Reactive Oxygen Species Distribution Involved in Stipe Gradient Elongation in the Mushroom Flammulina filiformis.

The mushroom stipe raises the pileus above the substrate into a suitable position for dispersing spores. The stipe elongates at different speeds along its length, with the rate of elongation decreasing in a gradient from the top to the base. However, the molecular mechanisms underlying stipe gradient elongation are largely unknown. Here, we used the model basidiomycete mushroom Flammulina filiformis to investigate the mechanism of mushroom stipe elongation and the role of reactive oxygen species (ROS) signaling in this process. Our results show that O2- and H2O2 exhibit opposite gradient distributions in the stipe, with higher O2- levels in the elongation region (ER), and higher H2O2 levels in the stable region (SR). Moreover, NADPH-oxidase-encoding genes are up-regulated in the ER, have a function in producing O2-, and positively regulate stipe elongation. Genes encoding manganese superoxide dismutase (MnSOD) are up-regulated in the SR, have a function in producing H2O2, and negatively regulate stipe elongation. Altogether, our data demonstrate that ROS (O2-/H2O2) redistribution mediated by NADPH oxidase and MnSODs is linked to the gradient elongation of the F. filiformis stipe.

Plant long non-coding RNAs in the regulation of transcription.

Eukaryotic genomes are pervasively transcribed, producing large numbers of non-coding RNAs (ncRNAs), including tens of thousands of long ncRNAs (lncRNAs), defined as ncRNAs longer than 200 nucleotides. Recent studies have revealed the important roles lncRNAs play in the regulation of gene expression at various levels in all eukaryotes; moreover, emerging research in plants has identified roles for lncRNAs in key processes such as flowering time control, root organogenesis, reproduction, and adaptation to environmental changes. LncRNAs participate in regulating most steps of gene expression, including reshaping nuclear organization and chromatin structure; governing multiple steps of transcription, splicing, mRNA stability, and translation; and affecting post-translational protein modifications. In this review, I present the latest progress on the lncRNA-mediated regulatory mechanisms modulating transcription in Arabidopsis thaliana, focusing on their functions in regulation of gene expression via chromatin structure and interactions with the transcriptional machinery.

Novel mRNAs 3' end-associated cis-regulatory elements with epigenomic signatures of mammalian enhancers in the Arabidopsis genome.

The precise spatial and temporal control of gene expression requires the coordinated action of genomic cis-regulatory elements (CREs), including transcriptional enhancers. However, our knowledge of enhancers in plants remains rudimentary and only a few plant enhancers have been experimentally defined. Here, we screened the Arabidopsis thaliana genome and identified >1900 unique candidate CREs that carry the genomic signatures of mammalian enhancers. These were termed putative enhancer-like elements (PEs). Nearly all PEs are intragenic and, unexpectedly, most associate with the 3' ends of protein-coding genes. PEs are hotspots for transcription factor binding and harbor motifs resembling cleavage/polyadenylation signals, potentially coupling 3' end processing to the transcriptional regulation of other genes. Hi-C data showed that 24% of PEs are located at regions that can interact intrachromosomally with other protein-coding genes and, surprisingly, many of these target genes interact with PEs through their 3' UTRs. Examination of the genomes of 1135 sequenced Arabidopsis accessions showed that PEs are conserved. Our findings suggest that the identified PEs may serve as transcriptional enhancers and sites for mRNA 3' end processing, and constitute a novel group of CREs in Arabidopsis.

An Overview of Methodologies in Studying lncRNAs in the High-Throughput Era: When Acronyms ATTACK!

The discovery of pervasive transcription in eukaryotic genomes provided one of many surprising (and perhaps most surprising) findings of the genomic era and led to the uncovering of a large number of previously unstudied transcriptional events. This pervasive transcription leads to the production of large numbers of noncoding RNAs (ncRNAs) and thus opened the window to study these diverse, abundant transcripts of unclear relevance and unknown function. Since that discovery, recent advances in high-throughput sequencing technologies have identified a large collection of ncRNAs, from microRNAs to long noncoding RNAs (lncRNAs). Subsequent discoveries have shown that many lncRNAs play important roles in various eukaryotic processes; these discoveries have profoundly altered our understanding of the regulation of eukaryotic gene expression. Although the identification of ncRNAs has become a standard experimental approach, the functional characterization of these diverse ncRNAs remains a major challenge. In this chapter, we highlight recent progress in the methods to identify lncRNAs and the techniques to study the molecular function of these lncRNAs and the application of these techniques to the study of plant lncRNAs.

Degradation of unmethylated miRNA/miRNA*s by a DEDDy-type 3' to 5' exoribonuclease Atrimmer 2 in Arabidopsis.

The 3' end methylation catalyzed by HUA Enhancer 1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that Atrimmer 2 (ATRM2), a DEDDy-type 3' to 5' exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis Loss-of-function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Dysfunction of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3' to 5' trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly correlated in hen1 atrm2 versus hen1 Unexpectedly, we observed a marked increase of 3' to 5' trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to Argonaute (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with Argonaute 1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RNA-induced silencing complex assembly.

Long Noncoding RNAs in Plants.

The eukaryotic genomes are pervasively transcribed. In addition to protein-coding RNAs, thousands of long noncoding RNAs (lncRNAs) modulate key molecular and biological processes. Most lncRNAs are found in the nucleus and associate with chromatin, but lncRNAs can function in both nuclear and cytoplasmic compartments. Emerging work has found that many lncRNAs regulate gene expression and can affect genome stability and nuclear domain organization both in plant and in the animal kingdom. Here, we describe the major plant lncRNAs and how they act, with a focus on research in Arabidopsis thaliana and our emerging understanding of lncRNA functions in serving as molecular sponges and decoys, functioning in regulation of transcription and silencing, particularly in RNA-directed DNA methylation, and in epigenetic regulation of flowering time.

Small RNAs: essential regulators of gene expression and defenses against environmental stresses in plants.

Eukaryotic genomes produce thousands of diverse small RNAs (smRNAs), which play vital roles in regulating gene expression in all conditions, including in survival of biotic and abiotic environmental stresses. SmRNA pathways intersect with most of the pathways regulating different steps in the life of a messenger RNA (mRNA), starting from transcription and ending at mRNA decay. SmRNAs function in both nuclear and cytoplasmic compartments; the regulation of mRNA stability and translation in the cytoplasm and the epigenetic regulation of gene expression in the nucleus are the main and best-known modes of smRNA action. However, recent evidence from animal systems indicates that smRNAs and RNA interference (RNAi) also participate in the regulation of alternative pre-mRNA splicing, one of the most crucial steps in the fast, efficient global reprogramming of gene expression required for survival under stress. Emerging evidence from bioinformatics studies indicates that a specific class of plant smRNAs, induced by various abiotic stresses, the sutr-siRNAs, has the potential to target regulatory regions within introns and thus may act in the regulation of splicing in response to stresses. This review summarizes the major types of plant smRNAs in the context of their mechanisms of action and also provides examples of their involvement in regulation of gene expression in response to environmental cues and developmental stresses. In addition, we describe current advances in our understanding of how smRNAs function in the regulation of pre-mRNA splicing. WIREs RNA 2016, 7:356-381. doi: 10.1002/wrna.1340 For further resources related to this article, please visit the WIREs website.

Long non-coding RNAs and their functions in plants.

Eukaryotic genomes encode thousands of long noncoding RNAs (lncRNAs), which play important roles in essential biological processes. Although lncRNAs function in the nuclear and cytoplasmic compartments, most of them occur in the nucleus, often in association with chromatin. Indeed, many lncRNAs have emerged as key regulators of gene expression and genome stability. Emerging evidence also suggests that lncRNAs may contribute to the organization of nuclear domains. This review briefly summarizes the major types of eukaryotic lncRNAs and provides examples of their mechanisms of action, with focus on plant lncRNAs, mainly in Arabidopsis thaliana, and describes current advances in our understanding of the mechanisms of lncRNA action and the roles of lncRNAs in RNA-dependent DNA methylation and in the regulation of flowering time.

Stress-induced endogenous siRNAs targeting regulatory intron sequences in Brachypodium.

Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3' UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr-siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes.

Arabidopsis RRP6L1 and RRP6L2 function in FLOWERING LOCUS C silencing via regulation of antisense RNA synthesis.

The exosome complex functions in RNA metabolism and transcriptional gene silencing. Here, we report that mutations of two Arabidopsis genes encoding nuclear exosome components AtRRP6L1 and AtRRP6L2, cause de-repression of the main flowering repressor FLOWERING LOCUS C (FLC) and thus delay flowering in early-flowering Arabidopsis ecotypes. AtRRP6L mutations affect the expression of known FLC regulatory antisense (AS) RNAs AS I and II, and cause an increase in Histone3 K4 trimethylation (H3K4me3) at FLC. AtRRP6L1 and AtRRP6L2 function redundantly in regulation of FLC and also act independently of the exosome core complex. Moreover, we discovered a novel, long non-coding, non-polyadenylated antisense transcript (ASL, for Antisense Long) originating from the FLC locus in wild type plants. The AtRRP6L proteins function as the main regulators of ASL synthesis, as these mutants show little or no ASL transcript. Unlike ASI/II, ASL associates with H3K27me3 regions of FLC, suggesting that it could function in the maintenance of H3K27 trimethylation during vegetative growth. AtRRP6L mutations also affect H3K27me3 levels and nucleosome density at the FLC locus. Furthermore, AtRRP6L1 physically associates with the ASL transcript and directly interacts with the FLC locus. We propose that AtRRP6L proteins participate in the maintenance of H3K27me3 at FLC via regulating ASL. Furthermore, AtRRP6Ls might participate in multiple FLC silencing pathways by regulating diverse antisense RNAs derived from the FLC locus.

The role of the Arabidopsis Exosome in siRNA-independent silencing of heterochromatic loci.

The exosome functions throughout eukaryotic RNA metabolism and has a prominent role in gene silencing in yeast. In Arabidopsis, exosome regulates expression of a "hidden" transcriptome layer from centromeric, pericentromeric, and other heterochromatic loci that are also controlled by small (sm)RNA-based de novo DNA methylation (RdDM). However, the relationship between exosome and smRNAs in gene silencing in Arabidopsis remains unexplored. To investigate whether exosome interacts with RdDM, we profiled Arabidopsis smRNAs by deep sequencing in exosome and RdDM mutants and also analyzed RdDM-controlled loci. We found that exosome loss had a very minor effect on global smRNA populations, suggesting that, in contrast to fission yeast, in Arabidopsis the exosome does not control the spurious entry of RNAs into smRNA pathways. Exosome defects resulted in decreased histone H3K9 dimethylation at RdDM-controlled loci, without affecting smRNAs or DNA methylation. Exosome also exhibits a strong genetic interaction with RNA Pol V, but not Pol IV, and physically associates with transcripts produced from the scaffold RNAs generating region. We also show that two Arabidopsis rrp6 homologues act in gene silencing. Our data suggest that Arabidopsis exosome may act in parallel with RdDM in gene silencing, by epigenetic effects on chromatin structure, not through siRNAs or DNA methylation.

Recognition of polyadenosine RNA by the zinc finger domain of nuclear poly(A) RNA-binding protein 2 (Nab2) is required for correct mRNA 3'-end formation.

Proteins bound to the poly(A) tail of mRNA transcripts, called poly(A)-binding proteins (Pabs), play critical roles in regulating RNA stability, translation, and nuclear export. Like many mRNA-binding proteins that modulate post-transcriptional processing events, assigning specific functions to Pabs is challenging because these processing events are tightly coupled to one another. To investigate the role that a novel class of zinc finger-containing Pabs plays in these coupled processes, we defined the mode of polyadenosine RNA recognition for the conserved Saccharomyces cerevisiae Nab2 protein and assessed in vivo consequences caused by disruption of RNA binding. The polyadenosine RNA recognition domain of Nab2 consists of three tandem Cys-Cys-Cys-His (CCCH) zinc fingers. Cells expressing mutant Nab2 proteins with decreased binding to polyadenosine RNA show growth defects as well as defects in poly(A) tail length but do not accumulate poly(A) RNA in the nucleus. We also demonstrate genetic interactions between mutant nab2 alleles and mutant alleles of the mRNA 3'-end processing machinery. Together, these data provide strong evidence that Nab2 binding to RNA is critical for proper control of poly(A) tail length.

Diverse poly(A) binding proteins mediate internal translational initiation by a plant viral IRES.

During 5'-cap-dependent translation, methylated 5'-cap and 3'-poly(A) tail work synergistically in a poly(A) binding protein (PABP)-dependent manner to facilitate translation via promoting the formation of a closed mRNA loop. On the other hand, during internal translation initiation, the requirement for and the roles of 3'-poly(A) tail and PABP vary depending on specific characteristics of each internal ribosomal entry site (IRES). In this study, we analyzed the effect of 3'-poly(A) tail and phylogenetically divergent PABPs on a polypurine tract-containing IRES element derived from the coat protein gene of crucifer-infecting tobamovirus (CrTMV IRES(CP)). We find that mutations in the internal polypurine tract decrease IRES activity in a heterologous (mammalian) system in vivo. Moreover, these mutations decrease the high-affinity binding of all phylogenetically divergent PABPs derived from Arabidopsis and yeast in electro mobility gel shift assays in vitro. Partial PABP depletion and reconstitution assays using Arabidopsis-derived PABP2, 3, 5, 8 and yeast Pab1p provide further evidence that CrTMV IRES(CP) requires PABP for maximal activity. Furthermore, stronger enhancement in the presence of 3'-poly(A) and the absence of 5'-methylated cap suggests a potential joint interaction between PABP, the CrTMV IRES(CP) and the 3'-poly(A).

Sus1, Sac3, and Thp1 mediate post-transcriptional tethering of active genes to the nuclear rim as well as to non-nascent mRNP.

Errors in the mRNP biogenesis pathway can lead to retention of mRNA in discrete, transcription-site-proximal foci. This RNA remains tethered adjacent to the transcription site long after transcriptional shutoff. Here we identify Sus1, Thp1, and Sac3 as factors required for the persistent tethering of such foci (dots) to their cognate genes. We also show that the prolonged association of previously activated GAL genes with the nuclear periphery after transcriptional shutoff is similarly dependent on the Sac3-Thp1-Sus1-Cdc31 complex. We suggest that the complex associates with nuclear mRNP and that mRNP properties influence the association of dot-confined mRNA with its gene of origin as well as the post-transcriptional retention of the cognate gene at the nuclear periphery. These findings indicate a coupling between the mRNA-to-gene and gene-to-nuclear periphery tethering. Taken together with other recent findings, these observations also highlight the importance of nuclear mRNP to the mobilization of active genes to the nuclear rim.

Genome-wide high-resolution mapping of exosome substrates reveals hidden features in the Arabidopsis transcriptome.

The exosome complex plays a central and essential role in RNA metabolism. However, comprehensive studies of exosome substrates and functional analyses of its subunits are lacking. Here, we demonstrate that as opposed to yeast and metazoans the plant exosome core possesses an unanticipated functional plasticity and present a genome-wide atlas of Arabidopsis exosome targets. Additionally, our study provides evidence for widespread polyadenylation- and exosome-mediated RNA quality control in plants, reveals unexpected aspects of stable structural RNA metabolism, and uncovers numerous novel exosome substrates. These include a select subset of mRNAs, miRNA processing intermediates, and hundreds of noncoding RNAs, the vast majority of which have not been previously described and belong to a layer of the transcriptome that can only be visualized upon inhibition of exosome activity. These first genome-wide maps of exosome substrates will aid in illuminating new fundamental components and regulatory mechanisms of eukaryotic transcriptomes.

MicroRNAs and messenger RNA turnover.

Although initially believed to act exclusively as translational repressors, microRNAs (miRNAs) are now known to target complementary messenger RNA (mRNA) transcripts for either translational repression or cleavage via the RNA-induced silencing complex (RISC) ([1], reviewed in ref. 2). The current model postulates that mature miRNAs are incorporated into the RISC, bind target mRNAs based on complementarity, and guide cleavage of mRNA targets with perfect or nearly perfect complementarity and translational repression of targets with lower complementarity (2). The translational repression mechanism of miRNA-mediated gene regulation, which is common in animals but also exists in plants, is not well understood mechanistically. Conversely, miRNA-directed mRNA cleavage by RISC is common in plants, but also occurs in animals (3). This chapter focuses on the mRNA cleavage by miRNA-programmed RISC, and, specifically, on characterizing the products of such cleavage.

3'-end formation signals modulate the association of genes with the nuclear periphery as well as mRNP dot formation.

Multiple studies indicate that mRNA processing defects cause mRNAs to accumulate in discrete nuclear foci or dots, in mammalian cells as well as yeast. To investigate this phenomenon, we have studied a series of GAL reporter constructs integrated into the yeast genome adjacent to an array of TetR-GFP-bound TetO sites. mRNA within dots is predominantly post-transcriptional, and dots are adjacent to but distinct from their transcription site. These reporter genes also localize to the nuclear periphery upon gene induction, like their endogenous GAL counterparts. Surprisingly, this peripheral localization persists long after transcriptional shutoff, and there is a comparable persistence of the RNA in the dots. Moreover, dot disappearance and gene delocalization from the nuclear periphery occur with similar kinetics after transcriptional shutoff. Both kinetics depend in turn on reporter gene 3'-end formation signals. Our experiments indicate that gene association with the nuclear periphery does not require ongoing transcription and suggest that the mRNPs within dots may make a major contribution to the gene-nuclear periphery tether.

mRNA deadenylation by PARN is essential for embryogenesis in higher plants.

Deadenylation of mRNA is often the first and rate-limiting step in mRNA decay. PARN, a poly(A)-specific 3' --> 5' ribonuclease which is conserved in many eukaryotes, has been proposed to be primarily responsible for such a reaction, yet the importance of the PARN function at the whole-organism level has not been demonstrated in any species. Here, we show that mRNA deadenylation by PARN is essential for viability in higher plants (Arabidopsis thaliana). Yet, this essential requirement for the PARN function is not universal across the phylogenetic spectrum, because PARN is dispensable in Fungi (Schizosaccharomyces pombe), and can be at least severely downregulated without any obvious consequences in Metazoa (Caenorhabditis elegans). Development of the Arabidopsis embryos lacking PARN (AtPARN), as well as of those expressing an enzymatically inactive protein, was markedly retarded, and ultimately culminated in an arrest at the bent-cotyledon stage. Importantly, only some, rather than all, embryo-specific transcripts were hyperadenylated in the mutant embryos, suggesting that preferential deadenylation of a specific select subset of mRNAs, rather than a general deadenylation of the whole mRNA population, by AtPARN is indispensable for embryogenesis in Arabidopsis. These findings indicate a unique, nonredundant role of AtPARN among the multiple plant deadenylases.

Evidence that poly(A) binding protein has an evolutionarily conserved function in facilitating mRNA biogenesis and export.

Eukaryotic poly(A) binding protein (PABP) is a ubiquitous, essential cellular factor with well-characterized roles in translational initiation and mRNA turnover. In addition, there exists genetic and biochemical evidence that PABP has an important nuclear function. Expression of PABP from Arabidopsis thaliana, PAB3, rescues an otherwise lethal phenotype of the yeast pab1Delta mutant, but it neither restores the poly(A) dependent stimulation of translation, nor protects the mRNA 5' cap from premature removal. In contrast, the plant PABP partially corrects the temporal lag that occurs prior to the entry of mRNA into the decay pathway in the yeast strains lacking Pab1p. Here, we examine the nature of this lag-correction function. We show that PABP (both PAB3 and the endogenous yeast Pab1p) act on the target mRNA via physically binding to it, to effect the lag correction. Furthermore, substituting PAB3 for the yeast Pab1p caused synthetic lethality with rna15-2 and gle2-1, alleles of the genes that encode a component of the nuclear pre-mRNA cleavage factor I, and a factor associated with the nuclear pore complex, respectively. PAB3 was present physically in the nucleus in the complemented yeast strain and was able to partially restore the poly(A) tail length control during polyadenylation in vitro, in a poly(A) nuclease (PAN)-dependent manner. Importantly, PAB3 in yeast also promoted the rate of entry of mRNA into the translated pool, rescued the conditional lethality, and alleviated the mRNA export defect of the nab2-1 mutant when overexpressed. We propose that eukaryotic PABPs have an evolutionarily conserved function in facilitating mRNA biogenesis and export.

Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3'-->5' exonuclease containing S1 and KH RNA-binding domains.

The exosome, an evolutionarily conserved complex of multiple 3'-->5' exoribonucleases, is responsible for a variety of RNA processing and degradation events in eukaryotes. In this report Arabidopsis thaliana AtRrp4p is shown to be an active 3'-->5' exonuclease that requires a free 3'-hydroxyl and degrades RNA hydrolytically and distributively, releasing nucleoside 5'-monophosphate products. AtRrp4p behaves as an approximately 500 kDa species during sedimentation through a 10-30% glycerol gradient, co-migrating with AtRrp41p, another exosome subunit, and it interacts in vitro with AtRrp41p, suggesting that it is also present in the plant cell as a subunit of the exosome. We found that, in addition to a previously reported S1-type RNA-binding domain, members of the Rrp4p family of proteins contain a KH-type RNA-binding domain in the C-terminal half and show that either domain alone can bind RNA. However, only the full-length protein is capable of degrading RNA and interacting with AtRrp41p.