutionary adaptation, that could be particularly relevant in the response to sudden and strong selective pressures.

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title: A High Quality Draft Consensus Sequence of the Genome of a Heterozygous Grapevine Variety
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title: The Grapevine Expression Atlas Reveals a Deep Transcriptome Shift Driving the Entire Plant into a Maturation Program[W][OA]
abstract: The authors developed a comprehensive transcriptome atlas in grapevine by comparing the genes expressed in 54 diverse samples accounting for ∼91% of all known grapevine genes. Using a panel of different statistical techniques, they found that the whole plant undergoes transcriptomic reprogramming, driving it towards maturity. We developed a genome-wide transcriptomic atlas of grapevine (Vitis vinifera) based on 54 samples representing green and woody tissues and organs at different developmental stages as well as specialized tissues such as pollen and senescent leaves. Together, these samples expressed ∼91% of the predicted grapevine genes. Pollen and senescent leaves had unique transcriptomes reflecting their specialized functions and physiological status. However, microarray and RNA-seq analysis grouped all the other samples into two major classes based on maturity rather than organ identity, namely, the vegetative/green and mature/woody categories. This division represents a fundamental transcriptomic reprogramming during the maturation process and was highlighted by three statistical approaches identifying the transcriptional relationships among samples (correlation analysis), putative biomarkers (O2PLS-DA approach), and sets of strongly and consistently expressed genes that define groups (topics) of similar samples (biclustering analysis). Gene coexpression analysis indicated that the mature/woody developmental program results from the reiterative coactivation of pathways that are largely inactive in vegetative/green tissues, often involving the coregulation of clusters of neighboring genes and global regulation based on codon preference. This global transcriptomic reprogramming during maturation has not been observed in herbaceous annual species and may be a defining characteristic of perennial woody plants.

title: De novo transcriptome characterization of Vitis vinifera cv. Corvina unveils varietal diversity
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title: Characterization of Transcriptional Complexity during Berry Development in Vitis vinifera Using RNA-Seq1[W]
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title: Transcriptome analysis at four developmental stages of grape berry (Vitis vinifera cv. Shiraz) provides insights into regulated and coordinated gene expression
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title: The plasticity of the grapevine berry transcriptome
abstract: BackgroundPhenotypic plasticity refers to the range of phenotypes a single genotype can express as a function of its environment. These phenotypic variations are attributable to the effect of the environment on the expression and function of genes influencing plastic traits. We investigated phenotypic plasticity in grapevine by comparing the berry transcriptome in a single clone of the vegetatively-propagated common grapevine species Vitis vinifera cultivar Corvina through 3 consecutive growth years cultivated in 11 different vineyards in the Verona area of Italy.ResultsMost of the berry transcriptome clustered by year of growth rather than common environmental conditions or viticulture practices, and transcripts related to secondary metabolism showed high sensitivity towards different climates, as confirmed also by metabolomic data obtained from the same samples. When analyzed in 11 vineyards during 1 growth year, the environmentally-sensitive berry transcriptome comprised 5% of protein-coding genes and 18% of the transcripts modulated during berry development. Plastic genes were particularly enriched in ontology categories such as transcription factors, translation, transport, and secondary metabolism. Specific plastic transcripts were associated with groups of vineyards sharing common viticulture practices or environmental conditions, and plastic transcriptome reprogramming was more intense in the year characterized by extreme weather conditions. We also identified a set of genes that lacked plasticity, showing either constitutive expression or similar modulation in all berries.ConclusionsOur data reveal candidate genes potentially responsible for the phenotypic plasticity of grapevine and provide the first step towards the characterization of grapevine transcriptome plasticity under different agricultural systems.

title: Plastic, fantastic! Phenotypic variance in the transcriptional landscape of the grape berry
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title: Genome-wide analysis of the grapevine stilbene synthase multigenic family: genomic organization and expression profiles upon biotic and abiotic stresses
abstract: BackgroundPlant stilbenes are a small group of phenylpropanoids, which have been detected in at least 72 unrelated plant species and accumulate in response to biotic and abiotic stresses such as infection, wounding, UV-C exposure and treatment with chemicals. Stilbenes are formed via the phenylalanine/polymalonate-route, the last step of which is catalyzed by the enzyme stilbene synthase (STS), a type III polyketide synthase (PKS). Stilbene synthases are closely related to chalcone synthases (CHS), the key enzymes of the flavonoid pathway, as illustrated by the fact that both enzymes share the same substrates. To date, STSs have been cloned from peanut, pine, sorghum and grapevine, the only stilbene-producing fruiting-plant for which the entire genome has been sequenced. Apart from sorghum, STS genes appear to exist as a family of closely related genes in these other plant species.ResultsIn this study a complete characterization of the STS multigenic family in grapevine has been performed, commencing with the identification, annotation and phylogenetic analysis of all members and integration of this information with a comprehensive set of gene expression analyses including healthy tissues at differential developmental stages and in leaves exposed to both biotic (downy mildew infection) and abiotic (wounding and UV-C exposure) stresses. At least thirty-three full length sequences encoding VvSTS genes were identified, which, based on predicted amino acid sequences, cluster in 3 principal groups designated A, B and C. The majority of VvSTS genes cluster in groups B and C and are located on chr16 whereas the few gene family members in group A are found on chr10. Microarray and mRNA-seq expression analyses revealed different patterns of transcript accumulation between the different groups of VvSTS family members and between VvSTSs and VvCHSs. Indeed, under certain conditions the transcriptional response of VvSTS and VvCHS genes appears to be diametrically opposed suggesting that flow of carbon between these two competing metabolic pathways is tightly regulated at the transcriptional level.ConclusionsThis study represents an overview of the expression pattern of each member of the STS gene family in grapevine under both constitutive and stress-induced conditions. The results strongly indicate the existence of a transcriptional subfunctionalization amongst VvSTSs and provide the foundation for further functional investigations about the role and evolution of this large gene family. Moreover, it represents the first study to clearly show the differential regulation of VvCHS and VvSTS genes, suggesting the involvement of transcription factors (TFs) in both the activation and repression of these genes.

title: Genome-wide mapping of alternative splicing in Arabidopsis thaliana.
abstract: Alternative splicing can enhance transcriptome plasticity and proteome diversity. In plants, alternative splicing can be manifested at different developmental stages, and is frequently associated with specific tissue types or environmental conditions such as abiotic stress. We mapped the Arabidopsis transcriptome at single-base resolution using the Illumina platform for ultrahigh-throughput RNA sequencing (RNA-seq). Deep transcriptome sequencing confirmed a majority of annotated introns and identified thousands of novel alternatively spliced mRNA isoforms. Our analysis suggests that at least approximately 42% of intron-containing genes in Arabidopsis are alternatively spliced; this is significantly higher than previous estimates based on cDNA/expressed sequence tag sequencing. Random validation confirmed that novel splice isoforms empirically predicted by RNA-seq can be detected in vivo. Novel introns detected by RNA-seq were substantially enriched in nonconsensus terminal dinucleotide splice signals. Alternative isoforms with premature termination codons (PTCs) comprised the majority of alternatively spliced transcripts. Using an example of an essential circadian clock gene, we show that intron retention can generate relatively abundant PTC(+) isoforms and that this specific event is highly conserved among diverse plant species. Alternatively spliced PTC(+) isoforms can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery or regulate the level of functional transcripts by the mechanism of regulated unproductive splicing and translation (RUST). We demonstrate that the relative ratios of the PTC(+) and reference isoforms for several key regulatory genes can be considerably shifted under abiotic stress treatments. Taken together, our results suggest that like in animals, NMD and RUST may be widespread in plants and may play important roles in regulating gene expression.

title: Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis.
abstract: Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. As very few genome-wide studies analyzing AS in plants are available, we have performed high-throughput sequencing of a normalized cDNA library which resulted in a high coverage transcriptome map of Arabidopsis. We detect ∼150,000 splice junctions derived mostly from typical plant introns, including an eightfold increase in the number of U12 introns (2069). Around 61% of multiexonic genes are alternatively spliced under normal growth conditions. Moreover, we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using high-resolution RT-PCR and Sanger sequencing. Intron retention (IR) is the most frequent AS event (∼40%), but many IRs have relatively low read coverage and are less well-represented in assembled transcripts. Additionally, ∼51% of Arabidopsis genes produce AS transcripts which do not involve IR. Therefore, the significance of IR in generating transcript diversity was generally overestimated in previous assessments. IR analysis allowed the identification of a large set of cryptic introns inside annotated coding exons. Importantly, a significant fraction of these cryptic introns are spliced out in frame, indicating a role in protein diversity. Furthermore, we show extensive AS coupled to nonsense-mediated decay in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, thus establishing a complex loop in AS regulation. We provide the most comprehensive analysis of AS to date which will serve as a valuable resource for the plant community to study transcriptome complexity and gene regulation.

title: Genome-Wide Analysis Uncovers Regulation of Long Intergenic Noncoding RNAs in Arabidopsis[C][W]
abstract: This work identifies 6480 long intergenic noncoding RNAs in Arabidopsis, many of which show organ-specific and stress-responsive expression. The biogenesis of a group of long intergenic noncoding RNAs is coregulated by the RNA processing proteins SERRATE, CBP20, and CBP80. Long intergenic noncoding RNAs (lincRNAs) transcribed from intergenic regions of yeast and animal genomes play important roles in key biological processes. Yet, plant lincRNAs remain poorly characterized and how lincRNA biogenesis is regulated is unclear. Using a reproducibility-based bioinformatics strategy to analyze 200 Arabidopsis thaliana transcriptome data sets, we identified 13,230 intergenic transcripts of which 6480 can be classified as lincRNAs. Expression of 2708 lincRNAs was detected by RNA sequencing experiments. Transcriptome profiling by custom microarrays revealed that the majority of these lincRNAs are expressed at a level between those of mRNAs and precursors of miRNAs. A subset of lincRNA genes shows organ-specific expression, whereas others are responsive to biotic and/or abiotic stresses. Further analysis of transcriptome data in 11 mutants uncovered SERRATE, CAP BINDING PROTEIN20 (CBP20), and CBP80 as regulators of lincRNA expression and biogenesis. RT-PCR experiments confirmed these three proteins are also needed for splicing of a small group of intron-containing lincRNAs.

title: Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing
abstract: BackgroundBiotic and abiotic stresses, such as powdery mildew infection and high temperature, are important limiting factors for yield and grain quality in wheat production. Emerging evidences suggest that long non-protein coding RNAs (npcRNAs) are developmentally regulated and play roles in development and stress responses of plants. However, identification of long npcRNAs is limited to a few plant species, such as Arabidopsis, rice and maize, no systematic identification of long npcRNAs and their responses to abiotic and biotic stresses is reported in wheat.ResultsIn this study, by using computational analysis and experimental approach we identified 125 putative wheat stress responsive long npcRNAs, which are not conserved among plant species. Among them, some were precursors of small RNAs such as microRNAs and siRNAs, two long npcRNAs were identified as signal recognition particle (SRP) 7S RNA variants, and three were characterized as U3 snoRNAs. We found that wheat long npcRNAs showed tissue dependent expression patterns and were responsive to powdery mildew infection and heat stress.ConclusionOur results indicated that diverse sets of wheat long npcRNAs were responsive to powdery mildew infection and heat stress, and could function in wheat responses to both biotic and abiotic stresses, which provided a starting point to understand their functions and regulatory mechanisms in the future.

title: Genomewide comparative analysis of alternative splicing in plants.
abstract: Alternative splicing (AS) has been extensively studied in mammalian systems but much less in plants. Here we report AS events deduced from EST/cDNA analysis in two model plants: Arabidopsis and rice. In Arabidopsis, 4,707 (21.8%) of the genes with EST/cDNA evidence show 8,264 AS events. Approximately 56% of these events are intron retention (IntronR), and only 8% are exon skipping. In rice, 6,568 (21.2%) of the expressed genes display 14,542 AS events, of which 53.5% are IntronR and 13.8% are exon skipping. The consistent high frequency of  suggests prevalence of splice site recognition by intron definition in plants. Different AS events within a given gene occur, for the most part, independently. In total, 36-43% of the AS events produce transcripts that would be targets of the non-sense-mediated decay pathway, if that pathway were to operate in plants as in humans. Forty percent of Arabidopsis AS genes are alternatively spliced also in rice, with some examples strongly suggesting a role of the AS event as an evolutionary conserved mechanism of posttranscriptional regulation. We created a comprehensive web-interfaced database to compile and visualize the evidence for alternative splicing in plants (Alternative Splicing in Plants, available at www.plantgdb.org/ASIP).

title: Comprehensive analysis of alternative splicing in rice and comparative analyses with Arabidopsis
abstract: BackgroundRecently, genomic sequencing efforts were finished for Oryza sativa (cultivated rice) and Arabidopsis thaliana (Arabidopsis). Additionally, these two plant species have extensive cDNA and expressed sequence tag (EST) libraries. We employed the Program to Assemble Spliced Alignments (PASA) to identify and analyze alternatively spliced isoforms in both species. comprehensive analysis of alternative splicing was performed in rice that started with >1.1 million publicly available spliced ESTs and over 30,000 full length cDNAs in conjunction with the newly enhanced PASA software. A parallel analysis was performed with Arabidopsis to compare and ascertain potential differences between monocots and dicots. Alternative splicing is a widespread phenomenon (observed in greater than 30% of the loci with transcript support) and we have described nine alternative splicing variations. While alternative splicing has the potential to create many RNA isoforms from a single locus, the majority of loci generate only two or three isoforms and transcript support indicates that these isoforms are generally not rare events. For the alternate donor (AD) and acceptor (AA) classes, the distance between the splice sites for the majority of events was found to be less than 50 basepairs (bp). In both species, the most frequent distance between AA is 3 bp, consistent with reports in mammalian systems. Conversely, the most frequent distance between AD is 4 bp in both plant species, as previously observed in mouse. Most alternative splicing variations are localized to the protein coding sequence and are predicted to significantly alter the coding sequence.ConclusionAlternative splicing is widespread in both rice and Arabidopsis and these species share many common features. Interestingly, alternative splicing may play a role beyond creating novel combinations of transcripts that expand the proteome. Many isoforms will presumably have negative consequences for protein structure and function, suggesting that their biological role involves post-transcriptional regulation of gene expression.

title: Complexity of the Alternative Splicing Landscape in Plants[C][W][OPEN]
abstract: 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.

title: Genome-wide analyses of alternative splicing in plants: opportunities and challenges.
abstract: Alternative splicing (AS) creates multiple mRNA transcripts from a single gene. While AS is known to contribute to gene regulation and proteome diversity in animals, the study of its importance in plants is in its early stages. However, recently available plant genome and transcript sequence data sets are enabling a global analysis of AS in many plant species. Results of genome analysis have revealed differences between animals and plants in the frequency of alternative splicing. The proportion of plant genes that have one or more alternative transcript isoforms is approximately 20%, indicating that AS in plants is not rare, although this rate is approximately one-third of that observed in human. The majority of plant AS events have not been functionally characterized, but evidence suggests that AS participates in important plant functions, including stress response, and may impact domestication and trait selection. The increasing availability of plant genome sequence data will enable larger comparative analyses that will identify functionally important plant AS events based on their evolutionary conservation, determine the influence of genome duplication on the evolution of AS, and discover plant-specific cis-elements that regulate AS. This review summarizes recent analyses of AS in plants, discusses the importance of further analysis, and suggests directions for future efforts.

title: Landscape of transcription in human cells
abstract: Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell’s regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.

title: Transcriptome analysis of human tissues and cell lines reveals one dominant transcript per gene
abstract: BackgroundRNA sequencing has opened new avenues for the study of transcriptome composition. Significant evidence has accumulated showing that the human transcriptome contains in excess of a hundred thousand different transcripts. However, it is still not clear to what extent this diversity prevails when considering the relative abundances of different transcripts from the same gene.ResultsHere we show that, in a given condition, most protein coding genes have one major transcript expressed at significantly higher level than others, that in human tissues the major transcripts contribute almost 85 percent to the total mRNA from protein coding loci, and that often the same major transcript is expressed in many tissues. We detect a high degree of overlap between the set of major transcripts and a recently published set of alternatively spliced transcripts that are predicted to be translated utilizing proteomic data. Thus, we hypothesize that although some minor transcripts may play a functional role, the major ones are likely to be the main contributors to the proteome. However, we still detect a non-negligible fraction of protein coding genes for which the major transcript does not code a protein.ConclusionsOverall, our findings suggest that the transcriptome from protein coding loci is dominated by one transcript per gene and that not all the transcripts that contribute to transcriptome diversity are equally likely to contribute to protein diversity. This observation can help to prioritize candidate targets in proteomics research and to predict the functional impact of the detected changes in variation studies.

title: The Transcriptional Landscape of the Mammalian Genome
abstract: This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5′ and 3′ boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

title: Noncoding RNA transcription beyond annotated genes.
abstract: Recent analyses based on high-throughput transcriptome data have revealed that the fraction of the genome that is transcribed largely exceeds the fraction encoding protein. Transcription of unconventional genes into noncoding RNAs is widespread and, in mammals, these RNAs comprise at least half the total number of RNAs transcribed by RNA polymerase II. Although the function of the majority of noncoding RNAs has yet to be discovered, many of them are transcribed from both strands of the genome, and evidence points towards a regulatory function for many noncoding RNAs in mammalian cells.

title: Long noncoding RNA: unveiling hidden layer of gene regulatory networks.
abstract: Long noncoding RNAs (lncRNAs) are increasingly recognized as functional regulatory components in eukaryotic gene regulation. Distinct classes of lncRNAs have been identified in eukaryotes and they play roles in various regulatory networks. Previously characterized lncRNAs include primary transcripts for small regulatory RNAs. In the era of deep sequencing, new classes of lncRNAs have emerged as potent regulatory components in gene regulation. Recent studies showed that many lncRNAs are potent cis- and trans-regulators of gene activity and they can function as scaffolds for chromatin-modifying complexes. Furthermore, differential expressions of lncRNAs suggest that transcription of lncRNAs can modulate gene activity during development and in response to external stimuli. Here, we summarize our current understanding on potential roles of lncRNAs in plants.

title: Widespread Long Noncoding RNAs as Endogenous Target Mimics for MicroRNAs in Plants1[W]
abstract: A computational method systematically identifies intergenic or noncoding gene-originated target mimics (eTMs) for 20 conserved microRNAs in Arabidopsis and rice and validates the biological functions of some eTMs. Target mimicry is a recently identified regulatory mechanism for microRNA (miRNA) functions in plants in which the decoy RNAs bind to miRNAs via complementary sequences and therefore block the interaction between miRNAs and their authentic targets. Both endogenous decoy RNAs (miRNA target mimics) and engineered artificial RNAs can induce target mimicry effects. Yet until now, only the Induced by Phosphate Starvation1 RNA has been proven to be a functional endogenous microRNA target mimic (eTM). In this work, we developed a computational method and systematically identified intergenic or noncoding gene-originated eTMs for 20 conserved miRNAs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). The predicted miRNA binding sites were well conserved among eTMs of the same miRNA, whereas sequences outside of the binding sites varied a lot. We proved that the eTMs of miR160 and miR166 are functional target mimics and identified their roles in the regulation of plant development. The effectiveness of eTMs for three other miRNAs was also confirmed by transient agroinfiltration assay.

title: Computational Identification and Functional Predictions of Long Noncoding RNA in Zea mays
abstract: Computational analysis of cDNA sequences from multiple organisms suggests that a large portion of transcribed DNA does not code for a functional protein. In mammals, noncoding transcription is abundant, and often results in functional RNA molecules that do not appear to encode proteins. Many long noncoding RNAs (lncRNAs) appear to have epigenetic regulatory function in humans, including HOTAIR and XIST. While epigenetic gene regulation is clearly an essential mechanism in plants, relatively little is known about the presence or function of lncRNAs in plants.  To explore the connection between lncRNA and epigenetic regulation of gene expression in plants, a computational pipeline using the programming language Python has been developed and applied to maize full length cDNA sequences to identify, classify, and localize potential lncRNAs. The pipeline was used in parallel with an SVM tool for identifying ncRNAs to identify the maximal number of ncRNAs in the dataset. Although the available library of sequences was small and potentially biased toward protein coding transcripts, 15% of the sequences were predicted to be noncoding. Approximately 60% of these sequences appear to act as precursors for small RNA molecules and may function to regulate gene expression via a small RNA dependent mechanism. ncRNAs were predicted to originate from both genic and intergenic loci. Of the lncRNAs that originated from genic loci, ∼20% were antisense to the host gene loci.  Consistent with similar studies in other organisms, noncoding transcription appears to be widespread in the maize genome. Computational predictions indicate that maize lncRNAs may function to regulate expression of other genes through multiple RNA mediated mechanisms.

title: Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses.
abstract: Large intergenic noncoding RNAs (lincRNAs) are emerging as key regulators of diverse cellular processes. Determining the function of individual lincRNAs remains a challenge. Recent advances in RNA sequencing (RNA-seq) and computational methods allow for an unprecedented analysis of such transcripts. Here, we present an integrative approach to define a reference catalog of >8000 human lincRNAs. Our catalog unifies previously existing annotation sources with transcripts we assembled from RNA-seq data collected from ∼4 billion RNA-seq reads across 24 tissues and cell types. We characterize each lincRNA by a panorama of >30 properties, including sequence, structural, transcriptional, and orthology features. We found that lincRNA expression is strikingly tissue-specific compared with coding genes, and that lincRNAs are typically coexpressed with their neighboring genes, albeit to an extent similar to that of pairs of neighboring protein-coding genes. We distinguish an additional subset of transcripts that have high evolutionary conservation but may include short ORFs and may serve as either lincRNAs or small peptides. Our integrated, comprehensive, yet conservative reference catalog of human lincRNAs reveals the global properties of lincRNAs and will facilitate experimental studies and further functional classification of these genes.

title: Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis.
abstract: Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies.

title: RNA-Seq: a revolutionary tool for transcriptomics
abstract: RNA-Seq is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies. Studies using this method have already altered our view of the extent and complexity of eukaryotic transcriptomes. RNA-Seq also provides a far more precise measurement of levels of transcripts and their isoforms than other methods. This article describes the RNA-Seq approach, the challenges associated with its application, and the advances made so far in characterizing several eukaryote transcriptomes.

title: RNA-Seq improves annotation of protein-coding genes in the cucumber genome
abstract: BackgroundAs more and more genomes are sequenced, genome annotation becomes increasingly important in bridging the gap between sequence and biology. Gene prediction, which is at the center of genome annotation, usually integrates various resources to compute consensus gene structures. However, many newly sequenced genomes have limited resources for gene predictions. In an effort to create high-quality gene models of the cucumber genome (Cucumis sativus var. sativus), based on the EVidenceModeler gene prediction pipeline, we incorporated the massively parallel complementary DNA sequencing (RNA-Seq) reads of 10 cucumber tissues into EVidenceModeler. We applied the new pipeline to the reassembled cucumber genome and included a comparison between our predicted protein-coding gene sets and a published set.ResultsThe reassembled cucumber genome, annotated with RNA-Seq reads from 10 tissues, has 23, 248 identified protein-coding genes. Compared with the published prediction in 2009, approximately 8, 700 genes reveal structural modifications and 5, 285 genes only appear in the reassembled cucumber genome. All the related results, including genome sequence and annotations, are available at http://cmb.bnu.edu.cn/Cucumis_sativus_v20/.ConclusionsWe conclude that RNA-Seq greatly improves the accuracy of prediction of protein-coding genes in the reassembled cucumber genome. The comparison between the two gene sets also suggests that it is feasible to use RNA-Seq reads to annotate newly sequenced or less-studied genomes.

title: RNA-Seq analysis reveals new gene models and alternative splicing in the fungal pathogen Fusarium graminearum
abstract: BackgroundThe genome of Fusarium graminearum has been sequenced and annotated previously, but correct gene annotation remains a challenge. In addition, posttranscriptional regulations, such as alternative splicing and RNA editing, are poorly understood in F. graminearum. Here we took advantage of RNA-Seq to improve gene annotations and to identify alternative splicing and RNA editing in F. graminearum.ResultsWe identified and revised 655 incorrectly predicted gene models, including revisions of intron predictions, intron splice sites and prediction of novel introns. 231 genes were identified with two or more alternative splice variants, mostly due to intron retention. Interestingly, the expression ratios between different transcript isoforms appeared to be developmentally regulated. Surprisingly, no RNA editing was identified in F. graminearum. Moreover, 2459 novel transcriptionally active regions (nTARs) were identified and our analysis indicates that many of these could be missed genes. Finally, we identified the 5′ UTR and/or 3′ UTR sequences of 7666 genes. A number of representative novel gene models and alternatively spliced genes were validated by reverse transcription polymerase chain reaction and sequencing of the generated amplicons.ConclusionsWe have developed novel and efficient strategies to identify alternatively spliced genes and incorrect gene models based on RNA-Seq data. Our study identified hundreds of alternatively spliced genes in F. graminearum and for the first time indicated that alternative splicing is developmentally regulated in filamentous fungi. In addition, hundreds of incorrect predicted gene models were identified and revised and thousands of nTARs were discovered in our study, which will be helpful for the future genomic and transcriptomic studies in F. graminearum.

title: Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome.
abstract: Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative splicing in the rice transcriptome revealed that alternative cis-splicing occurred in approximately 33% of all rice genes. This is far more than previously reported. In addition, we also identified 234 putative chimeric transcripts that seem to be produced by trans-splicing, indicating that transcript fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell. Taken together, our data provide extensive evidence that transcriptional regulation in rice is vastly more complex than previously believed.

title: Mapping and quantifying mammalian transcriptomes by RNA-Seq
abstract: We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41–52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 × 105 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices.

title: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.
abstract: High-throughput mRNA sequencing (RNA-Seq) promises simultaneous transcript discovery and abundance estimation. However, this would require algorithms that are not restricted by prior gene annotations and that account for alternative transcription and splicing. Here we introduce such algorithms in an open-source software program called Cufflinks. To test Cufflinks, we sequenced and analyzed >430 million paired 75-bp RNA-Seq reads from a mouse myoblast cell line over a differentiation time series. We detected 13,692 known transcripts and 3,724 previously unannotated ones, 62% of which are supported by independent expression data or by homologous genes in other species. Over the time series, 330 genes showed complete switches in the dominant transcription start site (TSS) or splice isoform, and we observed more subtle shifts in 1,304 other genes. These results suggest that Cufflinks can illuminate the substantial regulatory flexibility and complexity in even this well-studied model of muscle development and that it can improve transcriptome-based genome annotation.

title: Ab initio reconstruction of cell type–specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs
abstract: Massively parallel cDNA sequencing (RNA-Seq) provides an unbiased way to study a transcriptome, including both coding and noncoding genes. Until now, most RNA-Seq studies have depended crucially on existing annotations and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We applied it to mouse embryonic stem cells, neuronal precursor cells and lung fibroblasts to accurately reconstruct the full-length gene structures for most known expressed genes. We identified substantial variation in protein coding genes, including thousands of novel 5′ start sites, 3′ ends and internal coding exons. We then determined the gene structures of more than a thousand large intergenic noncoding RNA (lincRNA) and antisense loci. Our results open the way to direct experimental manipulation of thousands of noncoding RNAs and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes.

title: Biochemical and physiological responses of two grapevine rootstock genotypes to drought and salt treatments
abstract: In the light of possible climate change, a crucial aspect for future Mediterranean Europe viticulture is the selection of new rootstocks exhibiting adaptability to drought and salt stress. In this context and considering recent literature, it is fundamental to increase our knowledge of the biochemical and physiological events that characterise stress responses in grapevine roots. In this study, leaf and root responses induced by water stress (WS) and sodium chloride (NaCl) exposure in a new selected genotype, named M4 (Vitis vinifera × V. berlandieri) × V. berlandieri cv. Resseguier n. 1), were compared with those of the commercial rootstock 101.14.  The effect of progressive WS and NaCl exposure was studied under controlled environmental conditions on ungrafted plants grown in pots. Shoot growth, plant water status and leaf photosynthetic parameters were measured. The concentration of sugars, amino acids and total proteins, as well as the concentration of the more abundant ions, was determined in both leaf and root organs. The M4 genotype showed greater capacity to maintain photosynthetic activity, to accumulate osmotic compounds as well as to counteract Na and Cl accumulation.  The M4 genotype exhibited a greater capacity to tolerate both WS and exposure to an increasing concentration of Na and Cl, maintaining photosynthetic activity also under severe stress conditions. The root system appeared to play a central role in sustaining biochemical and physiological responses experienced under these adverse conditions.  This study showed that the tolerance to abiotic stress conditions, such as WS and NaCl exposure, depends on root integrity/functionality, confirming that these aspects must be considered in further selection programs.

title: Comparative analysis of grapevine whole-genome gene predictions, functional annotation, categorization and integration of the predicted gene sequences
abstract: BackgroundThe first draft assembly and gene prediction of the grapevine genome (8X base coverage) was made available to the scientific community in 2007, and functional annotation was developed on this gene prediction. Since then additional Sanger sequences were added to the 8X sequences pool and a new version of the genomic sequence with superior base coverage (12X) was produced.ResultsIn order to more efficiently annotate the function of the genes predicted in the new assembly, it is important to build on as much of the previous work as possible, by transferring 8X annotation of the genome to the 12X version. The 8X and 12X assemblies and gene predictions of the grapevine genome were compared to answer the question, “Can we uniquely map 8X predicted genes to 12X predicted genes?” The results show that while the assemblies and gene structure predictions are too different to make a complete mapping between them, most genes (18,725) showed a one-to-one relationship between 8X predicted genes and the last version of 12X predicted genes. In addition, reshuffled genomic sequence structures appeared. These highlight regions of the genome where the gene predictions need to be taken with caution. Based on the new grapevine gene functional annotation and in-depth functional categorization, twenty eight new molecular networks have been created for VitisNet while the existing networks were updated.ConclusionsThe outcomes of this study provide a functional annotation of the 12X genes, an update of VitisNet, the system of the grapevine molecular networks, and a new functional categorization of genes. Data are available at the VitisNet website (http://www.sdstate.edu/ps/research/vitis/pathways.cfm).

title: PASS: a program to align short sequences
abstract: Standard DNA alignment programs are inadequate to manage the data produced by new generation DNA sequencers. To answer this problem, we developed PASS with the objective of improving execution time and sensitivity when compared with other available programs. PASS performs fast gapped and ungapped alignments of short DNA sequences onto a reference DNA, typically a genomic sequence. It is designed to handle a huge amount of reads such as those generated by Solexa, SOLiD or 454 technologies. The algorithm is based on a data structure that holds in RAM the index of the genomic positions of 'seed' words (typically 11 and 12 bases) as well as an index of the precomputed scores of short words (typically seven and eight bases) aligned against each other. After building the genomic index, the program scans every query sequence performing three steps: (1) it finds matching seed words in the genome; (2) for every match checks the precomputed alignment of the short flanking regions; (3) if passes step 2, then it performs an exact dynamic alignment of a narrow region around the match. The performance of the program is very striking both for sensitivity and speed. For instance, gap alignment is achieved hundreds of times faster than BLAST and several times faster than SOAP, especially when gaps are allowed. Furthermore, PASS has a higher sensitivity when compared with the other available programs.



Source code and binaries are freely available for download at http://pass.cribi.unipd.it, implemented in C++and supported on Linux and Windows.

title: IsoLasso: A LASSO Regression Approach to RNA-Seq Based Transcriptome Assembly - (Extended Abstract)
abstract: The new second generation sequencing technology revolu- tionizes many biology related research fields, and posts various compu- tational biology challenges. One of them is transcriptome assembly based on RNA-Seq data, which aims at reconstructing all full-length mRNA transcripts simultaneously from millions of short reads. In this paper, we consider three objectives in transcriptome assembly: the maximization of prediction accuracy, minimization of interpretation, and maximization of completeness. The first objective, the maximization of prediction ac- curacy, requires that the estimated expression levels based on assembled transcripts should be as close as possible to the observed ones for ev- ery expressed region of the genome. The minimization of interpretation follows the parsimony principle to seek as few transcripts in the pre- diction as possible. The third objective, the maximization of complete- ness, requires that the maximum number of mapped reads (or "expressed segments" in gene models) be explained by (i.e., contained in) the pre- dicted transcripts in the solution. Based on the above three objectives, we present IsoLasso, a new RNA-Seq based transcriptome assembly tool. IsoLasso is based on the well-known LASSO algorithm, a multivariate regression method designated to seek a balance between the maximiza- tion of prediction accuracy and the minimization of interpretation. By including some additional constraints in the quadratic program involved in LASSO, IsoLasso is able to make the set of assembled transcripts as complete as possible. Experiments on simulated and real RNA-Seq datasets show that IsoLasso achieves higher sensitivity and precision si- multaneously than the state-of-art transcript assembly tools.

title: Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies.
abstract: The spliced alignment of expressed sequence data to genomic sequence has proven a key tool in the comprehensive annotation of genes in eukaryotic genomes. A novel algorithm was developed to assemble clusters of overlapping transcript alignments (ESTs and full-length cDNAs) into maximal alignment assemblies, thereby comprehensively incorporating all available transcript data and capturing subtle splicing variations. Complete and partial gene structures identified by this method were used to improve The Institute for Genomic Research Arabidopsis genome annotation (TIGR release v.4.0). The alignment assemblies permitted the automated modeling of several novel genes and >1000 alternative splicing variations as well as updates (including UTR annotations) to nearly half of the approximately 27 000 annotated protein coding genes. The algorithm of the Program to Assemble Spliced Alignments (PASA) tool is described, as well as the results of automated updates to Arabidopsis gene annotations.

title: Plant NBS-LRR proteins in pathogen sensing and host defense
abstract: Plant proteins belonging to the nucleotide-binding site–leucine-rich repeat (NBS-LRR) family are used for pathogen detection. Like the mammalian Nod-LRR protein 'sensors' that detect intracellular conserved pathogen-associated molecular patterns, plant NBS-LRR proteins detect pathogen-associated proteins, most often the effector molecules of pathogens responsible for virulence. Many virulence proteins are detected indirectly by plant NBS-LRR proteins from modifications the virulence proteins inflict on host target proteins. However, some NBS-LRR proteins directly bind pathogen proteins. Association with either a modified host protein or a pathogen protein leads to conformational changes in the amino-terminal and LRR domains of plant NBS-LRR proteins. Such conformational alterations are thought to promote the exchange of ADP for ATP by the NBS domain, which activates 'downstream' signaling, by an unknown mechanism, leading to pathogen resistance.

title: ASTALAVISTA: dynamic and flexible analysis of alternative splicing events in custom gene datasets
abstract: In the process of establishing more and more complete annotations of eukaryotic genomes, a constantly growing number of alternative splicing (AS) events has been reported over the last decade. Consequently, the increasing transcript coverage also revealed the real complexity of some variations in the exon–intron structure between transcript variants and the need for computational tools to address ‘complex’ AS events. ASTALAVISTA (alternative splicing transcriptional landscape visualization tool) employs an intuitive and complete notation system to univocally identify such events. The method extracts AS events dynamically from custom gene annotations, classifies them into groups of common types and visualizes a comprehensive picture of the resulting AS landscape. Thus, ASTALAVISTA can characterize AS for whole transcriptome data from reference annotations (GENCODE, REFSEQ, ENSEMBL) as well as for genes selected by the user according to common functional/structural attributes of interest: http://genome.imim.es/astalavista

title: Alternative splicing of pre-messenger RNAs in plants in the genomic era.
abstract: Primary transcripts (precursor-mRNAs) with introns can undergo alternative splicing to produce multiple transcripts from a single gene by differential use of splice sites, thereby increasing the transcriptome and proteome complexity within and between cells and tissues. Alternative splicing in plants is largely an unexplored area of gene expression, as this phenomenon used to be considered rare. However, recent genome-wide computational analyses have revealed that alternative splicing in flowering plants is far more prevalent than previously thought. Interestingly, pre-mRNAs of many spliceosomal proteins, especially serine/arginine-rich (SR) proteins, are extensively alternatively spliced. Furthermore, stresses have a dramatic effect on alternative splicing of pre-mRNAs including those that encode many spliceosomal proteins. Although the mechanisms that regulate alternative splicing in plants are largely unknown, several reports strongly suggest a key role for SR proteins in spliceosome assembly and regulated splicing. Recent studies suggest that alternative splicing in plants is an important posttranscriptional regulatory mechanism in modulating gene expression and eventually plant form and function.

title: Plant microRNA: a small regulatory molecule with big impact.
abstract: MicroRNAs (miRNAs) are an abundant new class of non-coding approximately 20-24 nt small RNAs. To date, 872 miRNAs, belonging to 42 families, have been identified in 71 plant species by genetic screening, direct cloning after isolation of small RNAs, computational strategy, and expressed sequence tag (EST) analysis. Many plant miRNAs are evolutionarily conserved from species to species, some from angiosperms to mosses. miRNAs may originate from inverted duplications of target gene sequences in plants. Although miRNA precursors display high variability, their mature sequences display extensive sequence complementarity to their target mRNA sequences. miRNAs play important roles in plant post-transcriptional gene regulation by targeting mRNAs for cleavage or repressing translation. miRNAs are involved in plant development, signal transduction, protein degradation, response to environmental stress and pathogen invasion, and regulate their own biogenesis. miRNAs regulate the expression of many important genes; a majority of these genes are transcriptional factors.

title: psRNATarget: a plant small RNA target analysis server.
abstract: Plant endogenous non-coding short small RNAs (20-24 nt), including microRNAs (miRNAs) and a subset of small interfering RNAs (ta-siRNAs), play important role in gene expression regulatory networks (GRNs). For example, many transcription factors and development-related genes have been reported as targets of these regulatory small RNAs. Although a number of miRNA target prediction algorithms and programs have been developed, most of them were designed for animal miRNAs which are significantly different from plant miRNAs in the target recognition process. These differences demand the development of separate plant miRNA (and ta-siRNA) target analysis tool(s). We present psRNATarget, a plant small RNA target analysis server, which features two important analysis functions: (i) reverse complementary matching between small RNA and target transcript using a proven scoring schema, and (ii) target-site accessibility evaluation by calculating unpaired energy (UPE) required to 'open' secondary structure around small RNA's target site on mRNA. The psRNATarget incorporates recent discoveries in plant miRNA target recognition, e.g. it distinguishes translational and post-transcriptional inhibition, and it reports the number of small RNA/target site pairs that may affect small RNA binding activity to target transcript. The psRNATarget server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster. The server front-end integrates three simplified user-friendly interfaces to accept user-submitted or preloaded small RNAs and transcript sequences; and outputs a comprehensive list of small RNA/target pairs along with the online tools for batch downloading, key word searching and results sorting. The psRNATarget server is freely available at

title: Alternative mRNA processing increases the complexity of microRNA-based gene regulation in Arabidopsis.
abstract: MicroRNAs (miRNAs) represent an important class of sequence-specific, trans-acting endogenous small RNA molecules that modulate gene expression at the post-transcriptional level. They function by binding to partial complementary cis-regulatory sites (miRNA binding sites) in their target mRNAs. Based on two recent observations from plant genome studies, namely that alternative splicing is a common phenomenon and that miRNA regulates a significant proportion of the transcriptome, we hypothesize that there may be a mechanism for gene regulation that involves both processes. In the present study, we performed a systemic search in the model plant Arabidopsis thaliana using annotated gene models as well as publically available high-throughput RNA sequencing data with a total of 570 million reads. Of the 354 high-confidence miRNA binding sites identified in Arabidopsis, at least 44 (12.4%) were affected by alternative splicing such that mRNA isoforms of the same miRNA target gene differ in the sequences encoding the miRNA binding sites. By simulation, we found that the frequency of alternative splicing at miRNA binding sites is significantly higher than at other regions. Comparative and functional analyses further indicated that the alternative splicing events are important for target gene expression and miRNA action. Together our results show that alternative splicing of miRNA binding sites is a plausible mechanism for attenuating miRNA-mediated gene regulation.

title: Plant serine/arginine-rich proteins and their role in pre-mRNA splicing.
abstract: Pre-messenger RNA (pre-mRNA) splicing, a process by which mature mRNAs are generated by excision of introns and ligation of exons, is an important step in the regulation of gene expression in all eukaryotes. Selection of alternative splice sites in a pre-mRNA generates multiple mRNAs from a single gene that encode structurally and functionally distinct proteins. Alternative splicing of pre-mRNAs contributes greatly to the proteomic complexity of plants and animals and increases the coding potential of a genome. However, the mechanisms that regulate constitutive and alternative splicing of pre-mRNA are not understood in plants. A serine/arginine-rich (SR) family of proteins is implicated in constitutive and alternative splicing of pre-mRNAs. Here I review recent progress in elucidating the roles of serine/arginine-rich proteins in pre-mRNA splicing.

title: The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression.
abstract: The human genome contains many thousands of long noncoding RNAs (lncRNAs). While several studies have demonstrated compelling biological and disease roles for individual examples, analytical and experimental approaches to investigate these genes have been hampered by the lack of comprehensive lncRNA annotation. Here, we present and analyze the most complete human lncRNA annotation to date, produced by the GENCODE consortium within the framework of the ENCODE project and comprising 9277 manually annotated genes producing 14,880 transcripts. Our analyses indicate that lncRNAs are generated through pathways similar to that of protein-coding genes, with similar histone-modification profiles, splicing signals, and exon/intron lengths. In contrast to protein-coding genes, however, lncRNAs display a striking bias toward two-exon transcripts, they are predominantly localized in the chromatin and nucleus, and a fraction appear to be preferentially processed into small RNAs. They are under stronger selective pressure than neutrally evolving sequences-particularly in their promoter regions, which display levels of selection comparable to protein-coding genes. Importantly, about one-third seem to have arisen within the primate lineage. Comprehensive analysis of their expression in multiple human organs and brain regions shows that lncRNAs are generally lower expressed than protein-coding genes, and display more tissue-specific expression patterns, with a large fraction of tissue-specific lncRNAs expressed in the brain. Expression correlation analysis indicates that lncRNAs show particularly striking positive correlation with the expression of antisense coding genes. This GENCODE annotation represents a valuable resource for future studies of lncRNAs.

title: A plethora of plant serine/arginine-rich proteins: redundancy or evolution of novel gene functions?
abstract: Precursor-mRNA (pre-mRNA) processing is an important step in gene expression and its regulation leads to the expansion of the gene product repertoire. SR (serine-arginine)-rich proteins are key players in intron recognition and spliceosome assembly and significantly contribute to the alternative splicing process. Due to several duplication events, at least 19 SR proteins are present in the Arabidopsis genome, which is almost twice as many as in humans. They fall into seven different subfamilies, three of them homologous with metazoan splicing factors, whereas the other four seem to be specific for plants. The current results show that most of the duplicated genes have different spatiotemporal expression patterns indicating functional diversification. Interestingly, most of the SR protein genes are alternatively spliced and in some cases this process was shown to be under developmental and/or environmental control. This might greatly influence gene expression of target genes as also exemplified by ectopic expression studies of particular SR proteins.

title: Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses.
abstract: Precursor mRNAs with introns can undergo alternative splicing (AS) to produce structurally and functionally different proteins from the same gene. Here, we show that the pre-mRNAs of Arabidopsis genes that encode serine/arginine-rich (SR) proteins, a conserved family of splicing regulators in eukaryotes, are extensively alternatively spliced. Remarkably about 95 transcripts are produced from only 15 genes, thereby increasing the complexity of the SR gene family transcriptome by six-fold. The AS of some SR genes is controlled in a developmental and tissue-specific manner. Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) dramatically altered the AS of pre-mRNAs of several SR genes, whereas hormones altered the splicing of only three SR genes. These results indicate that abiotic stresses regulate the AS of the pre-mRNAs of SR genes to produce different isoforms of SR proteins that are likely to have altered function(s) in pre-mRNA splicing. Sequence analysis of splice variants revealed that predicted proteins from a majority of these variants either lack one or more modular domains or contain truncated domains. Because of the modular nature of the various domains in SR proteins, the proteins produced from splice variants are likely to have distinct functions. Together our results indicate that Arabidopsis SR genes generate surprisingly large transcriptome complexity, which is altered by stresses and hormones.

title: Differential expression of alternatively spliced mRNAs of Arabidopsis SR protein homologs, atSR30 and atSR45a, in response to environmental stress.
abstract: Serine/arginine-rich (SR) proteins are associated with either the regulation or the execution of both constitutive splicing and the selection of alternative splice sites in animals and plants. We demonstrated the molecular characterization of a homolog of SR protein, atSR45a, in Arabidopsis plants. Six types of mRNA variants (atSR45a-1a-e and atSR45a-2) were generated by the alternative selection of transcriptional initiation sites and the alternative splicing of introns in atSR45a pre-mRNA. The atSR45a-1a and -2 proteins, presumed mature forms, were located in the nucleus and interacted with U1-70K, suggesting that these proteins function as a splicing factor in Arabidopsis. The levels of the transcripts atSR45a and atSR30, SF2/ASF-like SR proteins, were increased by various types of stress, such as high-light irradiation and salinity. Furthermore, the splicing patterns of atSR45a and atSR30 pre-mRNA themselves were altered under these stressful conditions. In particular, the expression of atSR45a-1a, atSR45a-2, atSR30 mRNA1 and atSR30 mRNA3 was greatly increased by high-light irradiation. These results indicate that the regulation of transcription and alternative splicing of atSR45a and atSR30 is responsive to various stressful conditions.

title: Alternative splicing in plants – coming of age
abstract: More than 60% of intron-containing genes undergo alternative splicing (AS) in plants. This number will increase when AS in different tissues, developmental stages, and environmental conditions are explored. Although the functional impact of AS on protein complexity is still understudied in plants, recent examples demonstrate its importance in regulating plant processes. AS also regulates transcript levels and the link with nonsense-mediated decay and generation of unproductive mRNAs illustrate the need for both transcriptional and AS data in gene expression analyses. AS has influenced the evolution of the complex networks of regulation of gene expression and variation in AS contributed to adaptation of plants to their environment and therefore will impact strategies for improving plant and crop phenotypes.

title: Multiple reference genomes and transcriptomes for Arabidopsis thaliana
abstract: Genetic differences between Arabidopsis thaliana accessions underlie the plant’s extensive phenotypic variation, and until now these have been interpreted largely in the context of the annotated reference accession Col-0. Here we report the sequencing, assembly and annotation of the genomes of 18 natural A. thaliana accessions, and their transcriptomes. When assessed on the basis of the reference annotation, one-third of protein-coding genes are predicted to be disrupted in at least one accession. However, re-annotation of each genome revealed that alternative gene models often restore coding potential. Gene expression in seedlings differed for nearly half of expressed genes and was frequently associated with cis variants within 5 kilobases, as were intron retention alternative splicing events. Sequence and expression variation is most pronounced in genes that respond to the biotic environment. Our data further promote evolutionary and functional studies in A. thaliana, especially the MAGIC genetic reference population descended from these accessions.

title: Grape berry ripening delay induced by a pre-véraison NAA treatment is paralleled by a shift in the expression pattern of auxin- and ethylene-related genes
abstract: BackgroundAuxins act as repressors of ripening inception in grape (véraison), while ethylene and abscisic acid (ABA) play a positive role as inducers of the syndrome. Despite the increasing amount of information made available on this topic, the complex network of interactions among these hormones remains elusive. In order to shed light on these aspects, a holistic approach was adopted to evaluate, at the transcriptomic level, the crosstalk between hormones in grape berries, whose ripening progression was delayed by applying naphtalenacetic acid (NAA) one week before véraison.ResultsThe NAA treatment caused significant changes in the transcription rate of about 1,500 genes, indicating that auxin delayed grape berry ripening also at the transcriptional level, along with the recovery of a steady state of its intracellular concentration. Hormone indices analysis carried out with the HORMONOMETER tool suggests that biologically active concentrations of auxins were achieved throughout a homeostatic recovery. This occurred within 7 days after the treatment, during which the physiological response was mainly unspecific and due to a likely pharmacological effect of NAA. This hypothesis is strongly supported by the up-regulation of genes involved in auxin conjugation (GH3-like) and action (IAA4- and IAA31-like). A strong antagonistic effect between auxin and ethylene was also observed, along with a substantial ‘synergism’ between auxins and ABA, although to a lesser extent.ConclusionsThis study suggests that, in presence of altered levels of auxins, the crosstalk between hormones involves diverse mechanisms, acting at both the hormone response and biosynthesis levels, creating a complex response network.

title: Basic local alignment search tool.
abstract: A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.

title: Automated generation of heuristics for biological sequence comparison
abstract: BackgroundExhaustive methods of sequence alignment are accurate but slow, whereas heuristic approaches run quickly, but their complexity makes them more difficult to implement. We introduce bounded sparse dynamic programming (BSDP) to allow rapid approximation to exhaustive alignment. This is used within a framework whereby the alignment algorithms are described in terms of their underlying model, to allow automated development of efficient heuristic implementations which may be applied to a general set of sequence comparison problems.ResultsThe speed and accuracy of this approach compares favourably with existing methods. Examples of its use in the context of genome annotation are given.ConclusionsThis system allows rapid implementation of heuristics approximating to many complex alignment models, and has been incorporated into the freely available sequence alignment program, exonerate.

title: Gene prediction with a hidden Markov model and a new intron submodel
abstract: The problem of finding the genes in eukaryotic DNA sequences by computational methods is still not satisfactorily solved. Gene finding programs have achieved relatively high accuracy on short genomic sequences but do not perform well on longer sequences with an unknown number of genes in them. Here existing programs tend to predict many false exons.



We have developed a new program, AUGUSTUS, for the ab initio prediction of protein coding genes in eukaryotic genomes. The program is based on a Hidden Markov Model and integrates a number of known methods and submodels. It employs a new way of modeling intron lengths. We use a new donor splice site model, a new model for a short region directly upstream of the donor splice site model that takes the reading frame into account and apply a method that allows better GC-content dependent parameter estimation. AUGUSTUS predicts on longer sequences far more human and drosophila genes accurately than the ab initio gene prediction programs we compared it with, while at the same time being more specific.



A web interface for AUGUSTUS and the executable program are located at http://augustus.gobics.de.

title: AUGUSTUS: a web server for gene finding in eukaryotes
abstract: We present a www server for AUGUSTUS, a novel software program for ab initio gene prediction in eukaryotic genomic sequences. Our method is based on a generalized Hidden Markov Model with a new method for modeling the intron length distribution. This method allows approximation of the true intron length distribution more accurately than do existing programs. For genomic sequence data from human and Drosophila melanogaster, the accuracy of AUGUSTUS is superior to existing gene-finding approaches. The advantage of our program becomes apparent especially for larger input sequences containing more than one gene. The server is available at http://augustus.gobics.de.

title: Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments
abstract: EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

title: CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine
abstract: Recent transcriptome studies have revealed that a large number of transcripts in mammals and other organisms do not encode proteins but function as noncoding RNAs (ncRNAs) instead. As millions of transcripts are generated by large-scale cDNA and EST sequencing projects every year, there is a need for automatic methods to distinguish protein-coding RNAs from noncoding RNAs accurately and quickly. We developed a support vector machine-based classifier, named Coding Potential Calculator (CPC), to assess the protein-coding potential of a transcript based on six biologically meaningful sequence features. Tenfold cross-validation on the training dataset and further testing on several large datasets showed that CPC can discriminate coding from noncoding transcripts with high accuracy. Furthermore, CPC also runs an order-of-magnitude faster than a previous state-of-the-art tool and has higher accuracy. We developed a user-friendly web-based interface of CPC at http://cpc.cbi.pku.edu.cn. In addition to predicting the coding potential of the input transcripts, the CPC web server also graphically displays detailed sequence features and additional annotations of the transcript that may facilitate users’ further investigation.

title: InterProScan - an integration platform for the signature-recognition methods in InterPro
abstract: InterProScan is a tool that scans given protein sequences against the protein signatures of the InterPro member databases, currently--PROSITE, PRINTS, Pfam, ProDom and SMART. The number of signature databases and their associated scanning tools as well as the further refinement procedures make the problem complex. InterProScan is designed to be a scalable and extensible system with a robust internal architecture.


AVAILABILITY
The Perl-based InterProScan implementation is available from the EBI ftp server (ftp://ftp.ebi.ac.uk/pub/software/unix/iprscan/) and the SRS-basedInterProScan is available upon request. We provide the public web interface (http://www.ebi.ac.uk/interpro/scan.html) as well as email submission server (interproscan@ebi.ac.uk).

title: EMBOSS: the European Molecular Biology Open Software Suite.
abstract: The European Molecular Biology Open Software Suite (EMBOSS) is a mature package of software tools developed for the molecular biology community. It includes a comprehensive set of applications for molecular sequence analysis and other tasks and integrates popular third-party software packages under a consistent interface. EMBOSS includes extensive C programming libraries and is a platform to develop and release software in the true open source spirit.

title: New Assembly, Reannotation and Analysis of the Entamoeba histolytica Genome Reveal New Genomic Features and Protein Content Information
abstract: In order to maintain genome information accurately and relevantly, original genome annotations need to be updated and evaluated regularly. Manual reannotation of genomes is important as it can significantly reduce the propagation of errors and consequently diminishes the time spent on mistaken research. For this reason, after five years from the initial submission of the Entamoeba histolytica draft genome publication, we have re-examined the original 23 Mb assembly and the annotation of the predicted genes.  The evaluation of the genomic sequence led to the identification of more than one hundred artifactual tandem duplications that were eliminated by re-assembling the genome. The reannotation was done using a combination of manual and automated genome analysis. The new 20 Mb assembly contains 1,496 scaffolds and 8,201 predicted genes, of which 60% are identical to the initial annotation and the remaining 40% underwent structural changes. Functional classification of 60% of the genes was modified based on recent sequence comparisons and new experimental data. We have assigned putative function to 3,788 proteins (46% of the predicted proteome) based on the annotation of predicted gene families, and have identified 58 protein families of five or more members that share no homology with known proteins and thus could be entamoeba specific. Genome analysis also revealed new features such as the presence of segmental duplications of up to 16 kb flanked by inverted repeats, and the tight association of some gene families with transposable elements.  This new genome annotation and analysis represents a more refined and accurate blueprint of the pathogen genome, and provides an upgraded tool as reference for the study of many important aspects of E. histolytica biology, such as genome evolution and pathogenesis.

title: Modelling and simulating generic RNA-Seq experiments with the flux simulator
abstract: High-throughput sequencing of cDNA libraries constructed from cellular RNA complements (RNA-Seq) naturally provides a digital quantitative measurement for every expressed RNA molecule. Nature, impact and mutual interference of biases in different experimental setups are, however, still poorly understood—mostly due to the lack of data from intermediate protocol steps. We analysed multiple RNA-Seq experiments, involving different sample preparation protocols and sequencing platforms: we broke them down into their common—and currently indispensable—technical components (reverse transcription, fragmentation, adapter ligation, PCR amplification, gel segregation and sequencing), investigating how such different steps influence abundance and distribution of the sequenced reads. For each of those steps, we developed universally applicable models, which can be parameterised by empirical attributes of any experimental protocol. Our models are implemented in a computer simulation pipeline called the Flux Simulator, and we show that read distributions generated by different combinations of these models reproduce well corresponding evidence obtained from the corresponding experimental setups. We further demonstrate that our in silico RNA-Seq provides insights about hidden precursors that determine the final configuration of reads along gene bodies; enhancing or compensatory effects that explain apparently controversial observations can be observed. Moreover, our simulations identify hitherto unreported sources of systematic bias from RNA hydrolysis, a fragmentation technique currently employed by most RNA-Seq protocols.