Chromatin-associated microprocessor assembly is regulated by the U1 snRNP auxiliary protein PRP40.

In plants, microRNA (miRNA) biogenesis involves cotranscriptional processing of RNA polymerase II (RNAPII)-generated primary transcripts by a multi-protein complex termed the microprocessor. Here, we report that Arabidopsis (Arabidopsis thaliana) PRE-MRNA PROCESSING PROTEIN 40 (PRP40), the U1 snRNP auxiliary protein, positively regulates the recruitment of SERRATE, a core component of the plant microprocessor, to miRNA genes. The association of DICER-LIKE1 (DCL1), the microprocessor endoribonuclease, with chromatin was altered in prp40ab mutant plants. Impaired cotranscriptional microprocessor assembly was accompanied by RNAPII accumulation at miRNA genes and retention of miRNA precursors at their transcription sites in the prp40ab mutant plants. We show that cotranscriptional microprocessor assembly, regulated by AtPRP40, positively affects RNAPII transcription of miRNA genes and is important to reach the correct levels of produced miRNAs.

Hyponastic Leaves 1 Interacts with RNA Pol II to Ensure Proper Transcription of MicroRNA Genes.

Hyponastic Leaves 1 (HYL1) [also known as Double-stranded RNA-Binding protein 1 (DRB1)] is a double-stranded RNA-binding protein involved in microRNA (miRNA) processing in plants. It is a core component of the Microprocessor complex and enhances the efficiency and precision of miRNA processing by the Dicer-Like 1 protein. In this work, we report a novel function of the HYL1 protein in the transcription of miRNA (MIR) genes. HYL1 colocalizes with RNA polymerase II and affects its distribution along MIR genes. Moreover, proteomic experiments revealed that the HYL1 protein interacts with many transcription factors. Finally, we show that the action of HYL1 is not limited to MIR genes and impacts the expression of many other genes, a majority of which are involved in plastid organization. These discoveries indicate HYL1 as an additional player in gene regulation at the transcriptional level, independent of its role in miRNA biogenesis.

The expanding role of RNA modifications in plant RNA polymerase II transcripts: highlights and perspectives.

Regulation of gene expression is a complicated process based on the coordination of many different pathways, including epigenetic control of chromatin state, transcription, RNA processing, export of mature transcripts to the cytoplasm, and their translation into proteins. In recent years, with the development of high-throughput sequencing techniques, the importance of RNA modifications in gene expression has added another layer to this regulatory landscape. To date, >150 different types of RNA modifications have been found. Most RNA modifications, such as N6-methyladenosine (m6A) and pseudouridine (Ψ), were initially identified in highly abundant structural RNAs, such as rRNAs, tRNAs, and small nuclear RNAs (snRNAs). Current methods provide the opportunity to identify new types of modifications and to precisely localize them not only in highly expressed RNAs but also in mRNA and small RNA molecules. The presence of modified nucleotides in protein-coding transcripts can affect their stability, localization, and further steps of pre-mRNA maturation. Finally, it may affect the quality and quantity of protein synthesis. In plants, the epitranscriptomic field is still narrow, but the number of reports is growing rapidly. This review presents highlights and perspectives of plant epitranscriptomic modifications, focusing on various aspects of modifications of RNA polymerase II transcripts and their influence on RNA fate.

mRNA adenosine methylase (MTA) deposits m6A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana.

In Arabidopsis thaliana, the METTL3 homolog, mRNA adenosine methylase (MTA) introduces N6-methyladenosine (m6A) into various coding and noncoding RNAs of the plant transcriptome. Here, we show that an MTA-deficient mutant (mta) has decreased levels of microRNAs (miRNAs) but accumulates primary miRNA transcripts (pri-miRNAs). Moreover, pri-miRNAs are methylated by MTA, and RNA structure probing analysis reveals a decrease in secondary structure within stem-loop regions of these transcripts in mta mutant plants. We demonstrate interaction between MTA and both RNA Polymerase II and TOUGH (TGH), a plant protein needed for early steps of miRNA biogenesis. Both MTA and TGH are necessary for efficient colocalization of the Microprocessor components Dicer-like 1 (DCL1) and Hyponastic Leaves 1 (HYL1) with RNA Polymerase II. We propose that secondary structure of miRNA precursors induced by their MTA-dependent m6A methylation status, together with direct interactions between MTA and TGH, influence the recruitment of Microprocessor to plant pri-miRNAs. Therefore, the lack of MTA in mta mutant plants disturbs pri-miRNA processing and leads to the decrease in miRNA accumulation. Furthermore, our findings reveal that reduced miR393b levels likely contributes to the impaired auxin response phenotypes of mta mutant plants.

MicroRNA biogenesis and activity in plant cell dedifferentiation stimulated by cell wall removal.

BACKGROUND: Despite the frequent use of protoplast-to-plant system in in vitro cultures of plants, the molecular mechanisms regulating the first and most limiting stages of this process, i.e., protoplast dedifferentiation and the first divisions leading to the formation of a microcallus, have not been elucidated. RESULTS: In this study, we investigated the function of miRNAs in the dedifferentiation of A. thaliana mesophyll cells in a process stimulated by the enzymatic removal of the cell wall. Leaf cells, protoplasts and CDPs (cells derived from protoplasts) cultured for 24, 72 and 120 h (first cell division). In protoplasts, a strong decrease in the amount of AGO1 in both the nucleus and the cytoplasm, as well as dicing bodies (DBs), which are considered to be sites of miRNA biogenesis, was shown. However during CDPs division, the amounts of AGO1 and DBs strongly increased. MicroRNA transcriptome studies demonstrated that lower amount of differentially expressed miRNAs are present in protoplasts than in CDPs cultured for 120 h. Then analysis of differentially expressed miRNAs, selected pri-miRNA and mRNA targets were performed. CONCLUSION: This result indicates that miRNA function is not a major regulation of gene expression in the initial but in later steps of dedifferentiation during CDPs divisions. miRNAs participate in organogenesis, oxidative stress, nutrient deficiencies and cell cycle regulation in protoplasts and CDPs. The important role played by miRNAs in the process of dedifferentiation of mesophyll cells was confirmed by the increased mortality and reduced cell division of CDPs derived from mutants with defective miRNA biogenesis and miR319b expression.

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis.

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis.

The interconnection between transcription and splicing is a subject of intense study. We report that Arabidopsis homologue of spliceosome disassembly factor NTR1 is required for correct expression and splicing of DOG1, a regulator of seed dormancy. Global splicing analysis in atntr1 mutants revealed a bias for downstream 5' and 3' splice site selection and an enhanced rate of exon skipping. A local reduction in PolII occupancy at misspliced exons and introns in atntr1 mutants suggests that directionality in splice site selection is a manifestation of fast PolII elongation kinetics. In agreement with this model, we found AtNTR1 to bind target genes and co-localise with PolII. A minigene analysis further confirmed that strong alternative splice sites constitute an AtNTR1-dependent transcriptional roadblock. Plants deficient in PolII endonucleolytic cleavage showed opposite effects for splice site choice and PolII occupancy compared to atntr1 mutants, and inhibition of PolII elongation or endonucleolytic cleavage in atntr1 mutant resulted in partial reversal of splicing defects. We propose that AtNTR1 is part of a transcription elongation checkpoint at alternative exons in Arabidopsis.

Positive cofactor 4 (PC4) contributes to the regulation of replication-dependent canonical histone gene expression.

BACKGROUND: Core canonical histones are required in the S phase of the cell cycle to pack newly synthetized DNA, therefore the expression of their genes is highly activated during DNA replication. In mammalian cells, this increment is achieved by both enhanced transcription and 3' end processing. In this paper, we described positive cofactor 4 (PC4) as a protein that contributes to the regulation of replication-dependent histone gene expression. RESULTS: We showed that PC4 influences RNA polymerase II recruitment to histone gene loci in a cell cycle-dependent manner. The most important effect was observed in S phase where PC4 knockdown leads to the elevated level of RNA polymerase II on histone genes, which corresponds to the increased total level of those gene transcripts. The opposite effect was caused by PC4 overexpression. Moreover, we found that PC4 has a negative effect on the unique 3' end processing of histone pre-mRNAs that can be based on the interaction of PC4 with U7 snRNP and CstF64. Interestingly, this effect does not depend on the cell cycle. CONCLUSIONS: We conclude that PC4 might repress RNA polymerase II recruitment and transcription of replication-dependent histone genes in order to maintain the very delicate balance between histone gene expression and DNA synthesis. It guards the cell from excess of histones in S phase. Moreover, PC4 might promote the interaction of cleavage and polyadenylation complex with histone pre-mRNAs, that might impede with the recruitment of histone cleavage complex. This in turn decreases the 3' end processing efficiency of histone gene transcripts.

Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels.

Plants as sessile organisms have developed prompt response mechanisms to react to rapid environmental changes. In addition to the transcriptional regulation of gene expression, microRNAs (miRNAs) are key posttranscriptional regulators of the plant stress response. We show here that the expression levels of many miRNAs were regulated under salt stress conditions. This regulation occurred at the transcriptional and posttranscriptional levels. During salinity stress, the levels of miRNA161 and miRNA173 increased, while the expression of pri-miRNA161 and pri-miRNA173 was down-regulated. Under salt stress conditions, miRNA161 and miRNA173 were stabilized in the cytoplasm, and the expressions of MIR161 and MIR173 were negatively regulated in the nucleus. ARGONAUTE1 (AGO1) participated in both processes. We demonstrated that AGO1 cotranscriptionally controlled the expression of MIR161 and MIR173 in the nucleus. Our results suggests that AGO1 interacts with chromatin at MIR161 and MIR173 loci and causes the disassembly of the transcriptional complex, releasing short and unpolyadenylated transcripts.

mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs.

BACKGROUND: MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism. DESCRIPTION: The mirEX 2.0 web portal ( http://www.combio.pl/mirex ) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page. CONCLUSIONS: The mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.

mirEX: a platform for comparative exploration of plant pri-miRNA expression data.

mirEX is a comprehensive platform for comparative analysis of primary microRNA expression data. RT-qPCR-based gene expression profiles are stored in a universal and expandable database scheme and wrapped by an intuitive user-friendly interface. A new way of accessing gene expression data in mirEX includes a simple mouse operated querying system and dynamic graphs for data mining analyses. In contrast to other publicly available databases, the mirEX interface allows a simultaneous comparison of expression levels between various microRNA genes in diverse organs and developmental stages. Currently, mirEX integrates information about the expression profile of 190 Arabidopsis thaliana pri-miRNAs in seven different developmental stages: seeds, seedlings and various organs of mature plants. Additionally, by providing RNA structural models, publicly available deep sequencing results, experimental procedure details and careful selection of auxiliary data in the form of web links, mirEX can function as a one-stop solution for Arabidopsis microRNA information. A web-based mirEX interface can be accessed at http://bioinfo.amu.edu.pl/mirex.

Down-regulation of CBP80 gene expression as a strategy to engineer a drought-tolerant potato.

Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down-regulation of nuclear cap-binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA-hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water-deficit tolerance of crop plants.

Pseudouridine guides germline small RNA transport and epigenetic inheritance.

Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes1. Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation2,3. In C. elegans, these inherited small RNAs have poly (UG) tails4, but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop novel assays to detect Ψ in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs and their precursors. We also detect substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in Arabidopsis pollen, and piwi-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are localized to sperm cells, and we found that PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with Ψ and is required for transport of easiRNAs into sperm cells from the vegetative nucleus. We further show that Exportin-t is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, Ψ has a conserved role in marking inherited small RNAs in the germline.

Excess nitrogen responsive HvMADS27 transcription factor controls barley root architecture by regulating abscisic acid level.

Nitrogen (N) is an important element for plant growth and development. Although several studies have examined plants' response to N deficiency, studies on plants' response to excess N, which is common in fertilizer-based agrosystems, are limited. Therefore, the aim of this study was to examine the response of barley to excess N conditions, specifically the root response. Additionally, genomic mechanism of excess N response in barley was elucidated using transcriptomic technologies. The results of the study showed that barley MADS27 transcription factor was mainly expressed in the roots and its gene contained N-responsive cis-regulatory elements in the promoter region. Additionally, there was a significant decrease in HvMADS27 expression under excess N condition; however, its expression was not significantly affected under low N condition. Phenotypic analysis of the root system of HvMADS27 knockdown and overexpressing barley plants revealed that HvMADS27 regulates barley root architecture under excess N stress. Further analysis of wild-type (WT) and transgenic barley plants (hvmads27 kd and hvmads27 c-Myc OE) revealed that HvMADS27 regulates the expression of HvBG1 ß-glucosidase, which in turn regulates abscisic acid (ABA) level in roots. Overall, the findings of this study showed that HvMADS27 expression is downregulated in barley roots under excess N stress, which induces HvBG1 expression, leading to the release of ABA from ABA-glucose conjugate, and consequent shortening of the roots.

Quantitative Analysis of Plant miRNA Primary Transcripts.

MicroRNAs control plant development and are key regulators of plant responses to biotic and abiotic stresses. Thus, their expression must be carefully controlled since both excess and deficiency of a given microRNA may be deleterious to plant cell. MicroRNA expression regulation can occur at several stages of their biogenesis pathway. One of the most important of these regulatory checkpoints is transcription efficiency. mirEX database is a tool for exploration and visualization of plant pri-miRNA expression profiles. It includes results obtained using high-throughput RT-qPCR platform designed to monitor pri-miRNA expression in different miRNA biogenesis mutants and developmental stages of Arabidopsis, barley, and Pellia plants. A step-by-step instruction for browsing the database and detailed protocol for high-throughput RT-qPCR experiments, including list of primers designed for the amplification of pri-miRNAs, are presented.

Arabidopsi s Spliceosome Factor SmD3 Modulates Immunity to Pseudomonas syringae Infection.

SmD3 is a core component of the small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. The role of Arabidopsis SmD3 in plant immunity was assessed by testing sensitivity of smd3a and smd3b mutants to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and its pathogenesis effectors flagellin (flg22), EF-Tu (elf18) and coronatine (COR). Both smd3 mutants exhibited enhanced susceptibility to Pst accompanied by marked changes in the expression of key pathogenesis markers. mRNA levels of major biotic stress response factors were also altered upon treatment with Pseudomonas effectors. Our genome-wide transcriptome analysis of the smd3b-1 mutant infected with Pst, verified by northern and RT-qPCR, showed that lack of SmD3-b protein deregulates defense against Pst infection at the transcriptional and posttranscriptional levels including defects in splicing and an altered pattern of alternative splicing. Importantly, we show that SmD3-b dysfunction impairs mainly stomatal immunity as a result of defects in stomatal development. We propose that it is the malfunction of the stomata that is the primary cause of an altered mutant response to the pathogen. Other changes in the smd3b-1 mutant involved enhanced elf18- and flg22-induced callose deposition, reduction of flg22-triggered production of early ROS and boost of secondary ROS caused by Pst infection. Together, our data indicate that SmD3 contributes to the plant immune response possibly via regulation of mRNA splicing of key pathogenesis factors.

Corrigendum: A Role of U12 Intron in Proper Pre-mRNA Splicing of Plant Cap Binding Protein 20 Genes.

[This corrects the article DOI: 10.3389/fpls.2018.00475.].

Regulation of Plant Microprocessor Function in Shaping microRNA Landscape.

MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem-loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed.

A Role of U12 Intron in Proper Pre-mRNA Splicing of Plant Cap Binding Protein 20 Genes.

The nuclear cap-binding complex (CBC) is composed of two cap-binding proteins: CBP20 and CBP80. The CBP20 gene structure is highly conserved across land plant species. All studied CBP20 genes contain eight exons and seven introns, with the fourth intron belonging to the U12 class. This highly conserved U12 intron always divides the plant CBP20 gene into two parts: one part encodes the core domain containing the RNA binding domain (RBD), and the second part encodes the tail domain with a nuclear localization signal (NLS). In this study, we investigate the importance of the U12 intron in the Arabidopsis thaliana CBP20 gene by moving it to different intron locations of the gene. Relocation of the U12 intron resulted in a significant decrease in the U12 intron splicing efficiency and the accumulation of wrongly processed transcripts. These results suggest that moving the U12 intron to any other position of the A. thaliana CBP20 gene disturbs splicing, leading to substantial downregulation of the level of properly spliced mRNA and CBP20 protein. Moreover, the replacement of the U12 intron with a U2 intron leads to undesired alternative splicing events, indicating that the proper localization of the U12 intron in the CBP20 gene secures correct CBP20 pre-mRNA maturation and CBP20 protein levels in a plant. Surprisingly, our results also show that the efficiency of U12 splicing depends on intron length. In conclusion, our study emphasizes the importance of proper U12 intron localization in plant CBP20 genes for correct pre-mRNA processing.