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.

MiRNAs differentially expressed in vegetative and reproductive organs of Marchantia polymorpha - insights into their expression pattern, gene structures and function.

MicroRNAs regulate gene expression affecting a variety of plant developmental processes. The evolutionary position of Marchantia polymorpha makes it a significant model to understand miRNA-mediated gene regulatory pathways in plants. Previous studies focused on conserved miRNA-target mRNA modules showed their critical role in Marchantia development. Here, we demonstrate that the differential expression of conserved miRNAs among land plants and their targets in selected organs of Marchantia additionally underlines their role in regulating fundamental developmental processes. The main aim of this study was to characterize selected liverwort-specific miRNAs, as there is a limited knowledge on their biogenesis, accumulation, targets, and function in Marchantia. We demonstrate their differential accumulation in vegetative and generative organs. We reveal that all liverwort-specific miRNAs examined are encoded by independent transcriptional units. MpmiR11737a, MpmiR11887 and MpmiR11796, annotated as being encoded within protein-encoding genes, have their own independent transcription start sites. The analysis of selected liverwort-specific miRNAs and their pri-miRNAs often reveal correlation in their levels, suggesting transcriptional regulation. However, MpmiR11796 shows a reverse correlation to its pri-miRNA level, suggesting post-transcriptional regulation. Moreover, we identify novel targets for selected liverwort-specific miRNAs and demonstrate an inverse correlation between their expression and miRNA accumulation. In the case of one miRNA precursor, we provide evidence that it encodes two functional miRNAs with two independent targets. Overall, our research sheds light on liverwort-specific miRNA gene structure, provides new data on their biogenesis and expression regulation. Furthermore, identifying their targets, we hypothesize the potential role of these miRNAs in early land plant development and functioning.

Conserved and non-conserved RNA-target modules in plants: lessons for a better understanding of Marchantia development.

A wide variety of functional regulatory non-coding RNAs (ncRNAs) have been identified as essential regulators of plant growth and development. Depending on their category, ncRNAs are not only involved in modulating target gene expression at the transcriptional and post-transcriptional levels but also are involved in processes like RNA splicing and RNA-directed DNA methylation. To fulfill their molecular roles properly, ncRNAs must be precisely processed by multiprotein complexes. In the case of small RNAs, DICER-LIKE (DCL) proteins play critical roles in the production of mature molecules. Land plant genomes contain at least four distinct classes of DCL family proteins (DCL1-DCL4), of which DCL1, DCL3 and DCL4 are also present in the genomes of bryophytes, indicating the early divergence of these genes. The liverwort Marchantia polymorpha has become an attractive model species for investigating the evolutionary history of regulatory ncRNAs and proteins that are responsible for ncRNA biogenesis. Recent studies on Marchantia have started to uncover the similarities and differences in ncRNA production and function between the basal lineage of bryophytes and other land plants. In this review, we summarize findings on the essential role of regulatory ncRNAs in Marchantia development. We provide a comprehensive overview of conserved ncRNA-target modules among M. polymorpha, the moss Physcomitrium patens and the dicot Arabidopsis thaliana, as well as Marchantia-specific modules. Based on functional studies and data from the literature, we propose new connections between regulatory pathways involved in Marchantia's vegetative and reproductive development and emphasize the need for further functional studies to understand the molecular mechanisms that control ncRNA-directed developmental processes.

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.

PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley.

MicroRNAs are small, noncoding RNA molecules that regulate the expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS-box transcription factors in rice and barley. We identified three barley MIR444 (MIR444a/b/c) genes and comprehensively characterised their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, generating functional and nonfunctional pri-miRNA444 isoforms. We show that barley pri-miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR-associated endonuclease 9 (Cas9)-mediated mutagenesis of the PEP444c-encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots and a 5-fold reduced level of mature microRNA444c in roots. Our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.

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.

Biogenesis, conservation, and function of miRNA in liverworts.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules, 18-24 nucleotides long, that control multiple gene regulatory pathways via post-transcriptional gene silencing in eukaryotes. To develop a comprehensive picture of the evolutionary history of miRNA biogenesis and action in land plants, studies on bryophyte representatives are needed. Here, we review current understanding of liverwort MIR gene structure, miRNA biogenesis, and function, focusing on the simple thalloid Pellia endiviifolia and the complex thalloid Marchantia polymorpha. We review what is known about conserved and non-conserved miRNAs, their targets, and the functional implications of miRNA action in M. polymorpha and P. endiviifolia. We note that most M. polymorpha miRNAs are encoded within protein-coding genes and provide data for 23 MIR gene structures recognized as independent transcriptional units. We identify M. polymorpha genes involved in miRNA biogenesis that are homologous to those identified in higher plants, including those encoding core microprocessor components and other auxiliary and regulatory proteins that influence the stability, folding, and processing of pri-miRNAs. We analyzed miRNA biogenesis proteins and found similar domain architecture in most cases. Our data support the hypothesis that almost all miRNA biogenesis factors in higher plants are also present in liverworts, suggesting that they emerged early during land plant evolution.

The MYB33, MYB65, and MYB101 transcription factors affect Arabidopsis and potato responses to drought by regulating the ABA signaling pathway.

Drought is one of the main climate threats limiting crop production. Potato is one of the four most important food crop species worldwide and is sensitive to water shortage. The CBP80 gene was shown to affect Arabidopsis and potato responses to drought by regulating the level of microRNA159 and, consequently, the levels of the MYB33 and MYB101 transcription factors (TFs). Here, we show that three MYB TFs, MYB33, MYB65, and MYB101, are involved in plant responses to water shortage. Their downregulation in Arabidopsis causes stomatal hyposensitivity to abscisic acid (ABA), leading to reduced tolerance to drought. Transgenic Arabidopsis and potato plants overexpressing these genes, with a mutated recognition site in miR159, show hypersensitivity to ABA and relatively high tolerance to drought conditions. Thus, the MYB33, MYB65, and MYB101 genes may be potential targets for innovative breeding to obtain crops with relatively high tolerance to drought.

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.

Core spliceosomal Sm proteins as constituents of cytoplasmic mRNPs in plants.

In recent years, research has increasingly focused on the key role of post-transcriptional regulation of messenger ribonucleoprotein (mRNP) function and turnover. As a result of the complexity and dynamic nature of mRNPs, the full composition of a single mRNP complex remains unrevealed and mRNPs are poorly described in plants. Here we identify canonical Sm proteins as part of the cytoplasmic mRNP complex, indicating their function in the post-transcriptional regulation of gene expression in plants. Sm proteins comprise an evolutionarily ancient family of small RNA-binding proteins involved in pre-mRNA splicing. The latest research indicates that Sm could also impact on mRNA at subsequent stages of its life cycle. In this work we show that in the microsporocyte cytoplasm of Larix decidua, the European larch, Sm proteins accumulate within distinct cytoplasmic bodies, also containing polyadenylated RNA. To date, several types of cytoplasmic bodies involved in the post-transcriptional regulation of gene expression have been described, mainly in animal cells. Their role and molecular composition in plants remain less well established, however. A total of 222 mRNA transcripts have been identified as cytoplasmic partners for Sm proteins. The specific colocalization of these mRNAs with Sm proteins within cytoplasmic bodies has been confirmed via microscopic analysis. The results from this work support the hypothesis, that evolutionarily conserved Sm proteins have been adapted to perform a whole repertoire of functions related to the post-transcriptional regulation of gene expression in Eukaryota. This adaptation presumably enabled them to coordinate the interdependent processes of splicing element assembly, mRNA maturation and processing, and mRNA translation regulation, and its degradation.

The identification of differentially expressed genes in male and female gametophytes of simple thalloid liverwort Pellia endiviifolia sp. B using an RNA-seq approach.

MAIN CONCLUSION: This study shows differences in gene expression between male and female gametophytes of the simple thalloid liverwort with a distinction between the vegetative and reproductive phases of growth. Pellia endiviifolia is a simple thalloid liverwort that, together with hornworts and mosses, represents the oldest living land plants. The limited taxon sampling for genomic and functional studies hampers our understanding of processes governing evolution of these plants. RNA sequencing represents an attractive way to elucidate the molecular mechanisms of non-model species development. In the present study, RNA-seq was used to profile the differences in gene expression between P. endiviifolia male and female gametophytes, with a distinction between the vegetative and reproductive phases of growth. By comparison of the gene expression profiles from individuals producing sex organs with the remaining thalli types, we have determined a set of genes whose expression might be important for the development of P. endiviifolia reproductive organs. The selected differentially expressed genes (DEGs) were categorized into five main pathways: metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems. A comparison of the obtained data with the Marchantia polymorpha transcriptome resulted in the identification of genes exhibiting a similar expression pattern during the reproductive phase of growth between members of the two distinct liverwort classes. The common expression profile of  87 selected genes suggests a common mechanism governing sex organ development in both liverwort species. The obtained RNA-seq results were confirmed by RT-qPCR for the DEGs with the highest differences in expression level. Five Pellia-female-specific and two Pellia-male-specific DEGs showed enriched expression in archegonia and antheridia, respectively. The identified genes are promising candidates for functional studies of their involvement in liverwort sexual reproduction.

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.

Active 5' splice sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from intron-containing genes.

Arabidopsis, miR402 that is encoded within the first intron of a protein-coding gene At1g77230, is induced by heat stress. Its upregulation correlates with splicing inhibition and intronic proximal polyA site selection. It suggests that miR402 is not processed from an intron, but rather from a shorter transcript after selection of the proximal polyA site within this intron. Recently, introns and active 5' splice sites (5'ss') have been shown to stimulate the accumulation of miRNAs encoded within the first exons of intron-containing MIR genes. In contrast, we have observed the opposite effect of splicing inhibition on intronic miR402 production. Transient expression experiments performed in tobacco leaves revealed a significant accumulation of the intronic mature miR402 when the 5'ss of the miR402-hosting intron was inactivated. In contrast, when the miR402 stem-loop structure was moved into the first exon, mutation of the first-intron 5'ss resulted in a decrease in the miRNA level. Thus, the 5'ss controls the efficiency of miRNA biogenesis. We also show that the SERRATE protein (a key component of the plant microprocessor) colocalizes and interacts with several U1 snRNP auxiliary proteins. We postulate that SERRATE-spliceosome connections have a direct effect on miRNA maturation.

tRex: A Web Portal for Exploration of tRNA-Derived Fragments in Arabidopsis thaliana.

tRNA-derived fragments (tRFs) constitute a new class of short regulatory RNAs that are a product of nascent or mature tRNA processing. tRF sequences have been identified in all domains of life; however, most published research pertains to human, yeast and some bacterial organisms. Despite growing interest in plant tRFs and accumulating evidence of their function in plant development and stress responses, no public, web-based repository dedicated to these molecules is currently available. Here, we introduce tRex (http://combio.pl/trex)-the first comprehensive data-driven online resource specifically dedicated to tRFs in the model plant Arabidopsis thaliana. The portal is based on verified Arabidopsis tRNA annotation and includes in-house-generated and publicly available small RNA sequencing experiments from various tissues, ecotypes, genotypes and stress conditions. The provided web-based tools are designed in a user-friendly manner and allow for seamless exploration of the data that are presented in the form of dynamic tables and cumulative coverage profiles. The tRex database is connected to external genomic and citation resources, which makes it a one-stop solution for Arabidopsis tRF-related research.

Genomewide identification of genes involved in the potato response to drought indicates functional evolutionary conservation with Arabidopsis plants.

Potato is one of the four most important food crop plants worldwide and is strongly affected by drought. The following two pairs of potato cultivars, which are related in ancestry but show different drought tolerances, were chosen for comparative gene expression studies: Gwiazda/Oberon and Tajfun/Owacja. Comparative RNA-seq analyses of gene expression differences in the transcriptomes obtained from drought-tolerant versus drought-sensitive plants during water shortage conditions were performed. The 23 top-ranking genes were selected, 22 of which are described here as novel potato drought-responsive genes. Moreover, all but one of the potato genes selected have homologues in the Arabidopsis genome. Of the seven tested A. thaliana mutants with altered expression of the selected homologous genes, compared to the wild-type Arabidopsis plants, six showed an improved tolerance to drought. These genes encode carbohydrate transporter, mitogen-activated protein kinase kinase kinase 15 (MAPKKK15), serine carboxypeptidase-like 19 protein (SCPL19), armadillo/beta-catenin-like repeat-containing protein, high-affinity nitrate transporter 2.7 and nonspecific lipid transfer protein type 2 (nsLPT). The evolutionary conservation of the functions of the selected genes in the plant response to drought confirms the importance of these identified potato genes in the ability of plants to cope with water shortage conditions. Knowledge regarding these gene functions can be used to generate potato cultivars that are resistant to unfavourable conditions. The approach used in this work and the obtained results allowed for the identification of new players in the plant response to drought.

A stable tRNA-like molecule is generated from the long noncoding RNA GUT15 in Arabidopsis.

The Arabidopsis GUT15 RNA belongs to a class of noncoding RNAs that are expressed from the intergenic regions of protein-coding genes. We show that the RNA polymerase II transcribed GUT15 transcript serves as a precursor for two stable RNA species, a tRNA-like molecule and GUT15-tRF-F5, which are both encoded by the final intron in the GUT15 gene. The GUT15-encoded tRNA-like molecule cannot be autonomously transcribed by RNA polymerase III. However, this molecule contains a CCA motif, suggesting that it may enter the tRNA maturation pathway. The GUT15-encoded tRNA-like sequence has an inhibiting effect on the splicing of its host intron. Moreover, we demonstrate that the canonical tRNA genes nested within introns do not affect the splicing patterns of their host protein-coding 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.