The XRN family of 5’→3’ exoribonucleases is critical for ensuring the fidelity of cellular RNA turnover in eukaryotes. Highly conserved across species, the family is typically represented by one cytoplasmic enzyme (XRN1/PACMAN or XRN4) and one or more nuclear enzymes (XRN2/RAT1 and XRN3). Cytoplasmic and/or nuclear XRNs have proven to be essential in all organisms tested, and deficiencies can have severe developmental phenotypes, demonstrating that XRNs are indispensable in fungi, plants and animals. XRNs degrade diverse RNA substrates during general RNA decay and function in specialized processes integral to RNA metabolism, such as nonsense-mediated decay (NMD), gene silencing, rRNA maturation, and transcription termination. Here, we review current knowledge of XRNs, highlighting recent work of high impact and future potential. One example is the breakthrough in our understanding of how XRN1 processively degrades 5’ monophosphorylated RNA, revealed by its crystal structure and mutational analysis. The expanding knowledge of XRN substrates and interacting partners is outlined and the functions of XRNs are interpreted at the organismal level using available mutant phenotypes. Finally, three case studies are discussed in more detail to underscore a few of the most exciting areas of research on XRN function: XRN4 involvement in small RNA-associated processes in plants, the roles of XRN1/PACMAN in Drosophila development, and the function of human XRN2 in nuclear transcriptional quality control. This article is part of a Special Issue entitled: RNA Decay Mechanisms.

Phosphorus availability is limited in many natural ecosystems. Plants adapt to phosphate (Pi) deficiency by complex molecular processes. There is growing evidence suggesting that transcription factors are key components in the regulation of these processes. In this study, we characterized the function of ZAT6 (zinc finger of Arabidopsis 6), a cysteine-2/histidine-2 zinc finger transcription factor that is responsive to Pi stress. ZAT6 is induced during Pi starvation and localizes to the nucleus. While the RNAi suppression of ZAT6 appeared to be lethal, its overexpression affects root development and retards seedling growth as a result of decreased Pi acquisition. The ZAT6 overexpression also resulted in altered root architecture of older plants, with consequent changes in Pi acquisition. These results indicate that ZAT6 regulates root development independent of the Pi status of the plant, thereby influencing Pi acquisition and homeostasis. In addition, the expression of several Pi starvation-responsive genes was decreased in ZAT6 overexpressing plants, thereby confirming the role of ZAT6 in regulating Pi homeostasis. This study thus indicates that ZAT6 is a repressor of primary root growth and regulates Pi homeostasis through the control of root architecture. To our knowledge, ZAT6 is the first cysteine-2/histidine-2 zinc finger transcription factor reported to regulate root development and nutrient stress responses.

Phosphorus (P) remobilization in plants is required for continuous growth and development. The Arabidopsis (Arabidopsis thaliana) inorganic phosphate (Pi) transporter Pht1;5 has been implicated in mobilizing stored Pi out of older leaves. In this study, we used a reverse genetics approach to study the role of Pht1;5 in Pi homeostasis. Under low-Pi conditions, Pht1;5 loss of function (pht1;5-1) resulted in reduced P allocation to shoots and elevated transcript levels for several Pi starvation-response genes. Under Pi-replete conditions, pht1;5-1 had higher shoot P content compared with the wild type but had reduced P content in roots. Constitutive overexpression of Pht1;5 had the opposite effect on P distribution: namely, lower P levels in shoots compared with the wild type but higher P content in roots. Pht1;5 overexpression also resulted in altered Pi remobilization, as evidenced by a greater than 2-fold increase in the accumulation of Pi in siliques, premature senescence, and an increase in transcript levels of genes involved in Pi scavenging. Furthermore, Pht1;5 overexpressors exhibited increased root hair formation and reduced primary root growth that could be rescued by the application of silver nitrate (ethylene perception inhibitor) or aminoethoxyvinylglycine (ethylene biosynthesis inhibitor), respectively. Together, these data indicate that Pht1;5 plays a critical role in mobilizing Pi from P source to sink organs in accordance with developmental cues and P status. The study also provides evidence for a link between Pi and ethylene signaling pathways.

Phosphate (Pi) availability is a major constraint to plant growth. Consequently, plants have evolved complex adaptations to tolerate low Pi conditions. Numerous genes implicated in these adaptations have been identified, but their chromatin-level regulation has not been investigated. The nuclear actin-related protein ARP6 is conserved among all eukaryotes and is an essential component of the SWR1 chromatin remodeling complex, which regulates transcription via deposition of the H2A.Z histone variant into chromatin. Here, we demonstrate that ARP6 is required for proper H2A.Z deposition at a number of Pi starvation response (PSR) genes in Arabidopsis (Arabidopsis thaliana). The loss of H2A.Z at these target loci results in their derepression in arp6 mutants and correlates with the presence of multiple Pi-starvation-related phenotypes, including shortened primary roots and increases in the number and length of root hairs, as well as increased starch accumulation and phosphatase activity in shoots. Our data suggest a model for chromatin-level control of Pi starvation responses in which ARP6-dependent H2A.Z deposition modulates the transcription of a suite of PSR genes.

The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5′-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5′-ribosome pausing leading to the XRN4-mediated 5′-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of ‘non-aberrant’ mRNAs.

Low inorganic phosphate (Pi) availability triggers an array of spatiotemporal adaptive responses in Arabidopsis (Arabidopsis thaliana). There are several reports on the effects of Pi deprivation on the root system that have been attributed to different growth conditions and/or inherent genetic variability. Here we show that the gelling agents, largely treated as inert components, significantly affect morphophysiological and molecular responses of the seedlings to deficiencies of Pi and other nutrients. Inductively coupled plasma-mass spectroscopy analysis revealed variable levels of elemental contaminants not only in different types of agar but also in different batches of the same agar. Fluctuating levels of phosphorus (P) in different agar types affected the growth of the seedlings under Pi-deprivation condition. Since P interacts with other elements such as iron, potassium, and sulfur, contaminating effects of these elements in different agars were also evident in the Pi-deficiency-induced morphological and molecular responses. P by itself acted as a contaminant when studying the responses of Arabidopsis to micronutrient (iron and zinc) deficiencies. Together, these results highlighted the likelihood of erroneous interpretations that could be easily drawn from nutrition studies when different agars have been used. As an alternative, we demonstrate the efficacy of a sterile and contamination-free hydroponic system for dissecting morphophysiological and molecular responses of Arabidopsis to different nutrient deficiencies.

Phosphate (Pi) is a common limiter of plant growth due to its low availability in most soils. Plants have evolved elaborate mechanisms for sensing Pi deficiency and for initiating adaptive responses to low Pi conditions. Pi signaling pathways are modulated by both local and long-distance, or systemic, sensing mechanisms. Local sensing of low Pi initiates major root developmental changes aimed at enhancing Pi acquisition, whereas systemic sensing governs pathways that modulate expression of numerous genes encoding factors involved in Pi transport and distribution. The gaseous phytohormone ethylene has been shown to play an integral role in regulating local, root developmental responses to Pi deficiency. Comparatively, a role for ethylene in systemic Pi signaling has been more circumstantial. However, recent studies have revealed that ethylene acts to modulate a number of systemically controlled Pi starvation responses. Herein we highlight the findings from these studies and offer a model for how ethylene biosynthesis and responsiveness are integrated into both local and systemic Pi signaling pathways.

XRN4, the plant cytoplasmic homolog of yeast and metazoan XRN1, catalyzes exoribonucleolytic degradation of uncapped mRNAs from the 5′ end. Most studies of cytoplasmic XRN substrates have focused on polyadenylated transcripts, although many substrates are likely first deadenylated. Here, we report the global investigation of XRN4 substrates in both polyadenylated and nonpolyadenylated RNA to better understand the impact of the enzyme in Arabidopsis. RNA degradome analysis demonstrated that xrn4 mutants overaccumulate many more decapped deadenylated intermediates than those that are polyadenylated. Among these XRN4 substrates that have 5′ ends precisely at cap sites, those associated with photosynthesis, nitrogen responses and auxin responses were enriched. Moreover, xrn4 was found to be defective in the dark stress response and lateral root growth during N resupply, demonstrating that XRN4 is required during both processes. XRN4 also contributes to nonsense-mediated decay (NMD) and xrn4 accumulates 3′ fragments of select NMD targets, despite the lack of the metazoan endoribonuclease SMG6 in plants. Beyond demonstrating that XRN4 is a major player in multiple decay pathways, this study identified intriguing molecular impacts of the enzyme, including those that led to new insights about mRNA decay and discovery of functional contributions at the whole-plant level.