A data-driven genome annotation approach for cassava.

Genome annotation files play a critical role in dictating the quality of downstream analyses by providing essential predictions for gene positions and structures. These files are pivotal in decoding the complex information encoded within DNA sequences. Here, we generated experimental data resolving RNA 5'- and 3'-ends as well as full-length RNAs for cassava TME12 sticklings in ambient temperature and cold. We used these data to generate genome annotation files using the TranscriptomeReconstructoR (TR) tool. A careful comparison to high-quality genome annotations suggests that our new TR genome annotations identified additional genes, resolved the transcript boundaries more accurately and identified additional RNA isoforms. We enhanced existing cassava genome annotation files with the information from TR that maintained the different transcript models as RNA isoforms. The resultant merged annotation was subsequently utilized for comprehensive analysis. To examine the effects of genome annotation files on gene expression studies, we compared the detection of differentially expressed genes during cold using the same RNA-seq data but alternative genome annotation files. We found that our merged genome annotation that included cold-specific TR gene models identified about twice as many cold-induced genes. These data indicate that environmentally induced genes may be missing in off-the-shelf genome annotation files. In conclusion, TR offers the opportunity to enhance crop genome annotations with implications for the discovery of differentially expressed candidate genes during plant-environment interactions.

LncRNA FLAIL affects alternative splicing and represses flowering in Arabidopsis.

How the noncoding genome affects cellular functions is a key biological question. A particular challenge is to distinguish the effects of noncoding DNA elements from long noncoding RNAs (lncRNAs) that coincide at the same loci. Here, we identified the flowering-associated intergenic lncRNA (FLAIL) in Arabidopsis through early flowering flail mutants. Expression of FLAIL RNA from a different chromosomal location in combination with strand-specific RNA knockdown characterized FLAIL as a trans-acting RNA molecule. FLAIL directly binds to differentially expressed target genes that control flowering via RNA-DNA interactions through conserved sequence motifs. FLAIL interacts with protein and RNA components of the spliceosome to affect target mRNA expression through co-transcriptional alternative splicing (AS) and linked chromatin regulation. In the absence of FLAIL, splicing defects at the direct FLAIL target flowering gene LACCASE 8 (LAC8) correlated with reduced mRNA expression. Double mutant analyses support a model where FLAIL-mediated splicing of LAC8 promotes its mRNA expression and represses flowering. Our study suggests lncRNAs as accessory components of the spliceosome that regulate AS and gene expression to impact organismal development.

Functional variation in the non-coding genome: molecular implications for food security.

The growing world population, in combination with the anticipated effects of climate change, is pressuring food security. Plants display an impressive arsenal of cellular mechanisms conferring resilience to adverse environmental conditions, and humans rely on these mechanisms for stable food production. The elucidation of the molecular basis of the mechanisms used by plants to achieve resilience promises knowledge-based approaches to enhance food security. DNA sequence polymorphisms can reveal genomic regions that are linked to beneficial traits of plants. However, our ability to interpret how a given DNA sequence polymorphism confers a fitness advantage at the molecular level often remains poor. A key factor is that these polymorphisms largely localize to the enigmatic non-coding genome. Here, we review the functional impact of sequence variations in the non-coding genome on plant biology in the context of crop breeding and agricultural traits. We focus on examples of non-coding with particularly convincing functional support. Our survey combines findings that are consistent with the view that the non-coding genome contributes to cellular mechanisms assisting many plant traits. Understanding how DNA sequence polymorphisms in the non-coding genome shape plant traits at the molecular level offers a largely unexplored reservoir of solutions to address future challenges in plant growth and resilience.

Cotranscriptional RNA processing and modification in plants.

The activities of RNA polymerases shape the epigenetic landscape of genomes with profound consequences for genome integrity and gene expression. A fundamental event during the regulation of eukaryotic gene expression is the coordination between transcription and RNA processing. Most primary RNAs mature through various RNA processing and modification events to become fully functional. While pioneering results positioned RNA maturation steps after transcription ends, the coupling between the maturation of diverse RNA species and their transcription is becoming increasingly evident in plants. In this review, we discuss recent advances in our understanding of the crosstalk between RNA Polymerase II, IV, and V transcription and nascent RNA processing of both coding and noncoding RNAs.

Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis.

Histone chaperones and chromatin remodelers control nucleosome dynamics, which are essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display a preference for the HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefers the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated histone deposition for proper epigenetic regulation of the genome.

R-loops at microRNA encoding loci promote co-transcriptional processing of pri-miRNAs in plants.

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency.

Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA and a divergent non-coding (DNC) transcript of unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. Using high-throughput reverse genetic screens based on quantitative single-cell fluorescence measurements, we identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC transcription. Nascent transcription profiling showed a genome-wide role of Hda1C in repression of DNC transcription. Live-cell imaging of transcription revealed that mutations in the Hda3 subunit increased the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC transcription regions, supporting DNC transcription repression by histone deacetylation. Our data support the interpretation that DNC transcription results as a consequence of the NDR-based architecture of eukaryotic promoters, but that it is governed by locus-specific repression to maintain genome fidelity.

TrancriptomeReconstructoR: data-driven annotation of complex transcriptomes.

BACKGROUND: The quality of gene annotation determines the interpretation of results obtained in transcriptomic studies. The growing number of genome sequence information calls for experimental and computational pipelines for de novo transcriptome annotation. Ideally, gene and transcript models should be called from a limited set of key experimental data. RESULTS: We developed TranscriptomeReconstructoR, an R package which implements a pipeline for automated transcriptome annotation. It relies on integrating features from independent and complementary datasets: (i) full-length RNA-seq for detection of splicing patterns and (ii) high-throughput 5' and 3' tag sequencing data for accurate definition of gene borders. The pipeline can also take a nascent RNA-seq dataset to supplement the called gene model with transient transcripts. We reconstructed de novo the transcriptional landscape of wild type Arabidopsis thaliana seedlings and Saccharomyces cerevisiae cells as a proof-of-principle. A comparison to the existing transcriptome annotations revealed that our gene model is more accurate and comprehensive than the most commonly used community gene models, TAIR10 and Araport11 for A.thaliana and SacCer3 for S.cerevisiae. In particular, we identify multiple transient transcripts missing from the existing annotations. Our new annotations promise to improve the quality of A.thaliana and S.cerevisiae genome research. CONCLUSIONS: Our proof-of-concept data suggest a cost-efficient strategy for rapid and accurate annotation of complex eukaryotic transcriptomes. We combine the choice of library preparation methods and sequencing platforms with the dedicated computational pipeline implemented in the TranscriptomeReconstructoR package. The pipeline only requires prior knowledge on the reference genomic DNA sequence, but not the transcriptome. The package seamlessly integrates with Bioconductor packages for downstream analysis.

Winter fields antisense RNAs to kick off flowering.

FLOWERING LOCUS C (FLC), a MADS-box transcription factor, plays a major role in determining flowering time in Arabidopsis In this issue of Genes & Development, Zhao and colleagues (pp. 888-898) elucidate the role of COOLAIR antisense noncoding RNAs in FLC regulation through field trials and laboratory experiments. COOLAIR-mediated FLC silencing is induced by the first seasonal frost in the field and thus acts as a key molecular indicator during autumn for winter arrival.

Light-Dependent Translation Change of Arabidopsis psbA Correlates with RNA Structure Alterations at the Translation Initiation Region.

mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5' UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes.

DNA Processing in the Context of Noncoding Transcription.

RNA polymerase (RNAP)II frequently transcribes non-protein-coding DNA sequences in eukaryotic genomes into long noncoding RNA (lncRNA). Distinct molecular mechanisms linked to the position of lncRNA relative to the coding gene illustrate how noncoding transcription controls gene expression. Here, we focus on the impact of the act of lncRNA transcription on nearby functional DNA units. We review the biological significance of the act of lncRNA transcription on DNA processing, highlighting common themes, such as mediating cellular responses to environmental changes. This review combines the background of chromatin signaling with examples in several organisms to clarify when functions of ncDNA can be interpreted through the act of RNAPII transcription.

A G(enomic)P(ositioning)S(ystem) for Plant RNAPII Transcription.

Post-translational modifications (PTMs) of histone residues shape the landscape of gene expression by modulating the dynamic process of RNA polymerase II (RNAPII) transcription. The contribution of particular histone modifications to the definition of distinct RNAPII transcription stages remains poorly characterized in plants. Chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) resolves the genomic distribution of histone modifications. Here, we review histone PTM ChIP-seq data in Arabidopsis thaliana and find support for a Genomic Positioning System (GPS) that guides RNAPII transcription. We review the roles of histone PTM 'readers', 'writers', and 'erasers', with a focus on the regulation of gene expression and biological functions in plants. The distinct functions of RNAPII transcription during the plant transcription cycle may rely, in part, on the characteristic histone PTM profiles that distinguish transcription stages.

Transcript isoform sequencing reveals widespread promoter-proximal transcriptional termination in Arabidopsis.

RNA polymerase II (RNAPII) transcription converts the DNA sequence of a single gene into multiple transcript isoforms that may carry alternative functions. Gene isoforms result from variable transcription start sites (TSSs) at the beginning and polyadenylation sites (PASs) at the end of transcripts. How alternative TSSs relate to variable PASs is poorly understood. Here, we identify both ends of RNA molecules in Arabidopsis thaliana by transcription isoform sequencing (TIF-seq) and report four transcript isoforms per expressed gene. While intragenic initiation represents a large source of regulated isoform diversity, we observe that ~14% of expressed genes generate relatively unstable short promoter-proximal RNAs (sppRNAs) from nascent transcript cleavage and polyadenylation shortly after initiation. The location of sppRNAs correlates with the position of promoter-proximal RNAPII stalling, indicating that large pools of promoter-stalled RNAPII may engage in transcriptional termination. We propose that promoter-proximal RNAPII stalling-linked to premature transcriptional termination may represent a checkpoint that governs plant gene expression.

Organismal benefits of transcription speed control at gene boundaries.

RNA polymerase II (RNAPII) transcription is crucial for gene expression. RNAPII density peaks at gene boundaries, associating these key regions for gene expression control with limited RNAPII movement. The connections between RNAPII transcription speed and gene regulation in multicellular organisms are poorly understood. Here, we directly modulate RNAPII transcription speed by point mutations in the second largest subunit of RNAPII in Arabidopsis thaliana. A RNAPII mutation predicted to decelerate transcription is inviable, while accelerating RNAPII transcription confers phenotypes resembling auto-immunity. Nascent transcription profiling revealed that RNAPII complexes with accelerated transcription clear stalling sites at both gene ends, resulting in read-through transcription. The accelerated transcription mutant NRPB2-Y732F exhibits increased association with 5' splice site (5'SS) intermediates and enhanced splicing efficiency. Our findings highlight potential advantages of RNAPII stalling through local reduction in transcription speed to optimize gene expression for the development of multicellular organisms.

Native elongation transcript sequencing reveals temperature dependent dynamics of nascent RNAPII transcription in Arabidopsis.

Temperature profoundly affects the kinetics of biochemical reactions, yet how large molecular complexes such as the transcription machinery accommodate changing temperatures to maintain cellular function is poorly understood. Here, we developed plant native elongating transcripts sequencing (plaNET-seq) to profile genome-wide nascent RNA polymerase II (RNAPII) transcription during the cold-response of Arabidopsis thaliana with single-nucleotide resolution. Combined with temporal resolution, these data revealed transient genome-wide reprogramming of nascent RNAPII transcription during cold, including characteristics of RNAPII elongation and thousands of non-coding transcripts connected to gene expression. Our results suggest a role for promoter-proximal RNAPII stalling in predisposing genes for transcriptional activation during plant-environment interactions. At gene 3'-ends, cold initially facilitated transcriptional termination by limiting the distance of read-through transcription. Within gene bodies, cold reduced the kinetics of co-transcriptional splicing leading to increased intragenic stalling. Our data resolved multiple distinct mechanisms by which temperature transiently altered the dynamics of nascent RNAPII transcription and associated RNA processing, illustrating potential biotechnological solutions and future focus areas to promote food security in the context of a changing climate.

Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis.

CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.

Author Correction: Transcriptional read-through of the long non-coding RNA SVALKA governs plant cold acclimation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcription-driven chromatin repression of Intragenic transcription start sites.

Progression of RNA polymerase II (RNAPII) transcription relies on the appropriately positioned activities of elongation factors. The resulting profile of factors and chromatin signatures along transcription units provides a "positional information system" for transcribing RNAPII. Here, we investigate a chromatin-based mechanism that suppresses intragenic initiation of RNAPII transcription. We demonstrate that RNAPII transcription across gene promoters represses their function in plants. This repression is characterized by reduced promoter-specific molecular signatures and increased molecular signatures associated with RNAPII elongation. The conserved FACT histone chaperone complex is required for this repression mechanism. Genome-wide Transcription Start Site (TSS) mapping reveals thousands of discrete intragenic TSS positions in fact mutants, including downstream promoters that initiate alternative transcript isoforms. We find that histone H3 lysine 4 mono-methylation (H3K4me1), an Arabidopsis RNAPII elongation signature, is enriched at FACT-repressed intragenic TSSs. Our analyses suggest that FACT is required to repress intragenic TSSs at positions that are in part characterized by elevated H3K4me1 levels. In sum, conserved and plant-specific chromatin features correlate with the co-transcriptional repression of intragenic TSSs. Our insights into TSS repression by RNAPII transcription promise to inform the regulation of alternative transcript isoforms and the characterization of gene regulation through the act of pervasive transcription across eukaryotic genomes.