A repertoire of intronic lariat RNAs reveals tissue-specific regulation and target mimicry potential in plants.

Lariat RNA is concomitantly produced by excised intron during RNA splicing, which is usually debranched by DBR1, an RNA debranching enzyme. However, increasing evidence showed that some lariat RNA could escape debranching. Little is known about how and why these lariat RNAs could be retained. By comparing the atlas of lariat RNAs between the non-dividing cell (mature pollen) and three actively dividing tissues (young shoot apex, young seeds, and young roots), we identified hundreds to thousands of lariat RNA naturally retained in each tissue, and the incidence of lariat RNA retention is much less in shoot apex while much more in pollen. Many lariat RNAs derived from the same intron or different lariat RNAs from the same pre-mRNA could be retained in one tissue while degraded in the other tissues. By deciphering lariat RNA sequences, we identified an AG-rich (RAAAAVAAAR) motif and a UC-rich (UCUCUYUCUC) motif for pollen-specific and the other three tissues-retained lariat RNAs, respectively. Reconstitution of the pollen-specific AG-rich motif indeed enhanced lariat RNA retention in plants. Biologically, hundreds of lariat RNAs harbored miRNA binding sites, and dual-luciferase reporter assay showed that these natural lariat RNAs had the potential to protect expression of miRNA target genes. Collectively, our results uncover that selective retention of lariat RNA is an actively regulatory process, and provide new insights into understanding how lariat RNA metabolism may impact miRNA activity.

Phased secondary small interfering RNAs in Camellia sinensis var. assamica.

Phased secondary small interfering RNAs (phasiRNAs) in plants play important roles in regulating genome stability, plant development and stress adaption. Camellia sinensis var. assamica has immense economic, medicinal and cultural significance. However, there are still no studies of phasiRNAs and their putative functions in this valuable plant. We identified 476 and 43 PHAS loci which generated 4290 twenty one nucleotide (nt) and 264 twenty four nt phasiRNAs, respectively. Moreover, the analysis of degradome revealed more than 35000 potential targets for these phasiRNAs. We identified several conserved 21 nt phasiRNA generation pathways in tea plant, including miR390 → TAS3, miR482/miR2118 → NB-LRR, miR393 → F-box, miR828 → MYB/TAS4, and miR7122 → PPR in this study. Furthermore, we found that some transposase and plant mobile domain genes could generate phasiRNAs. Our results show that phasiRNAs target genes in the same family in cis- or trans-manners, and different members of the same gene family may generate the same phasiRNAs. The phasiRNAs, generated by transposase and plant mobile domain genes, and their targets, suggest that phasiRNAs may be involved in the inhibition of transposable elements in tea plant. To summarize, these results provide a comprehensive view of phasiRNAs in Camellia sinensis var. assamica.

The plant FYVE domain-containing protein FREE1 associates with microprocessor components to repress miRNA biogenesis.

FYVE domain protein required for endosomal sorting 1 (FREE1), originally identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT) machinery, plays diverse roles either in endosomal sorting in the cytoplasm or in transcriptional regulation of abscisic acid signaling in the nucleus. However, to date, a role for FREE1 or other ESCRT components in the regulation of plant miRNA biology has not been discovered. Here, we demonstrate a nuclear function of FREE1 as a cofactor in miRNA biogenesis in plants. FREE1 directly interacts with the plant core microprocessor component CPL1 in nuclear bodies and disturbs the association between HYL1, SE and CPL1. Inactivation of FREE1 in the nucleus increases the binding affinity between HYL1, SE, and CPL1 and causes a transition of HYL1 from the inactive hyperphosphorylated version to the active hypophosphorylated form, thereby promoting miRNA biogenesis. Our results suggest that FREE1 has evolved as a negative regulator of miRNA biogenesis and provides evidence for a link between FYVE domain-containing proteins and miRNA biogenesis in plants.

SICKLE modulates lateral root development by promoting degradation of lariat intronic RNA.

Plant lateral roots (LRs) play vital roles in anchorage and uptake of water and nutrients. Here, we reveal that degradation of lariat intronic RNAs (lariRNAs) modulated by SICKLE (SIC) is required for LR development in Arabidopsis (Arabidopsis thaliana). Loss of SIC results in hyper-accumulation of lariRNAs and restricts the outgrowth of LR primordia, thereby reducing the number of emerged LRs. Decreasing accumulation of lariRNAs by over-expressing RNA debranching enzyme 1 (DBR1), a rate-limiting enzyme of lariRNA decay, restored LR defects in SIC-deficient plants. Mechanistically, SIC interacts with DBR1 and facilitates its nuclear accumulation, which is achieved through two functionally redundant regions (SIC1-244 and SIC252-319) for nuclear localization. Of the remaining amino acids in this region, six (SIC245-251) comprise a DBR1-interacting region while two (SICM246 and SICW251) are essential for DBR1-SIC interaction. Reducing lariRNAs restored microRNA (miRNA) levels and LR development in lariRNA hyper-accumulating plants, suggesting that these well-known regulators of LR development mainly function downstream of lariRNAs. Taken together, we propose that SIC acts as an enhancer of DBR1 nuclear accumulation by driving nuclear localization through direct interaction, thereby promoting lariRNA decay to fine-tune miRNA biogenesis and modulating LR development.

H3K9 demethylases IBM1 and JMJ27 are required for male meiosis in Arabidopsis thaliana.

Dimethylation of histone H3 lysine 9 (H3K9me2), a crucial modification for heterochromatin formation and transcriptional silencing, is essential for proper meiotic prophase progression in mammals. We analyzed meiotic defects and generated genome-wide profiles of H3K9me2 and transcriptomes for the mutants of H3K9 demethylases. Moreover, we also identified proteins interacting with H3K9 demethylases. H3K9me2 is usually found at transposable elements and repetitive sequences but is absent from the bodies of protein-coding genes. In this study, we show that the Arabidopsis thaliana H3K9 demethylases IBM1 and JMJ27 cooperatively regulate crossover formation and chromosome segregation. They protect thousands of protein-coding genes from ectopic H3K9me2, including genes essential for meiotic prophase progression. In addition to removing H3K9me2, IBM1 and JMJ27 interact with the Precocious Dissociation of Sisters 5 (PDS5) cohesin complex cofactors. The pds5 mutant shared similar transcriptional alterations with ibm1 jmj27, including meiosis-essential genes, yet without affecting H3K9me2 levels. Hence, PDS5s, together with IBM1 and JMJ27, regulate male meiosis and gene expression independently of H3K9 demethylation. These findings uncover a novel role of H3K9me2 removal in meiosis and a new function of H3K9 demethylases and cohesin cofactors in meiotic transcriptional regulation.

Identification of Intronic Lariat-Derived Circular RNAs in Arabidopsis by RNA Deep Sequencing.

Lariat RNAs are well-known by-products of pre-mRNA splicing in eukaryotes, which are produced by the excised introns when the 5' splice site (5' ss) joins with the branchpoint (BP) during splicing. In general, most of lariat RNAs are usually linearized by RNA debranching enzyme 1 (DBR1), followed by degradation for intron turnover. However, with the high-throughput RNA sequencing technology and bioinformatics methods, increasing evidences have shown that many lariat RNAs can stably accumulate under physiological conditions in both animals and plants. Here, we describe a large-scale analysis to systematically identify the lariat RNAs (i.e., intronic circular RNAs) in Arabidopsis by utilizing the RNA-sequencing data.

Brassinosteroids inhibit miRNA-mediated translational repression by decreasing AGO1 on the endoplasmic reticulum.

Translational repression is a conserved mechanism in microRNA (miRNA)-guided gene silencing. In Arabidopsis, ARGONAUTE1 (AGO1), the major miRNA effector, localizes in the cytoplasm for mRNA cleavage and at the endoplasmic reticulum (ER) for translational repression of target genes. However, the mechanism underlying miRNA-mediated translational repression is poorly understood. In particular, how the subcellular partitioning of AGO1 is regulated is largely unexplored. Here, we show that the plant hormone brassinosteroids (BRs) inhibit miRNA-mediated translational repression by negatively regulating the distribution of AGO1 at the ER in Arabidopsis thaliana. We show that the protein levels rather than the transcript levels of miRNA target genes were reduced in BR-deficient mutants but increased under BR treatments. The localization of AGO1 at the ER was significantly decreased under BR treatments while it was increased in the BR-deficient mutants. Moreover, ROTUNDIFOLIA3 (ROT3), an enzyme involved in BR biosynthesis, co-localizes with AGO1 at the ER and interacts with AGO1 in a GW motif-dependent manner. Complementation analysis showed that the AGO1-ROT3 interaction is necessary for the function of ROT3. Our findings provide new clues to understand how miRNA-mediated gene silencing is regulated by plant endogenous hormones.

The chromatin remodeling complex imitation of switch controls stamen filament elongation by promoting jasmonic acid biosynthesis in Arabidopsis.

Plant reproduction requires the coordinated development of both male and female reproductive organs. Jasmonic acid (JA) plays an essential role in stamen filament elongation. However, the mechanism by which the JA biosynthesis genes are regulated to promote stamen elongation remains unclear. Here, we show that the chromatin remodeling complex Imitation of Switch (ISWI) promotes stamen filament elongation by regulating JA biosynthesis. We show that AT-Rich Interacting Domain 5 (ARID5) interacts with CHR11, CHR17, and RLT1, several known subunits of ISWI. Mutations in ARID5 and RLTs caused a reduced seed set due to greatly shortened stamen filaments. RNA-seq analyses reveal that the expression of key genes responsible for JA biosynthesis is significantly down-regulated in the arid5 and rlt mutants. Consistently, the JA levels are drastically decreased in both arid5 and rlt mutants. Chromatin immunoprecipitation-quantitative PCR analyses further show that ARID5 is recruited to the chromatin of JA biosynthesis genes. Importantly, exogenous JA treatments can fully rescue the defects of stamen filament elongation in both arid5 and rlt mutants, leading to the partial recovery of fertility. Our results provide a clue how JA biosynthesisis positively regulated by the chromatin remodeling complex ISWI, thereby promoting stamen filament elongation in Arabidopsis.

Heterochromatic silencing is reinforced by ARID1-mediated small RNA movement in Arabidopsis pollen.

In the plant male germline, transposable elements (TEs) are reactivated in the companion vegetative nucleus, resulting in siRNA production and the intercellular movement of these siRNAs to reinforce TE silencing in sperm. However, the mechanism by which siRNA movement is regulated remains unexplored. Here we show that ARID1, a transcription factor which is constitutively expressed in the vegetative nucleus but dynamically accumulates in the generative cell (the progenitor of sperm) to promote the second pollen mitosis, mediates siRNA movement to reinforce heterochromatic silencing in the male germline. We looked for regulators involved in the accumulation of ARID1 in the generative cell, and found that AGO9, a germline-specific AGO in Arabidopsis, is required for the accumulation of ARID1 in the generative cell. Mutations in either ARID1 or AGO9 lead to the interruption of not only the second pollen mitosis but also the movement of siRNA from the vegetative nucleus to the male germline, resulting in the release of heterochromatic silencing in the male germline. Moreover, conditional knockdown of ARID1 in the generative cell causes reduced heterochromatic silencing in both bicellular and mature pollen. This study provides insights into how a spatiotemporal transcription factor coordinates heterochromatic silencing and male germline maturation.

Epigenetic Regulation of Megaspore Mother Cell Formation.

In flowering plants, the female gametophyte (FG) initiates from the formation of the megaspore mother cell (MMC). Among a pool of the somatic cells in the ovule primordium, only one hypodermal cell undergoes a transition of cell fate to become the MMC. Subsequently, the MMC undergoes a series of meiosis and mitosis to form the mature FG harboring seven cells with eight nuclei. Although SPL/NZZ, the core transcription factor for MMC formation, was identified several decades ago, which and why only one somatic cell is chosen as the MMC have long remained mysterious. A growing body of evidence reveal that MMC formation is associated with epigenetic regulation at multiple layers, including dynamic distribution of histone variants and histone modifications, small RNAs, and DNA methylation. In this review, we summarize the progress of epigenetic regulation in the MMC formation, emphasizing the roles of chromosome condensation, histone variants, histone methylation, small RNAs, and DNA methylation.

The transcriptome variations of Panaxnotoginseng roots treated with different forms of nitrogen fertilizers.

BACKGROUND: The sensitivity of plants to ammonia is a worldwide problem that limits crop production. Excessive use of ammonium as the sole nitrogen source results in morphological and physiological disorders, and retarded plant growth. RESULTS: In this study we found that the root growth of Panax notoginseng was inhibited when only adding ammonium nitrogen fertilizer, but the supplement of nitrate fertilizer recovered the integrity, activity and growth of root. Twelve RNA-seq profiles in four sample groups were produced and analyzed to identify deregulated genes in samples with different treatments. In comparisons to NH[Formula: see text] treated samples, ACLA-3 gene is up-regulated in samples treated with NO[Formula: see text] and with both NH[Formula: see text] and NO[Formula: see text], which is further validated by qRT-PCR in another set of samples. Subsequently, we show that the some key metabolites in the TCA cycle are also significantly enhanced when introducing NO[Formula: see text]. These potentially enhance the integrity and recover the growth of Panax notoginseng roots. CONCLUSION: These results suggest that the activated TCA cycle, as demonstrated by up-regulation of ACLA-3 and several key metabolites in this cycle, contributes to the increased Panax notoginseng root yield when applying both ammonium and nitrate fertilizer.

The Arabidopsis anaphase-promoting complex/cyclosome subunit 8 is required for male meiosis.

Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase-promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin-ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis. We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis. Meiocytes from apc8-1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8-1, including abnormal sister chromatid segregation and microtubule morphology. Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C-mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.

Spliceosome disassembly factors ILP1 and NTR1 promote miRNA biogenesis in Arabidopsis thaliana.

The intron-lariat spliceosome (ILS) complex is highly conserved among eukaryotes, and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, Increased Level of Polyploidy1-1D (ILP1) and NTC-Related protein 1 (NTR1), positively regulate microRNA (miRNA) biogenesis by facilitating transcriptional elongation of MIRNA (MIR) genes in Arabidopsis thaliana. ILP1 and NTR1 formed a stable complex and co-regulated alternative splicing of more than a hundred genes across the Arabidopsis genome, including some primary transcripts of miRNAs (pri-miRNAs). Intriguingly, pri-miRNAs, regardless of having introns or not, were globally down-regulated when the ILP1 or NTR1 function was compromised. ILP1 and NTR1 interacted with core miRNA processing proteins Dicer-like 1 and Serrate, and were required for proper RNA polymerase II occupancy at elongated regions of MIR chromatin, without affecting either MIR promoter activity or pri-miRNA decay. Our results provide further insights into the regulatory role of spliceosomal machineries in the biogenesis of miRNAs.

Intercellular delivery of small RNAs in plant gametes.

Small RNAs are 20-24 nucleotides in length. In plants, small RNAs are classified into microRNAs (miRNAs) and small interfering RNAs (siRNAs), based on their biogenesis and molecular features. In contrast to the extensive knowledge of the roles of small RNAs in sporophytic tissues, the distribution and function of small RNAs in gametophytic cells have been less well studied. However, with the improvement of single-cell sorting and RNA sequencing technologies, the distribution of small RNAs, especially siRNAs, between sperm cells and the vegetative cell, as well as the function of sperm-delivered small RNAs during early seed development have been elucidated. This review summarizes work from the past 5 years regarding small RNAs in male gametes, emphasizing the intercellular communication and biological significance of small RNAs in Arabidopsis.

A Comprehensive Map of Intron Branchpoints and Lariat RNAs in Plants.

Lariats are formed by excised introns, when the 5' splice site joins with the branchpoint (BP) during splicing. Although lariat RNAs are usually degraded by RNA debranching enzyme 1, recent findings in animals detected many lariat RNAs under physiological conditions. By contrast, the features of BPs and to what extent lariat RNAs accumulate naturally are largely unexplored in plants. Here, we analyzed 948 RNA sequencing data sets to document plant BPs and lariat RNAs on a genome-wide scale. In total, we identified 13,872, 5199, 29,582, and 13,478 BPs in Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa), and maize (Zea mays), respectively. Features of plant BPs are highly similar to those in yeast and human, in that BPs are adenine-preferred and flanked by uracil-enriched sequences. Intriguingly, ∼20% of introns harbor multiple BPs, and BP usage is tissue-specific. Furthermore, 10,580 lariat RNAs accumulate in wild-type Arabidopsis plants, and most of these lariat RNAs originate from longer or retroelement-depleted introns. Moreover, the expression of these lariat RNAs is accompanied by the incidence of back-splicing of parent exons. Collectively, our results provide a comprehensive map of intron BPs and lariat RNAs in four plant species and uncover a link between lariat turnover and splicing.

Anaphase-promoting complex/cyclosome regulates RdDM activity by degrading DMS3 in Arabidopsis.

During RNA-directed DNA methylation (RdDM), the DDR complex, composed of DRD1, DMS3, and RDM1, is responsible for recruiting DNA polymerase V (Pol V) to silence transposable elements (TEs) in plants. However, how the DDR complex is regulated remains unexplored. Here, we show that the anaphase-promoting complex/cyclosome (APC/C) regulates the assembly of the DDR complex by targeting DMS3 for degradation. We found that a substantial set of RdDM loci was commonly de-repressed in apc/c and pol v mutants, and that the defects in RdDM activity resulted from up-regulated DMS3 protein levels, which finally caused reduced Pol V recruitment. DMS3 was ubiquitinated by APC/C for degradation in a D box-dependent manner. Competitive binding assays and gel filtration analyses showed that a proper level of DMS3 is critical for the assembly of the DDR complex. Consistent with the importance of the level of DMS3, overaccumulation of DMS3 caused defective RdDM activity, phenocopying the apc/c and dms3 mutants. Moreover, DMS3 is expressed in a cell cycle-dependent manner. Collectively, these findings provide direct evidence as to how the assembly of the DDR complex is regulated and uncover a safeguarding role of APC/C in the regulation of RdDM activity.

Meiocyte-Specific and AtSPO11-1-Dependent Small RNAs and Their Association with Meiotic Gene Expression and Recombination.

Meiotic recombination ensures accurate chromosome segregation and results in genetic diversity in sexually reproducing eukaryotes. Over the last few decades, the genetic regulation of meiotic recombination has been extensively studied in many organisms. However, the role of endogenous meiocyte-specific small RNAs (ms-sRNAs; 21-24 nucleotide [nt]) and their involvement in meiotic recombination are unclear. Here, we sequenced the total small RNA (sRNA) and messenger RNA populations from meiocytes and leaves of wild type Arabidopsis (Arabidopsis thaliana) and meiocytes of spo11-1, a mutant defective in double-strand break formation, and we discovered 2,409 ms-sRNA clusters, 1,660 of which areSPORULATION 11-1 (AtSPO11-1)-dependent. Unlike mitotic small interfering RNAs that are enriched in intergenic regions and associated with gene silencing, ms-sRNAs are significantly enriched in genic regions and exhibit a positive correlation with genes that are preferentially expressed in meiocytes (i.e. Arabidopsis SKP1-LIKE1 and RAD51), in a fashion unrelated to DNA methylation. We also found that AtSPO11-1-dependent sRNAs have distinct characteristics compared with ms-sRNAs and tend to be associated with two known types of meiotic recombination hotspot motifs (i.e. CTT-repeat and A-rich motifs). These results reveal different meiotic and mitotic sRNA landscapes and provide new insights into how sRNAs relate to gene expression in meiocytes and meiotic recombination.

Reactive oxygen species-mediated BIN2 activity revealed by single-molecule analysis.

Much evidence has shown that reactive oxygen species (ROS) regulate several plant hormone signaling cascades, but little is known about the real-time kinetics and the underlying molecular mechanisms of the target proteins in the brassinosteroid (BR) signaling pathway. In this study, we used single-molecule techniques to investigate the true signaling timescales of the major BR signaling components BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINOSTEROID INSENSITIVE 2 (BIN2) of Arabidopsis thaliana. The rate constants of BIN2 associating with ATP and phosphorylating BES1 were determined to be 0.7 ± 0.4 mM-1  s-1 and 2.3 ± 1.4 s-1 , respectively. Interestingly, we found that the interaction of BIN2 and BES1 was oxygen-dependent, and oxygen can directly modify BIN2. The activity of BIN2 was switched on via modification of specific cysteine (Cys) residues, including C59, C95, C99 and C162. The mutation of these Cys residues inhibited the BR signaling outputs. These findings demonstrate the power of using single-molecule techniques to study the dynamic interactions of signaling components, which is difficult to be discovered by conventional physiological and biochemical methods.

jaw-1D: a gain-of-function mutation responsive to paramutation-like induction of epigenetic silencing.

The Arabidopsis thaliana gain-of-function T-DNA insertion mutant jaw-1D produces miR319A, a microRNA that represses genes encoding CIN-like TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), a family of transcription factors that play key roles in leaf morphogenesis. In this study, we show that jaw-1D is responsive to paramutation-like epigenetic silencing. A genetic cross of jaw-1D with the polycomb gene mutant curly leaf-29 (clf-29) leads to attenuation of the jaw-1D mutant plant phenotype. This induced mutation, jaw-1D*, was associated with down-regulation of miR319A, was heritable independently from clf-29, and displayed paramutation-like non-Mendelian inheritance. Down-regulation of miR319A in jaw-1D* was linked to elevated levels of histone H3 lysine 9 dimethylation and DNA methylation at the CaMV35S enhancer located within the activation-tagging T-DNA of the jaw-1D locus. Examination of 21 independent T-DNA insertion mutant lines revealed that 11 could attenuate the jaw-1D mutant phenotype in a similar way to the paramutation induced by clf-29. These paramutagenic mutant lines shared the common feature that their T-DNA insertion was present as multi-copy tandem repeats and contained high levels of CG and CHG methylation. Our results provide important insights into paramutation-like epigenetic silencing, and caution against the use of jaw-1D in genetic interaction studies.

Clearance of maternal barriers by paternal miR159 to initiate endosperm nuclear division in Arabidopsis.

Sperm entry triggers central cell division during seed development, but what factors besides the genome are inherited from sperm, and the mechanism by which paternal factors regulate early division events, are not understood. Here we show that sperm-transmitted miR159 promotes endosperm nuclear division by repressing central cell-transmitted miR159 targets. Disruption of paternal miR159 causes approximately half of the seeds to abort as a result of defective endosperm nuclear divisions. In wild-type plants, MYB33 and MYB65, two miR159 targets, are highly expressed in the central cell before fertilization, but both are rapidly abolished after fertilization. In contrast, loss of paternal miR159 leads to retention of MYB33 and MYB65 in the central cell after fertilization. Furthermore, ectopic expression of a miR159-resistant version of MYB33 (mMYB33) in the endosperm significantly inhibits initiation of endosperm nuclear division. Collectively, these results show that paternal miR159 inhibits its maternal targets to promote endosperm nuclear division, thus uncovering a previously unknown paternal effect on seed development.