Beixin Mo

MicroRNAs Inhibit the Translation of Target mRNAs on the Endoplasmic Reticulum in Arabidopsis

Translation inhibition is a major but poorly understood mode of action of microRNAs (miRNAs) 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.

The role of Mediator in small and long noncoding RNA production in Arabidopsis thaliana

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.

Distinct and cooperative activities of HESO1 and URT1 nucleotidyl transferases in microRNA turnover in Arabidopsis.

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.

Mechanisms that impact microRNA stability in plants

microRNAs (miRNAs) are 20–24 nucleotide RNAs that regulate a variety of developmental and metabolic processes. The accumulation of miRNAs in vivo can be controlled at multiple levels. In addition to miRNA biogenesis, mechanisms that lead to RNA degradation, such as 3′ uridylation and 3′ truncation, also affect the steady-state levels of miRNAs. On the other hand, 2’-O-methylation in plant miRNAs protects their 3′ ends from truncation and uridylation. The recent identification of HESO1 as the key enzyme responsible for miRNA uridylation in Arabidopsis was a first step toward a full understanding of the mechanisms underlying miRNA turnover. Analyses of the heso1 mutant predicted the existence of another uridylation activity and a previously unknown nuclease that act on miRNAs. The future identification of these enzymes will enrich our understanding of miRNA turnover.

Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis

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

ARGONAUTE10 promotes the degradation of miR165/6 through the SDN1 and SDN2 exonucleases in Arabidopsis

The degradation of small RNAs in plants and animals is associated with small RNA 3′ truncation and 3′ uridylation and thus relies on exonucleases and nucleotidyl transferases. ARGONAUTE (AGO) proteins associate with small RNAs in vivo and are essential for not only the activities but also the stability of small RNAs. AGO1 is the microRNA (miRNA) effector in Arabidopsis, and its closest homolog, AGO10, maintains stem cell homeostasis in meristems by sequestration of miR165/6, a conserved miRNA acting through AGO1. Here, we show that SMALL RNA DEGRADING NUCLEASES (SDNs) initiate miRNA degradation by acting on AGO1-bound miRNAs to cause their 3′ truncation, and the truncated species are uridylated and degraded. We report that AGO10 reduces miR165/6 accumulation by enhancing its degradation by SDN1 and SDN2 in vivo. In vitro, AGO10-bound miR165/6 is more susceptible to SDN1-mediated 3′ truncation than AGO1-bound miR165/6. Thus, AGO10 promotes the degradation of miR165/6, which is contrary to the stabilizing effect of AGO1. Our work identifies a class of exonucleases responsible for miRNA 3′ truncation in vivo and uncovers a mechanism of specificity determination in miRNA turnover. This work, together with previous studies on AGO10, suggests that spatially regulated miRNA degradation underlies stem cell maintenance in plants.

Trip to ER: MicroRNA-mediated translational repression in plants

miRNAs elicit gene silencing at the post-transcriptional level by several modes of action: translational repression, mRNA decay, and mRNA cleavage. Studies in animals have suggested that translational repression occurs at early steps of translation initiation, which can be followed by deadenylation and mRNA decay. Plant miRNAs were originally thought to solely participate in mRNA cleavage, but increasing evidence has indicated that they are also commonly involved in translational inhibition. Here we discuss recent findings on miRNA-mediated translational repression in plants. The identification of AMP1 in Arabidopsis as a protein required for the translational repression but not the mRNA cleavage activity of miRNAs links miRNA-based translational repression to the endoplasmic reticulum (ER). Future work is required to further elucidate the miRNA machinery on the ER.

Conservation and divergence of small RNA pathways and microRNAs in land plants

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.

Fast-suppressor screening for new components in protein trafficking, organelle biogenesis and silencing pathway in Arabidopsis thaliana using DEX-inducible FREE1-RNAi plants.

Membrane trafficking is essential for plant growth and responses to external signals. The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex (endosomal sorting complex required for transport). FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body (MVB), vacuolar protein transport and vacuole biogenesis, and autophagic degradation. FREE1 knockout plants show defective MVB formation, abnormal vacuolar transport, fragmented vacuoles, accumulated autophagosomes, and seedling lethality. To further uncover the underlying mechanisms of FREE1 function in plants, we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants. The obtained mutants are termed as suppressors of free1 (sof). To date, 229 putative sof mutants have been identified. Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors. Also 145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants. Through next-generation sequencing (NGS) of bulked DNA from F2 mapping population of two RNAi-related sof mutants, FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly. These FREE1- and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis, as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

POWERDRESS and HDA9 interact and promote histone H3 deacetylation at specific genomic sites in Arabidopsis.

Significance Histone deacetylases (HDACs) belong to a large protein family in plants, and little is known about how target specificity of each HDAC is achieved. We show that a paired SANT (SWI3/DAD2/N-CoR/TFIII-B) domain-containing protein, POWERDRESS, specifically acts with HDA9 to confer the deacetylation of histone H3 lysine residues at a set of genomic targets to regulate various biological processes. Our study elucidates the functional correlation between SANT domain-containing proteins and HDACs in plants. 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.