Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs). We have demonstrated recently that vacuolar sorting receptor (VSR) proteins are concentrated on PVCs. In this study, we generated transgenic Nicotiana tabacum (tobacco) BY-2 cell lines expressing two yellow fluorescent protein (YFP)-fusion reporters that mark PVC and Golgi organelles. Both transgenic cell lines exhibited typical punctate YFP signals corresponding to distinct PVC and Golgi organelles because the PVC reporter colocalized with VSR proteins, whereas the Golgi marker colocalized with mannosidase I in confocal immunofluorescence. Brefeldin A induced the YFP-labeled Golgi stacks but not the YFP-marked PVCs to form typical enlarged structures. By contrast, wortmannin caused YFP-labeled PVCs but not YFPlabeled Golgi stacks to vacuolate. VSR antibodies labeled multivesicular bodies (MVBs) on thin sections prepared from highpressure frozen/freeze substituted samples, and the enlarged PVCs also were indentified as MVBs. MVBs were further purified from BY-2 cells and found to contain VSR proteins via immunogold negative staining. Similar to YFP-labeled Golgi stacks, YFP-labeled PVCs are mobile organelles in BY-2 cells. Thus, we have unequivocally identified MVBs as PVCs in N. tabacum BY-2 cells. Uptake studies with the styryl dye FM4-64 strongly indicate that PVCs also lie on the endocytic pathway of BY-2 cells.

Translation inhibition is a major but poorly understood mode of action of micro(mi)RNAs in plants and animals. In particular, the subcellular location where this process takes place is unknown. Here we show that the translation inhibition but not the mRNA cleavage activity of Arabidopsis miRNAs requires ALTERED MERISTEM PROGRAM1 (AMP1). AMP1 encodes an integral membrane protein associated with endoplasmic reticulum (ER) and ARGONAUTE1, the miRNA effector and a peripheral ER membrane protein. Large differences in polysome association of miRNA target RNAs are found between wild type and the amp1 mutant for membrane-bound but not total polysomes. This, together with AMP1-independent recruitment of miRNA target transcripts to membrane fractions, shows that miRNAs inhibit the translation of target RNAs on the ER. This study demonstrates that translation inhibition is an important activity of plant miRNAs, reveals the subcellular location of this activity, and uncovers a previously unknown function of the ER.

SUMMARY microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs) impact numerous biological processes in eukaryotes. In addition to biogenesis, turnover contributes to the steady-state levels of small RNAs. One major factor that stabilizes miRNAs and siRNAs in plants as well as siRNAs and piRNAs in animals is 2′;-O-methylation on the 3′; terminal ribose by the methyltransferase HUA ENHANCER1 (HEN1) [1–6]. Genetic studies with Arabidopsis, Drosophila and zebrafish hen1 mutants show that 2′-O-methylation protects small RNAs from 3′-to-5′ truncation and 3′ uridylation, the addition of non-templated nucleotides, predominantly uridine [2, 7, 8]. Uridylation is a widespread phenomenon that is not restricted to small RNAs in hen1 mutants, and is often associated with their reduced accumulation ([7, 9, 10]; reviewed in [11]). The enzymes responsible for 3′ uridylation of small RNAs when they lack methylation in plants or animals have remained elusive. Here, we identify the Arabidopsis HEN1 SUPPRESSOR1 (HESO1) gene as responsible for small RNA uridylation in hen1 mutants. HESO1 exhibits terminal nucleotidyl transferase activity, prefers uridine as the substrate nucleotide, and is completely inhibited by 2′-O-methylation. We show that uridylation leads to miRNA degradation, and the degradation is most likely through an enzyme that is distinct from that causing the 3′ truncation in hen1 mutants.

Mediator is a conserved multi-subunit complex known to promote the transcription of protein-coding genes by RNA polymerase II (Pol II) in eukaryotes. It has been increasingly realized that Pol II transcribes a large number of intergenic loci to generate noncoding RNAs, but the role of Mediator in Pol II-mediated noncoding RNA production has been largely unexplored. The role of Mediator in noncoding RNA production in plants is particularly intriguing given that plants have evolved from Pol II two additional polymerases, Pol IV and Pol V, to specialize in noncoding RNA production and transcriptional gene silencing at heterochromatic loci. Here, we show that Mediator is required for microRNA (miRNA) biogenesis by recruiting Pol II to promoters of miRNA genes. We also show that several well-characterized heterochromatic loci are de-repressed in Mediator mutants and that Mediator promotes Pol II-mediated production of long noncoding scaffold RNAs, which serve to recruit Pol V to these loci. This study expands the function of Mediator to include Pol II-mediated intergenic transcription and implicates a role of Mediator in genome stability.

Small RNAs are central players in RNA silencing, yet their cytoplasmic compartmentalization and the effects it may have on their activities have not been studied at the genomic scale. Here we report that Arabidopsis microRNAs (miRNAs) and small interfering RNAs (siRNAs) are distinctly partitioned between the endoplasmic reticulum (ER) and cytosol. All miRNAs are associated with membrane-bound polysomes (MBPs) as opposed to polysomes in general. The MBP association is functionally linked to a deeply conserved and tightly regulated activity of miRNAs – production of phased siRNAs (phasiRNAs) from select target RNAs. The phasiRNA precursor RNAs, thought to be noncoding, are on MBPs and are occupied by ribosomes in a manner that supports miRNA-triggered phasiRNA production, suggesting that ribosomes on the rough ER impact siRNA biogenesis. This study reveals global patterns of cytoplasmic partitioning of small RNAs and expands the known functions of ribosomes and ER.DOI: http://dx.doi.org/10.7554/eLife.22750.001

Histone acetylation is a major epigenetic control mechanism that is tightly linked to the promotion of gene expression. Histone acetylation levels are balanced through the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Arabidopsis HDAC genes (AtHDACs) compose a large gene family, and distinct phenotypes among AtHDAC mutants reflect the functional specificity of individual AtHDACs. However, the mechanisms underlying this functional diversity are largely unknown. Here, we show that POWERDRESS (PWR), a SANT (SWI3/DAD2/N-CoR/TFIII-B) domain protein, interacts with HDA9 and promotes histone H3 deacetylation, possibly by facilitating HDA9 function at target regions. The developmental phenotypes of pwr and hda9 mutants were highly similar. Three lysine residues (K9, K14, and K27) of H3 retained hyperacetylation status in both pwr and hda9 mutants. Genome-wide H3K9 and H3K14 acetylation profiling revealed elevated acetylation at largely overlapping sets of target genes in the two mutants. Highly similar gene-expression profiles in the two mutants correlated with the histone H3 acetylation status in the pwr and hda9 mutants. In addition, PWR and HDA9 modulated flowering time by repressing AGAMOUS-LIKE 19 expression through histone H3 deacetylation in the same genetic pathway. Finally, PWR was shown to physically interact with HDA9, and its SANT2 domain, which is homologous to that of subunits in animal HDAC complexes, showed specific binding affinity to acetylated histone H3. We therefore propose that PWR acts as a subunit in a complex with HDA9 to result in lysine deacetylation of histone H3 at specific genomic targets.SANT domain | POWERDRESS | HDA9 | histone deacetylation | AGL19 P osttranslational modifications of histones-including acetylation, methylation, phosphorylation, and ubiquitinationplay important roles in plant development, genome integrity, and stress responses. Histone acetylation/deacetylation, a reversible process, promotes/represses gene expression (1) and occurs at lysine residues within histone N-terminal tails. The histone acetylation status is regulated by counteracting enzymes: histone acetyltransferases (HATs) and histone deacetylases (HDACs). The 18 HDACs identified in Arabidopsis (2) can be categorized into three groups based on phylogenetic analysis: reduced potassium dependency-3/histone deacetylase-1 (RPD3/HDA1), histone deacetylase-2 (HD2), and silent information regulator-2 (SIR2)-like (3). Twelve HDACs belong to the RPD3/HDA1 group (3) and are involved in various biological processes, such as organ development, reproductive processes, hormone signaling, and DNA methylation (4-9). They can be further classified into three classes based on sequence homology (3). The HD2 group is plant-specific and includes four HDACs that act in plant development and stress responses (10-13). The two HDACs encoded by the SIR2 family genes in Arabidopsis, SRT1 and SRT2, regulate mitochondrial energy metabolism and cellular dedifferentiation, respectively (14,15).I...

BackgroundAs key regulators of gene expression in eukaryotes, small RNAs have been characterized in many seed plants, and pathways for their biogenesis, degradation, and action have been defined in model angiosperms. However, both small RNAs themselves and small RNA pathways are not well characterized in other land plants such as lycophytes and ferns, preventing a comprehensive evolutionary perspective on small RNAs in land plants.ResultsUsing 25 representatives from major lineages of lycophytes and ferns, most of which lack sequenced genomes, we characterized small RNAs and small RNA pathways in these plants. We identified homologs of DICER-LIKE (DCL), ARGONAUTE (AGO), and other genes involved in small RNA pathways, predicted over 2600 conserved microRNA (miRNA) candidates, and performed phylogenetic analyses on small RNA pathways as well as miRNAs. Pathways underlying miRNA biogenesis, degradation, and activity were established in the common ancestor of land plants, but the 24-nucleotide siRNA pathway that guides DNA methylation is incomplete in sister species of seed plants, especially lycophytes. We show that the functional diversification of key gene families such as DCL and AGO as observed in angiosperms occurred early in land plants followed by parallel expansion of the AGO family in ferns and angiosperms. We uncovered a conserved AGO subfamily absent in angiosperms.ConclusionsOur phylogenetic analyses of miRNAs in bryophytes, lycophytes, ferns, and angiosperms refine the time-of-origin for conserved miRNA families as well as small RNA machinery in land plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1291-2) contains supplementary material, which is available to authorized users.

3’ uridylation is increasingly recognized as a conserved RNA modification process associated with RNA turnover in eukaryotes. 2’-O-methylation on the 3’ terminal ribose protects micro(mi)RNAs from 3’ truncation and 3’ uridylation in Arabidopsis. Previously, we identified HESO1 as the nucleotidyl transferase that uridylates most unmethylated miRNAs in vivo, but substantial 3’ tailing of miRNAs still remains in heso1 loss-of-function mutants. In this study, we found that among nine other potential nucleotidyl transferases, UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) is the single most predominant nucleotidyl transferase that tails miRNAs. URT1 and HESO1 prefer substrates with different 3’ end nucleotides in vitro and act cooperatively to tail different forms of the same miRNAs in vivo. Moreover, both HESO1 and URT1 exhibit nucleotidyl transferase activity on AGO1-bound miRNAs. Although these enzymes are able to add long tails to AGO1-bound miRNAs, the tailed miRNAs remain associated with AGO1. Moreover, tailing of AGO1-bound miRNA165/6 drastically reduces the slicing activity of AGO1-miR165/6, suggesting that tailing reduces miRNA activity. However, monouridylation of miR171a by URT1 endows the miRNA the ability to trigger the biogenesis of secondary siRNAs. Therefore, 3’ tailing could affect the activities of miRNAs in addition to leading to miRNA degradation.