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SummaryEukaryotic genes are interrupted by introns that are removed from transcribed RNAs by splicing. The extent of alternative splicing is the best genomic predictor of developmental complexity, yet it is unclear what mediates change in patterns of splicing complexity between species. Here we show that variation in 5’ splice site sequence preferences correlate with the presence of the U6 snRNA methyltransferase METTL16 and the splicing factor SNRNP27K. We used inter-species association mapping with Saccharomycotina species to correlate splicing phenotypes with the presence or absence of splicing factors. The greatest variation in 5’ splice site sequence occurred at the +4 position and involved a preference switch between adenosine and uridine. Loss of METTL16 and SNRNP27K orthologs, or a single SNRNP27K methionine residue, was associated with a preference for +4U. These findings are consistent with splicing analyses of mutants defective in either METTL16 or SNRNP27K orthologs and models derived from spliceosome structures, demonstrating that inter-species association mapping is a powerful orthogonal approach to molecular studies. We conclude that variation in concerted processes of 5’ splice site selection by U6 snRNA is crucial to evolutionary change in splicing complexity.

Plants coordinate their growth and development through complex regulatory networks involving changes in the expression of thousands of genes. Many developmental pathways are regulated at the level of messenger RNA (mRNA) through alternative choices in mRNA processing. These choices can have consequences for the localization, stability or translatability of mRNAs. One of the key ways in which RNAs are processed is by the methylation of the RNA base adenosine – a modification known as m6A. Even though it was first discovered in the 1970s, the biological significance of m6A marks has only recently become clear. In this feature article, we identify the factors controlling the writing and reading of m6A modifications in plants. We also highlight some of the features of plant development that depend on m6A and explore the recently discovered molecular mechanisms that use m6A to control development or response to environmental stress.

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5' splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAll the information necessary to build the proteins that perform the biological processes required for life is encoded in the DNA of an organism. Making these proteins requires the DNA sequence of a gene to be transcribed into a ‘messenger RNA’ (mRNA), which is then processed into a final, mature form. This blueprint is then translated to assemble the corresponding protein. When an mRNA is processed, segments of the sequence that do not code for protein are removed and the remaining coding sequences are joined together in the right order. An intricate molecular machine known as the spliceosome controls this mechanism by recognising the ‘splice sites’ where coding and non-coding sequences meet. Depending on external conditions, the spliceosome can ‘pick-and-mix’ the coding sequences to create different processed mRNAs (and therefore proteins) from a single gene. This alternative splicing mechanism is often used to regulate when certain biological processes take place based on environmental cues; for example, the splicing of genes which control the timing of plant flowering is sensitive to ambient temperatures. To investigate this mechanism, Parker et al. focused on

The methyltransferase complex (m6A writer), which catalyzes the deposition of N6-methyladenosine (m6A) in mRNAs, is highly conserved across most eukaryotic organisms, but its components and interactions between them are still far from fully understood. Here, using in vivo interaction proteomics, two HAKAI-interacting zinc finger proteins, HIZ1 and HIZ2, are discovered as components of the Arabidopsis m6A writer complex. HAKAI is required for the interaction between HIZ1 and MTA (mRNA adenosine methylase A). Whilst HIZ1 knockout plants have normal levels of m6A, plants in which it is overexpressed show reduced methylation and decreased lateral root formation. Mutant plants lacking HIZ2 are viable but have an 85% reduction in m6A abundance and show severe developmental defects. Our findings suggest that HIZ2 is likely the plant equivalent of ZC3H13 (Flacc) of the metazoan m6A-METTL Associated Complex.

The methyltransferase complex (m

SummaryYanocomp is a tool for predicting the positions and stoichiometries of RNA modifications in Nanopore direct RNA sequencing data. It uses general mixture models to identify differentially modified sites between two conditions, with good support for replicates. Yanocomp models across adjacent kmers and uses a uniform component to account for outliers, improving the accuracy of single molecule predictions. Consequently, Yanocomp can be used to measure modification stoichiometry, and correlate modifications with other RNA processing events.AvailabilityYanocomp is available under an MIT license at www.github.com/bartongroup/yanocomp.

Genes involved in disease resistance are some of the fastest evolving and most diverse components of genomes. Large numbers ofnucleotide-binding,leucine-rich repeatreceptor (NLR) genes are found in plant genomes and are required for disease resistance. However, NLRs can trigger autoimmunity, disrupt beneficial microbiota or reduce fitness. It is therefore crucial to understand how NLRs are controlled. Here we show that the RNA-binding protein FPA mediates widespread premature cleavage and polyadenylation of NLR transcripts, thereby controlling their functional expression and impacting immunity. Using long-read Nanopore direct RNA sequencing, we resolved the complexity of NLR transcript processing and gene annotation. Our results uncover a co-transcriptional layer of NLR control with implications for understanding the regulatory and evolutionary dynamics of NLRs in the immune responses of plants.

The nutrient-rich tubers of the greater yam Dioscorea alata L. provide food and income security for millions of people around the world. Despite its global importance, however, greater yam remains an “orphan crop.” Here we address this resource gap by presenting a highly-contiguous chromosome-scale genome assembly of greater yam combined with a dense genetic map derived from African breeding populations. The genome sequence reveals an ancient lineage-specific genome duplication, followed by extensive genome-wide reorganization. Using our new genomic tools we find quantitative trait loci for susceptibility to anthracnose, a damaging fungal pathogen of yam, and several tuber quality traits. Genomic analysis of breeding lines reveals both extensive inbreeding as well as regions of extensive heterozygosity that may represent interspecific introgression during domestication. These tools and insights will enable yam breeders to unlock the potential of this staple crop and take full advantage of its adaptability to varied environments.

Transcription of eukaryotic genomes involves complex alternative processing of RNAs. Sequencing of full-length RNAs using long reads reveals the true complexity of processing. However, the relatively high error rates of long-read sequencing technologies can reduce the accuracy of intron identification. Here we apply alignment metrics and machine-learning-derived sequence information to filter spurious splice junctions from long-read alignments and use the remaining junctions to guide realignment in a two-pass approach. This method, available in the software package 2passtools (https://github.com/bartongroup/2passtools), improves the accuracy of spliced alignment and transcriptome assembly for species both with and without existing high-quality annotations.

The nutrient-rich tubers of the greater yam, Dioscorea alata L., provide food and income security for millions of people around the world. Despite its global importance, however, greater yam remains an orphan crop. Here, we address this resource gap by presenting a highly contiguous chromosome-scale genome assembly of D. alata combined with a dense genetic map derived from African breeding populations. The genome sequence reveals an ancient allotetraploidization in the Dioscorea lineage, followed by extensive genome-wide reorganization. Using the genomic tools, we find quantitative trait loci for resistance to anthracnose, a damaging fungal pathogen of yam, and several tuber quality traits. Genomic analysis of breeding lines reveals both extensive inbreeding as well as regions of extensive heterozygosity that may represent interspecific introgression during domestication. These tools and insights will enable yam breeders to unlock the potential of this staple crop and take full advantage of its adaptability to varied environments.

Genes involved in disease resistance are some of the fastest evolving and most diverse components of genomes. Large numbers of nucleotide-binding, leucine-rich repeat (NLR) genes are found in plant genomes and are required for disease resistance. However, NLRs can trigger autoimmunity, disrupt beneficial microbiota or reduce fitness. It is therefore crucial to understand how NLRs are controlled. Here, we show that the RNA-binding protein FPA mediates widespread premature cleavage and polyadenylation of NLR transcripts, thereby controlling their functional expression and impacting immunity. Using long-read Nanopore direct RNA sequencing, we resolved the complexity of NLR transcript processing and gene annotation. Our results uncover a co-transcriptional layer of NLR control with implications for understanding the regulatory and evolutionary dynamics of NLRs in the immune responses of plants.

Transcription of eukaryotic genomes involves complex alternative processing of RNAs. Sequencing of full-length RNAs using long reads reveals the true complexity of processing. However, the relatively high error rates of long-read sequencing technologies can reduce the accuracy of intron identification. Here we apply alignment metrics and machine-learning-derived sequence information to filter spurious splice junctions from long read alignments and use the remaining junctions to guide realignment in a two-pass approach. This method, available in the software package 2passtools (https://github.com/bartongroup/2passtools), improves the accuracy of spliced alignment and transcriptome assembly for species both with and without existing high-quality annotations.

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

MotivationAntisense transcription is known to have a range of impacts on sense gene expression, including (but not limited to) impeding transcription initiation, disrupting post-transcriptional processes, and enhancing, slowing, or even preventing transcription of the sense gene. Strand-specific RNA-Seq protocols preserve the strand information of the original RNA in the data, and so can be used to identify where antisense transcription may be implicated in regulating gene expression. However, our analysis of 199 strand-specific RNA-Seq experiments reveals that spurious antisense reads are often present in these datasets at levels greater than 1% of sense gene expression levels. Furthermore, these levels can vary substantially even between replicates in the same experiment, potentially disrupting any downstream analysis, if the incorrectly assigned antisense counts dominate the set of genes with high antisense transcription levels. Currently, no tools exist to detect or correct for this spurious antisense signal.ResultsOur tool, RoSA (Removal of Spurious Antisense), detects the presence of high levels of spurious antisense read alignments in strand-specific RNA-Seq datasets. It uses incorrectly spliced reads on the antisense strand and/or ERCC spike-ins (if present in the data) to calculate both global and gene-specific antisense correction factors. We demonstrate the utility of our tool to filter out spurious antisense transcript counts in an Arabidopsis thaliana RNA-Seq experiment.AvailabilityRoSA is open source software available under the GPL licence via the Barton Group GitHub page https://github.com/bartongroup.Contactg.j.barton@dundee.ac.uk, g.g.simpson@dundee.ac.uk

Motivation RNA-seq experiments are usually carried out in three or fewer replicates. In order to work well with so few samples, differential gene expression (DGE) tools typically assume the form of the underlying gene expression distribution. In this paper, the statistical properties of gene expression from RNA-seq are investigated in the complex eukaryote, Arabidopsis thaliana, extending and generalizing the results of previous work in the simple eukaryote Saccharomyces cerevisiae. Results We show that, consistent with the results in S.cerevisiae, more gene expression measurements in A.thaliana are consistent with being drawn from an underlying negative binomial distribution than either a log-normal distribution or a normal distribution, and that the size and complexity of the A.thaliana transcriptome does not influence the false positive rate performance of nine widely used DGE tools tested here. We therefore recommend the use of DGE tools that are based on the negative binomial distribution. Availability and implementation The raw data for the 17 WT Arabidopsis thaliana datasets is available from the European Nucleotide Archive (E-MTAB-5446). The processed and aligned data can be visualized in context using IGB (Freese et al., 2016), or downloaded directly, using our publicly available IGB quickload server at https://compbio.lifesci.dundee.ac.uk/arabidopsisQuickload/public_quickload/ under ‘RNAseq>Froussios2019’. All scripts and commands are available from github at https://github.com/bartongroup/KF_arabidopsis-GRNA. Supplementary information Supplementary data are available at Bioinformatics online.

Antisense transcription is known to have a range of impacts on sense gene expression, including (but not limited to) impeding transcription initiation, disrupting post-transcriptional processes, and enhancing, slowing, or even preventing transcription of the sense gene. Strand-specific RNA-Seq protocols preserve the strand information of the original RNA in the data, and so can be used to identify where antisense transcription may be implicated in regulating gene expression. However, our analysis of 199 strand-specific RNA-Seq experiments reveals that spurious antisense reads are often present in these datasets at levels greater than 1% of sense gene expression levels. Furthermore, these levels can vary substantially even between replicates in the same experiment, potentially disrupting any downstream analysis, if the incorrectly assigned antisense counts dominate the set of genes with high antisense transcription levels. Currently, no tools exist to detect or correct for this spurious antisense signal. Our tool, RoSA (Removal of Spurious Antisense), detects the presence of high levels of spurious antisense read alignments in strand-specific RNA-Seq datasets. It uses incorrectly spliced reads on the antisense strand and/or ERCC spikeins (if present in the data) to calculate both global and gene-specific antisense correction factors. We demonstrate the utility of our tool to filter out spurious antisense transcript counts in an Arabidopsis thaliana RNA-Seq experiment. Availability: RoSA is open source software available under the GPL licence via the Barton Group GitHub page https://github.com/bartongroup.

Plants control the time at which they flower in order to ensure reproductive success. This control is underpinned by precision in gene regulation acting through genetically separable pathways. The genetic dissection of this process in the model plant Arabidopsis thaliana has led to the recurrent identification of plant-specific and highly conserved RNA 3' end processing factors required to control flowering by specifically controlling transcription of mRNA encoding the floral repressor FLOWERING LOCUS C (FLC). Here, we review the features of these RNA-processing and RNA-associated proteins, and the complex architecture of coding and non-coding RNA transcription at the FLC locus. We discuss alternative concepts that might explain how these RNA-processing events regulate FLC transcription and hence control flowering time.

Lignin Biosynthesis Complications Lignin is a polymer that lends its sturdy properties to wood and makes plant cell walls tougher, which creates problems for chemists converting cellulosic plant biomass into biofuels. Vanholme et al. (p. 1103 , published online 15 August; see the cover) have identified a new step in the biosynthetic pathway of lignin in Arabidopsis in which caffeoyl shikimate esterase catalyzes synthesis of caffeate. Cellulose from mutant plants, which had reduced amounts of lignin, was more efficiently processed into glucose.

RNA-seq experiments are usually carried out in three or fewer replicates. In order to work well with so few samples, Differential Gene Expression (DGE) tools typically assume the form of the underlying distribution of gene expression. A recent highly replicated study revealed that RNA-seq gene expression measurements in yeast are best represented as being drawn from an underlying negative binomial distribution. In this paper, the statistical properties of gene expression in the higher eukaryote Arabidopsis thaliana are shown to be essentially identical to those from yeast despite the large increase in the size and complexity of the transcriptome: Gene expression measurements from this model plant species are consistent with being drawn from an underlying negative binomial or log-normal distribution and the false positive rate performance of nine widely used DGE tools is not strongly affected by the additional size and complexity of the A. thaliana transcriptome. For RNA-seq data, we therefore recommend the use of DGE tools that are based on the negative binomial distribution.

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%–40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.

The Arabidopsis protein FPA controls flowering time by regulating the alternative 3'-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3'-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.

Flowering time is controlled by precision in gene regulation mediated by different pathways. Two Arabidopsis thaliana components of the autonomous flowering pathway, FCA and FPA, function as genetically independent trans-acting regulators of alternative cleavage and polyadenylation. FCA and FPA directly associate with chromatin at the locus encoding the floral repressor FLC, but appear to control FLC transcription by mediating alternative polyadenylation of embedded non-coding antisense RNAs. These findings prompt the re-examination of how other factors control FLC expression, as it is formally possible that they function primarily to control alternative processing of antisense RNAs. As co-expressed sense and antisense gene pairs are widespread in eukaryotes, alternative processing of antisense RNAs may represent a significant form of gene regulation.

SummaryThe spen family protein FPA is required for flowering time control and has been implicated in RNA silencing. The mechanism by which FPA carries out these functions is unknown. We report the identification of an activity for FPA in controlling mRNA 3′ end formation. We show that FPA functions redundantly with FCA, another RNA binding protein that controls flowering and RNA silencing, to control the expression of alternatively polyadenylated antisense RNAs at the locus encoding the floral repressor FLC. In addition, we show that defective 3′ end formation at an upstream RNA polymerase II-dependent gene explains the apparent derepression of the AtSN1 retroelement in fpa mutants. Transcript readthrough accounts for the absence of changes in DNA methylation and siRNA abundance at AtSN1 in fpa mutants, and this may explain other examples of epigenetic transitions not associated with chromatin modification.

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%-40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to85% for the subset of SDE genes changing in expression by more than fourfold. To achieve85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.

Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a “splicing memory” hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events.

Spring has arrived as I sit down to write this article in the garden of my Dundee home. Next to the dry-stane dyke and ahead of my Aquilegia, Arabidopsis plants have come into flower. They must think there is something special about this time of year, as I've seen them first come into flower here in April of the last 3 years (Figure 1). How do they know that April has arrived?

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCAtranscript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fymutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3′-end processing.

Mechanisms that mediate the control of flowering time have been accessed through a molecular genetic approach in Arabidopsis. Flowering is regulated by different pathways and, in the past year, all of the known components of the so-called autonomous pathway have been identified. The autonomous pathway comprises a combination of factors involved in RNA processing and epigenetic regulation that downregulate the floral repressor, FLOWERING LOCUS C (FLC). However, components of the autonomous pathway are more widely conserved in plant species other than Arabidopsis than is FLC. Therefore, the broadest lessons we learn from dissecting the function of the autonomous pathway may be in revealing how precision in regulated gene expression is delivered.

Multiple environmental and endogenous inputs regulate when plants flower. The molecular genetic dissection of flowering time control in Arabidopsis has identified an integrated network of pathways that quantitatively control the timing of this developmental switch. This framework provides the basis to understand the evolution of different reproductive strategies and how floral pathways interact through seasonal progression.

▪ Abstract At a certain stage in their life cycle, plants switch from vegetative to reproductive development. This transition is regulated by multiple developmental and environmental cues. These ensure that the plant switches to flowering at a time when sufficient internal resources have been accumulated and the environmental conditions are favorable. The use of a molecular genetic approach in Arabidopsis has resulted in the identification and cloning of many of the genes involved in regulating floral transition. The current view on the molecular function of these genes, their division into different genetic pathways, and how the pathways interact in a complex regulatory network are summarized.

The complex control of flowering time ensures that plants flower in conditions favourable for reproductive success. A recent study adds another dimension to this established complexity by revealing that nitric oxide (NO) represses flowering in Arabidopsis. The analysis of recently identified mutants that either overproduce or are compromised in endogenous NO production has identified NO-sensitive features of the circuitry of flowering time control: NO acts to repress the amplification of gene expression dependent on the circadian clock and promotes the accumulation of mRNA encoding a key repressor of flowering, FLC.

The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3′ untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3′ polyadenylation sites to within +/− 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3′ UTR re-annotation (including extension of one 3′ UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data.

RNA-binding proteins (RBPs) play an important role in plant host-microbe interactions. In this study, we show that the plant RBP known as FPA, which regulates 3'-end mRNA polyadenylation, negatively regulates basal resistance to bacterial pathogen Pseudomonas syringae in Arabidopsis. A custom microarray analysis reveals that flg22, a peptide derived from bacterial flagellins, induces expression of alternatively polyadenylated isoforms of mRNA encoding the defence-related transcriptional repressor ETHYLENE RESPONSE FACTOR 4 (ERF4), which is regulated by FPA. Flg22 induces expression of a novel isoform of ERF4 that lacks the ERF-associated amphiphilic repression (EAR) motif, while FPA inhibits this induction. The EAR-lacking isoform of ERF4 acts as a transcriptional activator in vivo and suppresses the flg22-dependent reactive oxygen species burst. We propose that FPA controls use of proximal polyadenylation sites of ERF4, which quantitatively limit the defence response output.

It has recently been shown that RNA 3'-end formation plays a more widespread role in controlling gene expression than previously thought. To examine the impact of regulated 3'-end formation genome-wide, we applied direct RNA sequencing to A. thaliana. Here we show the authentic transcriptome in unprecedented detail and describe the effects of 3'-end formation on genome organization. We reveal extreme heterogeneity in RNA 3' ends, discover previously unrecognized noncoding RNAs and propose widespread reannotation of the genome. We explain the origin of most poly(A)(+) antisense RNAs and identify cis elements that control 3'-end formation in different registers. These findings are essential to understanding what the genome actually encodes, how it is organized and how regulated 3'-end formation affects these processes.