MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). ...Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE). We have combined a luciferase-based genetic screen with whole-genome sequencing for rapid identification of new regulators of miRNA biogenesis and action. Among the first six mutants analyzed were three alleles of C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1)/FIERY2 (FRY2). In the miRNA processing complex, SE functions as a scaffold to mediate CPL1 interaction with HYL1, which needs to be dephosphorylated for optimal activity. In the absence of CPL1, HYL1 dephosphorylation and hence accurate processing and strand selection from miRNA duplexes are compromised. Our findings thus define a new regulatory step in plant miRNA biogenesis.
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► CPL1 is required for accurate miRNA processing and strand selection ► Excess phosphorylation of HYL1 compromises miRNA processing and strand selection ► CPL1 dephosphorylates HYL1 in the microprocessor to ensure full HYL1 function
A genetic screen for new regulators of miRNA biogenesis in plants identifies CPL1. By dephosphorylating a core miRNA processing factory, HYL1, CPL1 controls strand selection and accurate processing from miRNA duplexes.
Summary
MicroRNAs (miRNAs) regulate plant development by post‐transcriptional regulation of target genes. In Arabidopsis thaliana, DCL1 processes precursors (pri‐miRNAs) to miRNA duplexes, which ...associate with AGO1. Additional proteins act in concert with DCL1 (e.g. HYL1 and SERRATE) or AGO1 to facilitate efficient and precise pri‐miRNA processing and miRNA loading, respectively. In this study, we show that the accumulation of plant microRNAs depends on RECEPTOR FOR ACTIVATED C KINASE 1 (RACK1), a scaffold protein that is found in all higher eukaryotes. miRNA levels are reduced in rack1 mutants, and our data suggest that RACK1 affects the microRNA pathway via several distinct mechanisms involving direct interactions with known microRNA factors: RACK1 ensures the accumulation and processing of some pri‐miRNAs, directly interacts with SERRATE and is part of an AGO1 complex. As a result, mutations in RACK1 lead to over‐accumulation of miRNA target mRNAs, which are important for ABA responses and phyllotaxy, for example. In conclusion, our study identified complex functioning of RACK1 proteins in the Arabidopsis miRNA pathway; these proteins are important for miRNA production and therefore plant development.
The responses of plants to abiotic stresses are accompanied by massive changes in transcriptome composition. To provide a comprehensive view of stress-induced changes in the Arabidopsis thaliana ...transcriptome, we have used whole-genome tiling arrays to analyze the effects of salt, osmotic, cold and heat stress as well as application of the hormone abscisic acid (ABA), an important mediator of stress responses. Among annotated genes in the reference strain Columbia we have found many stress-responsive genes, including several transcription factor genes as well as pseudogenes and transposons that have been missed in previous analyses with standard expression arrays. In addition, we report hundreds of newly identified, stress-induced transcribed regions. These often overlap with known, annotated genes. The results are accessible through the Arabidopsis thaliana Tiling Array Express (At-TAX) homepage, which provides convenient tools for displaying expression values of annotated genes, as well as visualization of unannotated transcribed regions along each chromosome.
The processing of Arabidopsis thaliana microRNAs (miRNAs) from longer primary transcripts (pri-miRNAs) requires the activity of several proteins, including DICER-LIKE1 (DCL1), the double-stranded ...RNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). It has been noted before that the morphological appearance of weak se mutants is reminiscent of plants with mutations in ABH1/CBP80 and CBP20, which encode the two subunits of the nuclear cap-binding complex. We report that, like SE, the cap-binding complex is necessary for proper processing of pri-miRNAs. Inactivation of either ABH1/CBP80 or CBP20 results in decreased levels of mature miRNAs accompanied by apparent stabilization of pri-miRNAs. Whole-genome tiling array analyses reveal that se, abh1/cbp80, and cbp20 mutants also share similar splicing defects, leading to the accumulation of many partially spliced transcripts. This is unlikely to be an indirect consequence of improper miRNA processing or other mRNA turnover pathways, because introns retained in se, abh1/cbp80, and cbp20 mutants are not affected by mutations in other genes required for miRNA processing or for nonsense-mediated mRNA decay. Taken together, our results uncover dual roles in splicing and miRNA processing that distinguish SE and the cap-binding complex from specialized miRNA processing factors such as DCL1 and HYL1.
Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing ...technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis.
We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage.
AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.
Intron splicing increases proteome complexity, promotes RNA stability, and enhances transcription. However, introns and the concomitant need for splicing extend the time required for gene expression ...and can cause an undesirable delay in the activation of genes. Here, we show that the plant microRNA processing factor SERRATE (SE) plays an unexpected and pivotal role in the regulation of intronless genes. Arabidopsis SE associated with more than 1000, mainly intronless, genes in a transcription-dependent manner. Chromatin-bound SE liaised with paused and elongating polymerase II complexes and promoted their association with intronless target genes. Our results indicate that stress-responsive genes contain no or few introns, which negatively affects their expression strength, but that some genes circumvent this limitation via a novel SE-dependent transcriptional activation mechanism. Transcriptome analysis of a Drosophila mutant defective in ARS2, the metazoan homologue of SE, suggests that SE/ARS2 function in regulating intronless genes might be conserved across kingdoms.
MicroRNAs (miRNAs) are short regulatory RNAs processed from partially self-complementary foldbacks within longer MIRNA primary transcripts. Several MIRNA families are conserved deeply through land ...plants, but many are present only in closely related species or are species specific. The finding of numerous evolutionarily young MIRNA, many with low expression and few if any targets, supports a rapid birth-death model for MIRNA evolution. A systematic analysis of MIRNA genes and families in the close relatives, Arabidopsis thaliana and Arabidopsis lyrata, was conducted using both whole-genome comparisons and high-throughput sequencing of small RNAs. Orthologs of 143 A. thaliana MIRNA genes were identified in A. lyrata, with nine having significant sequence or processing changes that likely alter function. In addition, at least 13% of MIRNA genes in each species are unique, despite their relatively recent speciation (~10 million years ago). Alignment of MIRNA foldbacks to the Arabidopsis genomes revealed evidence for recent origins of 32 families by inverted or direct duplication of mostly protein-coding gene sequences, but less than half of these yield miRNA that are predicted to target transcripts from the originating gene family. miRNA nucleotide divergence between A. lyrata and A. thaliana orthologs was higher for young MIRNA genes, consistent with reduced purifying selection compared with deeply conserved MIRNA genes. Additionally, target sites of younger miRNA were lost more frequently than for deeply conserved families. In summary, our systematic analyses emphasize the dynamic nature of the MIRNA complement of plant genomes.
The four-member SPA protein family of Arabidopsis acts in concert with the E3 ubiquitin ligase COP1 to suppress photomorphogenesis in dark-grown seedlings. Here, we demonstrate that SPA proteins are, ...moreover, essential for photoperiodic flowering. Mutations in SPA1 cause phyA-independent early flowering under short day (SD) but not long day (LD) conditions, and this phenotype is enhanced by additional loss of SPA3 and SPA4 function. These spa1 spa3 spa4 triple mutants flower at the same time in LD and SD, indicating that the SPA gene family is essential for the inhibition of flowering under non-inductive SD. Among the four SPA genes, SPA1 is necessary and sufficient for normal photoperiodic flowering. Early flowering of SD-grown spa mutant correlates with strongly increased FT transcript levels, whereas CO transcript levels are not altered. Epistasis analysis demonstrates that both early flowering and FT induction in spa1 mutants is fully dependent on CO . Consistent with this finding, SPA proteins interact physically with CO in vitro and in vivo, suggesting that SPA proteins regulate CO protein function. Domain mapping shows that the SPA1-CO interaction requires the CCT-domain of CO, but is independent of the B-box type Zn fingers of CO. We further show that spa1 spa3 spa4 mutants exhibit strongly increased CO protein levels, which are not caused by a change in CO gene expression. Taken together, our results suggest, that SPA proteins regulate photoperiodic flowering by controlling the stability of the floral inducer CO.
The RNA-binding proteins FCA and FPA were identified based on their repression of the flowering time regulator FLC but have since been shown to have widespread roles in the Arabidopsis thaliana ...genome. Here, we use whole-genome tiling arrays to show that a wide spectrum of genes and transposable elements are misexpressed in the fca-9 fpa-7 (fcafpa) double mutant at two stages of seedling development. There was a significant bias for misregulated genomic segments mapping to the 3' region of genes. In addition, the double mutant misexpressed a large number of previously unannotated genomic segments corresponding to intergenic regions. We characterized a subset of these misexpressed unannotated segments and established that they resulted from extensive transcriptional read-through, use of downstream polyadenylation sites, and alternative splicing. In some cases, the transcriptional read-through significantly reduced expression of the associated genes. FCA/FPA-dependent changes in DNA methylation were found at several loci, supporting previous associations of FCA/FPA function with chromatin modifications. Our data suggest that FCA and FPA play important roles in the A. thaliana genome in RNA 3' processing and transcription termination, thus limiting intergenic transcription.
In Arabidopsis thaliana, four different dicer-like (DCL) proteins have distinct but partially overlapping functions in the biogenesis of microRNAs (miRNAs) and siRNAs from longer, noncoding precursor ...RNAs. To analyze the impact of different components of the small RNA biogenesis machinery on the transcriptome, we subjected dcl and other mutants impaired in small RNA biogenesis to whole-genome tiling array analysis. We compared both protein-coding genes and noncoding transcripts, including most pri-miRNAs, in two tissues and several stress conditions. Our analysis revealed a surprising number of common targets in dcl1 and dcl2 dcl3 dcl4 triple mutants. Furthermore, our results suggest that the DCL1 is not only involved in miRNA action but also contributes to silencing of a subset of transposons, apparently through an effect on DNA methylation.
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