Thesis
Reference
Biochemical and physiological studies on polyphosphate metabolism in plants
LORENZO-ORTS, Laura
Abstract
As other living organisms, plants rely on phosphate (P i ) as an essential macronutrient required for growth. One way to store P i in both prokaryotic and eukaryotic cells is in form of inorganic polyphosphates (polyP). PolyP are linear polymers formed by P i subunits linked by high energy phosphoanhydride bonds. PolyP are versatile molecules which can adopt different chain lengths, bind to cations and other positively charged molecules and accumulate in various subcellular compartments. Although polyP are very ancient polymers, they have evolved to have many different functions. In bacteria, polyP are mainly involved in stress responses, as for instance nutrient deprivation or metal toxicity. In yeast cells, polyP represent the main P i pool and maintain both P i and ion homeostasis. In humans, polyP regulate blood clotting, bone calcification, rRNA biogenesis and the cell cycle. While polyP have been found in bacterial, fungal and human cells, it is not yet known whether plants can accumulate polyP. The enzymes involved in polyP synthesis and breakdown are known in bacteria and lower eukaryotes, including yeast, amoeba, [...]
LORENZO-ORTS, Laura. Biochemical and physiological studies on polyphosphate metabolism in plants. Thèse de doctorat : Univ. Genève, 2019, no. Sc. 5359
DOI : 10.13097/archive-ouverte/unige:121353 URN : urn:nbn:ch:unige-1213533
Available at:
http://archive-ouverte.unige.ch/unige:121353
Disclaimer: layout of this document may differ from the published version.
Département de botanique et biologie végétale Professeur Michael Hothorn
Biochemical and physiological studies on polyphosphate metabolism in plants THÈSE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Laura Lorenzo Orts
de
Valencia (Spain)
Thèse Nº 5359
GENÈVE
Centre d’impression Uni Mail 2019
First of all, I would like to thank Michael for giving me the opportunity of joining his lab.
He trusted me both personally and professionally, giving me a very exciting but challenging project.
Then, he trusted me again when I decided to continue working on mRNA in his lab. I am aware that many supervisors would not allow their PhD students to work on something completely far from the lab’s core expertise. He gave me all the freedom (and money) to do all what I wanted to do, and he acknowledged my work by making me a corresponding author in my first publication. Then, he also pushed me encouraged me to learn some biochemistry and structural biology. I am still a passionated biologist, but I think that learning some biochemistry was definitely a good idea.
Second, I want to thank all my colleagues, former and current ones. ‘Especial’ mention to Jacobo, who listened very patiently to all my crazy hypothesis about the role of TTM3 and helped me to accommodate myself in Europe and in the lab; Julia, who helped me with many diverse things and kept my closet always full; Satohiro, who was always willing to give me a hand; and Kelvin, who made my back feel better. Thanks to Andrea, Daniel, Jin, Martina, Elsa, Joel, Larissa, Ben, Rebekka, Florian and Julie for the nice lab atmosphere. Special thanks to Florian to translate my abstract into French. I have enjoyed very much working with all of you at both personal and professional levels.
Next, I would like to thank the members of my thesis advisory committee (Prof. Niko Geldner and Prof. Sophie Martin) for their really nice advices. Thanks to Prof. Roman Ulm, Prof.
Robbie Loewith and Prof. Christian Fankhauser for accepting to be members of my thesis jury.
Last but not least, I would like to thank my family for always supporting me and for bringing me tons of olive oil, ham and oranges from Valencia. Special thanks to my dad, who did 13 h by car to help me moving. A BIG thanks to Ulrich for being my colleague during 4 years and my partner for a bit less. He thus deserves a double acknowledgement: as my colleague, helping me with biochemical experiments and having great scientific discussions with me, and as my partner,
Abstract / Résumé 1 / 3
1. INTRODUCTION 5
1.1. Inorganic polyphosphates (polyP) 5
1.1.1. Subcellular localization of polyP 6
1.1.2. PolyP functions 7
1.1.3. PolyP metabolism – polyP kinases and polyphosphatases 9
1.2. Phosphate – an essential nutrient in plants 10
1.2.1. Plant phosphate metabolism and signaling 11
1.2.2. Do polyP exist in plants? 12
1.2.2.1. Plant orthologs of polyP-binding proteins 12
1.3. mRNA translation 15
1.3.1. Alternative translation in eukaryotes 16
1.3.1.1. Polycistronic transcripts in animals 17
1.3.1.2. Polycistronic transcripts in plants 18
1.3.1.3. Mechanisms of translation of eukaryotic polycistronic transcripts 18
1.4. Aims of this work 25
1.5. References 26
2. RESULTS 36
2.1. Concerted expression of a cell cycle regulator and a metabolic enzyme from a
bicistronic transcript in plants 38
2.2. The Arabidopsis ttm3-1 mutant displays strong developmental defects which are
suppressed in the offspring 69
2.3. Molecular characterization of CHAD domains as inorganic polyphosphate binding
modules 77
3. DISCUSSION 92
3.1. TTM3 function remains unknown 92
3.2. The CDC26-TTM3 transcript suggests a link between polyP and the cell cycle 93 3.3. A long intron may control CDC26 expression in other eukaryotes 94 3.4. Further epigenetic modifications may control CDC26 expression in ttm3-1 94
3.6 Other polycistronic transcripts may exist in plants 96 3.6.1 Polycistronic mRNAs may have a regulatory role during embryogenesis 98 3.7. RcCHAD might also be part of a bicistronic transcript in Ricinus 99
3.8. RcCHAD might bind polyP in the nucleolus of plant cells 99
3.9. PolyP inhibits rRNA biogenesis in eukaryotes and bacteria 100 3.9.1. CHAD may regulate polyP-mediated inhibition of RNA polymerase I 101 3.9.2. CHAD may increase the processivity of TTM catalytic domains 102
3.10. Conclusions and perspectives 103
3.11. References 104
As other living organisms, plants rely on phosphate (Pi) as an essential macronutrient required for growth. One way to store Pi in both prokaryotic and eukaryotic cells is in form of inorganic polyphosphates (polyP). PolyP are linear polymers formed by Pi subunits linked by high energy phosphoanhydride bonds. PolyP are versatile molecules which can adopt different chain lengths, bind to cations and other positively charged molecules and accumulate in various subcellular compartments. Although polyP are very ancient polymers, they have evolved to have many different functions. In bacteria, polyP are mainly involved in stress responses, as for instance nutrient deprivation or metal toxicity. In yeast cells, polyP represent the main Pi pool and maintain both Pi and ion homeostasis. In humans, polyP regulate blood clotting, bone calcification, rRNA biogenesis and the cell cycle.
While polyP have been found in bacterial, fungal and human cells, it is not yet known whether plants can accumulate polyP. The enzymes involved in polyP synthesis and breakdown are known in bacteria and lower eukaryotes, including yeast, amoeba, protozoans and algae. Although plants do not have homologs of polyP biosynthetic enzymes, they contain structurally-related proteins. Here I report two plant proteins with features similar to polyP metabolizing enzymes. One of them, the TRIPHOSPHATE TUNNEL METALLOENZYME 3 (TTM3) harbors a domain which is similar in structure to the yeast polyP kinase Vtc4 (Hothorn et al., 2009). In vitro characterization of TTM3 by our lab and others (Moeder et al., 2012; Martinez et al., 2015) demonstrated that Arabidopsis TTM3 is not producing polyP, as previously thought, but rather cleaves tripolyphosphate into pyrophosphate and Pi. My work shows that TTM3 is expressed together with the CELL DIVISION CYCLE PROTEIN 26 (CDC26) from a plant polycistronic transcript.
Polycistronic transcripts are rare in eukaryotes and had not been reported in plants thus far. I characterize CDC26 as a member of the plant Anaphase Promoting Complex/Cyclosome, an E3 ubiquitin ligase involved in cell cycle progression. CDC26 has an essential role in embryo
conserved in the entire plant lineage for over 700 million years (from algae to dicotyledon plants), suggesting an important role of this transcript in cell cycle regulation, as well as a putative connection between the cell cycle and polyP metabolism in plants.
A protein from the plant Ricinus communis L. (castorbean) was found to harbor a domain of unknown function named CHAD (conserved histidine α-helical domain). In bacteria, CHADs are often fused to TTM domains, or expressed as stand-alone domains from operons encoding polyP metabolizing enzymes. I obtained a crystal structure of the CHAD-containing protein from castorbean, which revealed a basic surface patch common in polyP-metabolizing enzymes. Next I found that CHAD-containing proteins from all kingdoms of life specifically bind polyP with μM to nM affinity in grating-coupled interferometry assays. A complex structure of a bacterial CHAD and polyP showed that CHAD interacts with polyP through its basic surface patch. A fluorescent-tagged version of CHAD localized in the nucleus and nucleolus of plant cells. Co-expression of CHAD with a bacterial polyP kinase demonstrated that CHAD can bind polyP in vivo, as previously observed in bacteria. My work defines CHAD as a novel polyP-binding module which may serve as a tool to further investigate the presence of polyP in plants.
Comme tous les organismes vivants, les plantes ont besoin du phosphate (Pi), celui-ci étant un macronutriment essentiel pour leur croissance. Les cellules eucaryotiques et procaryotiques peuvent stocker le Pi sous forme de polyphosphates inorganiques (PolyP). Les PolyP sont des polymères formés par des sous-unités de Pi reliés entre elles par les liaisons phosphoanhydrides, de haute énergie, pour former de longues chaînes linéaires. Les PolyP sont des molécules modulables qui peuvent adopter différentes longueurs de chaîne, capter des cations et autres molécules chargées et s’accumuler dans divers compartiments subcellulaires. Même si les PolyP sont des polymères très anciens, ils ont évolué pour participer à des fonctions cellulaires très variées. Chez les bactéries, les PolyP sont majoritairement impliqués dans les réponses aux stress, par exemple avec les stress provoqués par des carences en nutriments ou la toxicité des métaux. Chez les levures, les PolyP représentent la réserve principale de Pi et maintiennent à la fois l’homéostasie du Pi et aussi l’homéostasie ionique dans les cellules. Chez les humains, les PolyP régulent la biosynthèse des ARN ribosomaux et le cycle cellulaire.
La présence des PolyP a été mise en évidence chez les bactéries, les champignons et les cellules humaines, mais il n’a pas encore été démontré que les plantes puissent aussi les accumuler.
Les enzymes impliquées dans la synthèse des PolyP et leur dégradation sont connues seulement chez les bactéries et quelques eucaryotes, incluant levures, amibes, protozoaires et algues. Malgré le fait que les plantes ne possèdent pas de protéines homologues aux enzymes de biosynthèse des PolyP, elles possèdent des protéines avec une structure similaire. Je me suis intéressée à deux protéines végétales qui possèdent des fonctions similaires aux enzymes de dégradation des PolyP.
L’une d’elles, la protéine TRIPHOSPHATE TUNNEL METALLOENZYME 3 (TTM3) possède un domaine qui est structuralement similaire à la protéine kinase de levure Vtc4 (Hothorn et al., 2009).
La caractérisation in vitro de TTM3 par notre labo et d’autres (Moeder et al., 2012; Martinez et al., 2015) ont montré que la protéine d’Arabidopsis TTM3 ne produit pas de PolyP, comme nous le
(soit deux Pi) et un Pi. Mon travail montre que TTM3 est exprimée avec la protéine CELL DIVISION CYCLE 26 (CDC26) à partir d’un transcrit polycistronique. Les transcrits polycistroniques sont rares chez les eucaryotes et c’est la première fois que quelqu’un en trouve un chez les plantes. J’ai caractérisé CDC26 chez les plantes, cette protéine faisant partie du complexe de promotion de l’anaphase, aussi appelé Cyclosome. CDC26 est une protéine ligase impliquée dans la progression du cycle cellulaire. Cette protéine a un rôle essentiel dans le développement des embryons et peut réguler la croissance des plantes. Le transcrit bicistronique CDC26-TTM3 a été conservé au cours des 700 derniers millions d’années, ce qui suggère un rôle important de ce transcrit dans la régulation du cycle cellulaire, mais aussi un possible lien entre le cycle cellulaire et le métabolisme des PolyP chez les plantes.
Une protéine de la plante Ricinus communis L. a été trouvée pour avoir un domaine de fonction inconnue appelé CHAD (Conserved Histidine α -helical Domain). Les domaines CHAD sont souvent fusionnés aux domaines TTM, ou exprimés seuls comme les domaines fonctionnels des opérons bactériens qui encodent les enzymes qui métabolisent les PolyP. Mon travail a démontré via la structure moléculaire que la protéine de ricin contenant un domaine CHAD révèle qu’à la surface de la protéine il y a une partie très basique, laquelle est présente chez les protéines du métabolisme des PolyP. Les protéines qui contiennent un domaine CHAD dans tous les règnes de la vie peuvent fixer les PolyP lors d’expériences biochimiques quantitatives. Un complexe structural de la protéine CHAD bactérienne et de PolyP montrent que CHAD peut interagir avec les PolyP au niveau de sa partie de surface basique. L’expression de CHAD dans différents tissus de plante révèle une localisation nucléaire et nucléolaire. La co-expression de CHAD avec une kinase de PolyP démontre que CHAD peut aussi fixer les PolyP in vivo, comme il a été observé chez les bactéries. Mon travail révèle un nouveau domaine fixateur de PolyP et suggère que les PolyP pourraient s’accumuler dans les noyaux et les nucléoles des cellules végétales.
1. INTRODUCTION
Phosphorus (P) is one of the six elements on which life is based. Phosphate (PO43−or Pi), a chemical derivative of phosphoric acid, is a major building block of DNA, RNA, ATP and cellular membranes. Maintaining intracellular Pi levels constant is thus required for proper cell functioning.
A primordial way to store Pi in both prokaryotic and eukaryotic cells is in form of inorganic polyphosphates (polyP). In the first sections of this introduction (1.1 to 1.2), I will report on the localization, metabolism and functions of polyP in the cell. I will discuss the existence of polyP in plants, describing possible polyP subcellular localizations and putative plant orthologs of polyP metabolizing enzymes.
While investigating the in vivo function of a plant polyphosphatase, I came across a plant polycistronic transcript with uncommon features in eukaryotes. Protein translation in eukaryotes has been long considered as monocistronic, with one messenger RNA (mRNA) coding for a single protein. In contrast, some bacterial and viral mRNAs, known as polycistronic transcripts, can code for more than one protein. In the section 1.3 of this introduction, I will describe the presence of alternative translation events in eukaryotes.
1.1. Inorganic polyphosphates (polyP)
Inorganic polyphosphates (polyP) are linear polymers formed by 3 (tripolyphosphate) to many hundred orthophosphate subunits (Pi) (Rao et al., 2009). Like other Pi-containing molecules, polyP are negatively charged, acting thus as chelating agents of cations and other positively charged molecules. They have been proposed to play a role in prebiotic evolution – being originated in volcanic eruptions (Yamagata et al., 1991) – and in early life evolution (Gibard et al., 2018), acting as primordial Pi-storage molecules. PolyP are currently present in all living organisms, from bacteria to higher eukaryotes, and can have diverse and important functions beyond their role as a Pi
source (Kornberg et al., 1999).
1.1.1. Subcellular localization of polyP
PolyP are large, ion-chelating molecules. PolyP binding to positively charged molecules in the cytosol (as, for instance, basic amino acids, calcium or magnesium) could impact several metabolic reactions and signaling pathways. Therefore, cells sequester polyP in specific organelles or granules (Fig. 1).
In bacteria, polyP form electrodense granules in the nucleoid region (Babes, 1895). Some bacteria can accumulate polyP granules constitutively, while other bacteria do only form polyP granules in response to nutrient deprivation (Ault-Riché et al., 1998). Bacterial polyP granules are spatially restricted, maintaining a certain distance from the cell poles (Racki et al., 2017), and do not only contain polyP molecules but also proteins and cations (Tumlirsch and Jendrossek, 2017).
Also, polyP can associate with calcium ions (Ca2+) and polyhydroxybutyrate (PHB) in bacterial membranes, forming DNA-entry channels involved in bacterial competence (Castuma et al., 1995).
In lower eukaryotes, polyP accumulate in acidic compartments, including acidocalcisomes (acidic organelles conserved from bacteria to humans (Ruiz et al., 2004; Docampo et al., 2010)), vacuoles (Ruiz et al., 2001; Hothorn et al., 2009) or the cell wall (Werner et al., 2007a; Werner et al., 2007b). In yeast cells, polyP can contribute to ~ 20 % of the total dry weight (Christ and Blank, 2019). Less than 10 % of the total polyP content found in yeast cells (Kulakovskaya et al., 2010) is present in the mitochondria (Pestov et al., 2004). In humans, polyP have been detected in lysosomes of fibroblasts (Pisoni and Lindley, 1992), in the nucleolus of myeloma cells (Jimenez-Nuñez et al., 2012; Azevedo et al., 2015), and in acidocalcisomes of platelet cells (Ruiz et al., 2004).
Figure 1. Overview of polyP localizations and functions in pro- and eukaryotic cells. Representations of prokaryotic (top), animal/fungal (middle) and plant (bottom) cells are shown on the left. Subcellular compartments where polyP have been located are indicated with an arrow. A table summarizing the functions reported for the different cellular pools of polyP is shown on the right.
1.1.2. PolyP functions
PolyP can form chains of different lengths, bind to diverse compounds and localize to various subcellular compartments. This versatility allows for diverse cellular functions (Fig. 1) which I will summarize in the following section.
In bacteria, polyP can act as a Pi source, facilitating processes such as sporulation (Shi et al., 2004) or resistance to nutrient deprivation (Kuroda et al., 1999). PolyP can also modulate other biological processes, including virulence (controlling cell motility and biofilm formation (Rashid et al., 2000; Shi et al., 2004)) and the cell cycle (Racki et al., 2017). PolyP are also involved in stress tolerance (Gray and Jakob, 2015), binding to metal ions (Remonsellez et al., 2006; Ruiz et al., 2011) or to miss-folded proteins generated during stress and preventing their aggregation (Gray et al., 2014).
In contrast to some bacteria which accumulate polyP under nutrient deprivation, yeast over- accumulates polyP when grown in Pi-rich medium and can metabolize polyP under Pi starvation (Shirahama et al., 1996). In yeast cells, polyP act mainly as a Pi buffer, maintaining internal Pi
concentrations independent from the environment. In yeast, polyP can control the cell cycle by providing the Pi necessary for the synthesis of nucleotides during DNA duplication (Bru et al., 2016). Furthermore, polyP can modulate calcium signaling (Dunn et al., 1994) as well as ion and pH homeostasis (Rosenfeld et al., 2010; Canadell et al., 2015; Eskes et al., 2018). In mitochondria, polyP can form polyP/Ca2+/PHB complexes, giving raise to the mitochondrial permeability transition pore (mPTP) involved in cell death (Abramov et al., 2007; Elustondo et al., 2016).
In algae, polyP can regulate both Pi and ion homeostasis (as seen in yeast) (Yagisawa et al., 2009) and confer resistance to nutrient deprivation (as in bacteria) (Aksoy et al., 2014). PolyP also accumulate in the cell wall during cytokenesis, protecting algae from pathogens or toxic compounds during mitosis (Werner et al., 2007a).
In humans, polyP have been found in lysosomes of fibroblast cells, where they have been proposed to maintain an acidic pH, or to protect compounds from hydrolysis (Pisoni and Lindley, 1992). In the human brain, polyP might mediate communication between astrocytes (Holmström et al., 2013). PolyP accumulate in platelets and osteoblasts cells (Leyhausen et al., 1998; Ruiz et al., 2004), suggesting a role of this polymer in coagulation and bone formation, respectively (Hacchou et al., 2007; Travers et al., 2015). It has also been proposed that polyP can control cell proliferation by stimulating mTOR kinase activity (Wang et al., 2003). PolyP localize to the nucleolus of myeloma cells and modulate RNA polymerase I activity (Jimenez-Nuñez et al., 2012) or bind to nucleolar proteins and regulate rRNA biogenesis (Azevedo et al., 2015).
1.1.3. PolyP metabolism – polyP kinases and polyphosphatases
Although polyP are an ancient form to store Pi in all living cells, the enzymes and protein domains involved in their synthesis and breakdown have not been conserved across evolution.
Bacteria
In bacteria, there are two enzymes involved in polyP synthesis: PPK1 and PPK2 (from PolyPhosphate Kinase 1/2). PPK1 contains a protein domain similar to the catalytic domain of the phospholipase D (PLD) and catalyzes the synthesis of polyP from ATP (Kornberg, 1957; Ahn and Kornberg, 1990). Under certain conditions, PPK1 can also catalyze the reverse reaction, producing ATP from polyP (Kornberg, 1957). PPK2 is related to thimidine kinases, can use either ATP or GTP as substrates (Zhang et al., 2002), and favors the production of GTP from polyP (Zhang et al., 2002).
Regarding polyP breakdown, bacteria have exopolyphosphatase proteins (PPX) which catalyze the release of terminal Pi from polyP chains (Akiyama et al., 1993). PPX enzymes belong to the same protein superfamily than actin, HSP70 chaperones and sugar kinases. Besides their role in polyP catalysis, PPX proteins can also hydrolyze guanosine pentaphosphate (pppGpp), a signaling molecule controlling the bacterial stringent response (Kuroda et al., 1997). Other bacterial proteins have been reported to cleave short polyP molecules. For instance, the CYTH domain (CYaB THiamine triphosphatase) ygiF cleaves tripolyphosphate molecules into pyrophosphate and Pi (Kohn et al., 2012; Martinez et al., 2015).
Slime molds
The amoeba Dictyostelium discoideum has two enzymes involved in polyP synthesis: a homolog of the bacterial PPK1 (Zhang et al., 2005), and an actin-like protein (named as DdPPK2), which forms filaments concurrently with the synthesis of polyP (Gómez-García and Kornberg, 2004).
Yeast
In yeast, polyP synthesis is carried out by the VTC complex (from Vacuolar Transporter Chaperone) (Hothorn et al., 2009), which is composed of at least three protein subunits. The catalytic subunit, Vtc4, contains a tunnel-shaped TTM domain that catalyzes the synthesis of polyP from ATP and a N-terminal SPX domain. While Vtc2 and Vtc3 contain a catalytically inactive TTM domain, Vtc1 only contains a transmembrane domain . PolyP synthesis and translocation to the vacuolar lumen occurs concomitantly, since polyP accumulation in the cytosol results toxic (Gerasimaitė et al., 2014). The VTC complex is also conserved in algae (Aksoy et al., 2014).
Yeast contain several enzymes which can release terminal and/or internal Pi from polyP chains of different length. Yeast PPX1 belongs to the superfamily of DHH phosphoesterases and catalyzes the release of terminal Pi from short-chain polyP (Wurst and Kornberg, 1994). PPN1 (Endopolyphosphatase) is related to calcineurin-like phosphoesterases and catalyzes the hydrolysis of either terminal or internal Pi from long chain-polyP (Kumble and Kornberg, 1996). PPN2, a protein belonging to the PPP-superfamily of metallophosphatases, was recently discovered to exclusively cleave internal Pi from polyP (Gerasimaitė and Mayer, 2017).
Humans
No human polyP polymerases have been characterized thus far. Regarding polyphosphatases, the phosphodiesterase PRUNE1 has been reported to cleave preferentially short chain polyP (Tammenkoski et al., 2008). Also, three members of the nudix hydrolase family of proteins, DIPP1, DIPP2 and DIPP3, are capable of degrading polyP in vitro (Lonetti et al., 2011).
1.2. Phosphate – an essential nutrient in plants
In plants, Pi is one of the 17 essential nutrients required for growth (López-Arredondo et al., 2014). Although P is an abundant element on earth, it is mainly found in apatite and phosphorite rocks. P bioavailability is thus very limited, and current agricultural practices rely on the use of P
fertilizers to ensure good yields. However, the use of fertilizers often causes environmental problems (such as eutrophication), and the fertilizer prizes are raising due to an increase in their demand and a decrease in their production (Elser, 2012). Therefore, understanding how plants can uptake, transport, store and use Pi might be of potential interest for agriculture.
1.2.1. Plant phosphate metabolism and signaling
Plant roots uptake P from the soil mainly as Pi, and relocate it to different plant tissues by Pi
membrane transporters (Shin et al., 2004; Lapis-Gaza et al., 2014). Most plant roots (around 90 % of all plant species) are associated with mycorrhizal fungi, establishing a symbiotic relationship in which the plant provides carbohydrates to the fungus, while the fungus transports nutrients (mainly nitrogen and Pi) into the plant root (Bonfante and Genre, 2010). Interestingly, mycorrhizal fungi accumulate polyP to levels that exceed 60 % of the total cellular Pi content. Thus, most of the mycorrhizal Pi transported to plant roots comes from polyP pools (Hijikata et al., 2010).
Pi participates in many plant metabolic processes, including photosynthesis and carbohydrate metabolism (Rouached et al., 2010), for instance by regulating the activity of metabolic enzymes (Plaxton, 1996). While yeast uses polyP to buffer internal Pi levels (see section 1.1.2.), plants can store free Pi in vacuoles and relocate it to the cytosol when needed (Liu et al., 2016). Seeds and fruits, however, accumulate Pi as phytic acid, the free-acid form of myo- inositolhexakiphosphate (InsP6), which is then hydrolyzed in sequential steps by phytases into lower inositol phosphate molecules and Pi (Secco et al., 2017).
Plants respond to low Pi levels with metabolic and developmental changes aimed to maintain intracellular Pi levels constant. However, how plants sense Pi levels is not totally understood. It has been recently shown that inositol pyrophosphates (InsPs) can act as Pi-sensor molecules (Wild et al., 2016). Depletion of two bifunctional inositol kinases leads to Pi starvation
responses in Arabidopsis, providing additional biochemical and in vivo evidence that InsPs act as Pi
sensor molecules in plants (Zhu et al., 2018).
1.2.2. Do polyP exist in plants?
PolyP exist in pro- and eukaryotic cells, including algae. It is however unclear whether higher plants can accumulate polyP, or whether they rely on Pi and InsPs as the only Pi- storage/sensing molecules. Early reports describe the presence of polyP in spinach leaves (Miyachi, 1961) and parasitic plants (Tewari and Singh, 1964) using acid polyP hydrolisis, which can lead to detection of other Pi species. Plant seeds accumulate Pi mainly in the form of InsP6, which localizes in electrodense organelles similar to acidocalcisomes (known as globoids (Greenwood and Bewley, 1984; Otegui et al., 2002)). Electrodense vacuoles have also been detected in meiotic anthers (Mamun et al., 2005). However, it is currently unknown whether these vacuoles can accumulate polyP.
1.2.2.1. Plant orthologs of polyP-metabolizing enzymes
So far, no enzyme involved in polyP synthesis or breakdown has been described in plants.
However, it has been recently reported that the Arabidopsis Vacuolar proton-Pyrophosphatase 1 (AVP1) can synthesize pyrophosphate using a transmembrane proton gradient depending on cytosolic Pi concentration (Scholz-Starke et al., 2019). Indeed, it has been speculated that higher eukaryotes could generate polyP using a proton motif force. In agreement with this idea, depolarization of the mitochondrial membrane results in decreased polyP synthesis in mammalian cells (Pavlov et al., 2010). Also, mammalian cell lysates only contain residual polyP kinase activity (Kumble and Kornberg, 1995), which cannot explain the polyP concentrations estimated in mammalian cells.
Some of the protein domains present in the polyP metabolizing enzymes from bacteria and other eukaryotes (see section 1.1.3.) are also present in plants (Table 1). For instance, the model
plant Arabidopsis thaliana contains 39 PLD domain proteins (with homology to the bacterial polyP polymerase PPK1), 1 PPX domain protein (with homology to the bacterial polyphosphatase PPX), 7 DHHA1 domain proteins (with homology to the yeast polyphosphatase PPX) and 190 proteins annotated as calcineurin-like metallo-phosphoesterases (with homology to the yeast polyphosphatase PPN) according to Interpro (EMBL-EBI, https://www.ebi.ac.uk/interpro/). In addition, the CYTH protein domain, present in all kingdoms of life, adopts a fold similar to the yeast polyP polymerase Vtc4. Arabidopsis contains 3 CYTH-containing proteins (named as TTM1- 3, from triphosphate tunnel metalloenzyme).
Table 1. Table containing polyP metabolizing enzymes from bacteria, yeast and amoeba. Protein domains and plant protein orthologs (based on primary sequence and secondary structure) of the corresponding polyP metabolizing enzymes are listed.
TTM proteins
In Arabidopsis, there are 3 TTM proteins (TTM1/2/3) harboring a CYTH domain. The CYTH domain is characteristic of enzymes acting on triphosphorylated substrates, as for instance
the human thiamine-triphosphatase (Martinez et al., 2015) or the yeast RNA 5’-triphosphatase Cet1p (Ho et al., 1998). In addition to the CYTH domain, TTM1 and TTM2 proteins contain a N- terminal uridine kinase domain.
Although TTM proteins are similar in structure to the yeast polyP polymerase Vtc4, none of the three Arabidopsis TTM proteins catalyze polyP synthesis in vitro. While TTM1 and TTM2 show pyrophosphatase activity in vitro (Ung et al., 2014; Ung et al., 2017), TTM3 cleaves preferentially inorganic tripolyphosphate into pyrophosphate and Pi (Moeder et al., 2013; Martinez et al., 2015).
The three TTM Arabidopsis proteins do not only show different activities and domain architectures, but also different functions: while TTM1 and TTM2 regulate plant senescence (Ung et al., 2014; Ung et al., 2017), TTM3 was reported to modulate organ growth (Moeder et al., 2013).
CHAD
CHAD (from conserved histidine alpha-helical domain) is a domain of unknown function with conserved histidines and other positively charged residues. Proteins harboring a CHAD motif were recently shown to co-localize in vivo with polyP granules in the bacteria Ralstonia eutropha (Tumlirsch and Jendrossek, 2017). CHAD-containing proteins are often expressed from operons encoding polyP metabolizing enzymes (Iyer and Aravind, 2002), or expressed as fusion proteins harboring a N-terminal CYTH domain. Although CHAD-containing proteins are mainly present in bacteria, there are 10 proteins belonging to eukaryotes (including the plant Ricinus communis L.).
Direct binding of polyP to CHAD has recently been demonstrated (Lorenzo-Orts et al., 2019a).
PPX
Arabidopsis contains a single gene encoding an uncharacterized PPX-containing protein.
PPX proteins do not only cleave polyP but also guanosine pentaphosphate (pppGpp) (see section 1.1.3 above) (Kuroda et al., 1997), which is present in the chloroplast of plant cells. RSH proteins are responsible for the synthesis and catalysis of pppGpp in the chloroplast (Boniecka et al., 2017).
However, it has been proposed that pppGpp might also play a role in the cytoplasm, inhibiting plant growth (Boniecka et al., 2017). Hence, the role of PPX in planta could be to convert pppGpp to ppGpp instead of cleaving polyP.
1.3. mRNA translation
The analysis of a TTM-containing protein from Arabidopsis led me to the discovery of a bicistronic transcript in plants. In the next chapters of this section, I will thus describe the basis of mRNA translation in eukaryotes, emphasizing on the mechanism that allow polycistronic mRNA translation.
Translation is the process in which ribosomes can build proteins using the genetic information encoded in a mRNA. Eukaryotic transcripts are normally formed by a 5’ cap of 7- methylguanosine, a 5’ untranslated region (5’ UTR), a coding sequence (CDS), and a 3‘ UTR, followed by a 3’ polyadenine tail (polyA) (Fig. 2A). Normally, eukaryotic transcripts are monocistronic – containing only one CDS per molecule – while bacterial and viral transcripts are often polycistronic, encoding the genetic information for multiple proteins.
In eukaryotes, multiple factors are involved in canonical translation (Browning and Bailey- Serres, 2015). First, translation initiation requires binding of the cap-binding complex eiF4F and of the polyadenine-binding protein PABP to the 5’and 3’ ends of the mRNA, respectively (Fig. 2B).
eiF4F and PABP recruit initiation factors that unwind the mRNA, promoting the formation of a pre- initiation complex (43S PIC) composed by the 40 S ribosome and multiple initiation factors. A ternary complex (48S PIC) is completed upon binding of eIF2·Met-tRNAi·GTP, and can scan the 5’
UTR region of the mRNA until finding the translation initiation codon. Binding of the 60 S subunit to the 48S ribosome complex (forming the 80 S ribosome) occurs with the exchange of multiple initiation factors. The 80 S ribosome can then translate a protein or peptide until reaching a stop
codon. Then, eukaryotic release factors promote translation termination with the subsequent release of the protein, ribosomal subunits, the tRNA and the mRNA.
Figure 2. Cannonical translation of a eukaryotic transcript. A, A eukaryotic messenger RNA (mRNA) normally contains a 5’ cap of 7-methylguanosine (m7G), a coding sequence (CDS) including an ATG translation initiation codon, 5’ and 3’ untranslated regions (5’ and 3’ UTRs), and a polyadenine tail (polyA). B, Translation initiation requires binding of the eukaryotic initiation factor 4E (eiF4E) to the 5’ cap, and of the polyA binding protein (PABP) to the polyA tail. Binding of both protein complexes through eiF4G promotes ribosome binding and translation.
Unlike eukaryotic mRNAs, bacterial transcripts do not contain a 5’ cap or a polyA tail.
While monocistronic mRNAs contain a single ribosomal binding site (RBS) located in the 5’UTR, bacterial polycistronic transcripts normally harbor independent RBS for each gene. While in eukaryotes, protein translation occurs in the cytosol and endoplasmic reticulus (mRNAs need to be exported from the nucleus), bacterial transcription and translation occur simultaneously in the cytoplasm. In bacteria, long polycistronic mRNAs are thus translated more efficiently than monocistronic transcripts, since the ribosome can access for a longer time to different RBS (Lim et al., 2011).
1.3.1. Alternative translation in eukaryotes
In the past few years, the vision of eukaryotic mRNAs being monocistronic has been challenged with the discovery of more and more eukaryotic transcripts harboring upstream open reading frames (uORFs) (von Arnim et al., 2014; Wethmar, 2014). uORFs encode small proteins or peptides which normally regulate the translation of the downstream open reading frames (also
called main ORFs, or mORFs) (Medenbach et al., 2011). uORF thus compete for the translational machinery required to translate the mORF. Furthermore, the uORF-stop codon is often interpreted as a premature stop codon, leading to the degradation of the transcript by non-sense mediated decay (NMD) (Nyikó et al., 2009).
In addition to uORFs, there is evidence of eukaryotic transcripts containing the genetic information for two or more functional proteins. Some transcripts can harbor alternative translational initiation sites, resulting in protein isoforms with different N-terminal residues. For instance, the LOS2/ENO2 locus in Arabidopsis contains two translational initiation sites (Eremina et al., 2015). If translation starts in the upstream initiation codon, the protein contains an additional N-terminal fragment which is absent in the protein translated from the downstream translational initiation site. While the long CDS encodes an enolase, the short CDS encodes a transcription factor which regulates the expression of its own transcript.
Few additional transcripts have been reported in eukaryotes to encode two or more functional proteins with a complete different amino acid sequence (Mouilleron et al., 2016). In the next sections, I will exclusively refer to this last kind of transcripts, reminiscent to bacterial operons, as polycistronic transcripts.
1.3.1.1. Polycistronic transcripts in animals
As in bacterial operons, the proteins encoded in eukaryotic polycistronic transcripts are often functionally connected. For example, a tricistronic mRNA from silkworm encodes two cytokine precursor-like proteins and a cytokine paralytic peptide involved in immunity (Kanamori et al., 2010). Another example is the human bicistronic transcript coding for the extra-large G- protein Xlαs and its ligand (ALEX), both proteins with a role in signal transduction at the plasma membrane (Klemke et al., 2001). In both examples, the mRNAs are conserved in insects and
vertebrates, respectively, suggesting a biological advantage of the polycistronic configuration of these transcripts across evolution.
1.3.1.2. Polycistronic transcripts in plants
While polycistronic mRNAs have been reported in humans, zebrafish and insects; the presence of transcripts containing multiple coding sequences in plants is not clear. In 1997, Garcia- Rios et al. reported a cDNA from tomato encoding two enzymes from the proline-synthesis pathway (García-Ríos et al., 1997). The coding sequences of both enzymes, present in a single mRNA, are separated by 4 base-pairs. However, western blots in different plant tissues against one of the proteins revealed a translational product migrating at a higher molecular weight than predicted and matching the size of a fusion protein. In other plant species, as in Arabidipsis thaliana and Vigna aconitifolia, the two catalytic domains present as two independent ORFs in tomato are expressed as a single protein. Garcia-Rios et al. thus speculate that, in tomato cells, the ribosome might overpass the first stop codon of this transcript, translating a single protein. We cannot thus consider this transcript as as polycistronic since only one protein is translated in plant cells.
1.3.1.3. Mechanisms of translation of eukaryotic polycistronic transcripts
Translation of polycistronic mRNAs can represent major challenges for the eukaryotic translational machinery. As mentioned before, recognition of a stop codon normally triggers the release of the ribosome from the mRNA, making it difficult to restart translation of a second downstream ORF. Also, canonical translation initiation requires binding of eukaryotic initiation factors to the 5’cap, and starts upon the recognition of the most upstream start codon. Thus, translation of a downstream ORF may require overpassing the first translation initiation codon, or the start of translation of the mORF in a cap-independent manner.
Recently, an operon from E. coli encoding genes from the enterobactin biosynthetic pathway has been found in a budding yeast taxa (Kominek et al., 2018). Interestingly, the operon-like mRNA
has been modified to include eukaryotic mRNA features, such as polyadenylation sites and promoters, which can drive the expression of monocistronic transcripts. To overcome the challenge of polycistronic translation, eukaryotic cells have transformed this operon into monocistronic mRNAs which can be translated in a canonical cap-dependent, polyA-dependent manner.
In the following section, I will discuss non-canonical translation events (summarized in Fig.
3) which might allow eukaryotic cells to translate more than one protein from a single transcript.
Figure 3. Possible mechanisms of translation of polycistronic mRNAs. Schematic representation of translation via an internal ribosomal entry site (IRES), leaky scanning, ribosome reinitiation, N6-methylated adenosine residues (m6a), 3’ cap-independent translation elements (3’CITEs) or circular mRNAs (circRNAs). Translation of polycistronic transcripts forming circRNAs or containing m6A residues has not been reported thus far.
Internal ribosome entry site (IRES)
Translation initiation requires the recognition of the mRNA 5’ cap-structure by the cap- binding complex eIF4F (the eIF4F is depicted by eiF4E and eIF4G in Fig. 2B). In some situations, for instance during cell division or under certain stresses, eIF4F formation is compromised and cap- dependent translation is thus inhibited (Pyronnet et al., 2001; Sonenberg and Hinnebusch, 2009).
Some viral and eukaryotic transcripts contain internal ribosome entry sites (IRES) to which ribosomes can bind in a cap-independent manner (Mailliot and Martin, 2018). Eukaryotic IRES are thus common in transcripts encoding proteins involved in cell division. For instance, the mammalian target of rapamycin (mTOR) protein kinase, which controls cell growth and proliferation, can be translated cap-independently by an IRES located in the 5’ UTR of its transcript (Marques-Ramos et al., 2017). Also, a human transcript encoding a cyclin-dependent kinase contains an IRES located in the CDS (Cornelis et al., 2000). Binding of the ribosome to the IRES promotes translational initiation from an in-frame starting codon. In this case, mRNA translation can be thus initiated at two different positions, giving raise to two different cyclin-dependent kinase isoforms with different N-terminal residues.
IRES also aids translation of polycistronic mRNAs encoding for distinct proteins. For instance, the human leucine zipper protein 6 (LUZP6/MPD6) was identified in the 3’ UTR of myotrophin mRNA (Xiong et al., 2006). In this case, MPD6 can be translated by an IRES located 109 base-pairs upstream of the start codon.
In plants, the presence of IRES is controversial, and only few examples have been reported to date. The maize mRNA encoding the heat-shock protein Hsp101 contains an IRES which allows Hsp101 cap-independent translation under heat stress (Dinkova et al., 2005). Also, the plant homeodomain protein WUSCHEL (WUS) is encoded in a mRNA containing an IRES in its 5’ UTR
(Cui et al., 2015). IRES-dependent translation of the WUS transcript can be activated by La protein under stress conditions, when cap-dependent translation is inhibited.
In vertebrates, the death-associated protein 5 (DAP5/eIF4G2/NAT1) is involved in IRES- dependent translation (Yoffe et al., 2016), and essential for cell differentiation during embryogenesis (Yamanaka et al., 2000). In Arabidopsis, the proteins with the closest sequence- similarity to DAP5 are the translation initiation factors eIFiso4G1 and eIFiso4G2. Double mutant eIFiso4G1/eIFiso4G2 plants show severe defects in growth and development (Lellis et al., 2010), although the potential role of these proteins in IRES-mediated translation is not known.
Leaky scanning
In eukaryotes, the small 40S ribosomal subunit scans the mRNA until reaching the start codon. In case of transcripts containing more than one ORF, the ribosome can bypass the first start codon and initiate translation at a further downstream start codon. This mechanism of protein translation, known as leaky scanning, is thus dependent on the efficiency with which the ribosome can recognize a certain start codon (Kozak, 1999). This efficiency is normally determined by the nucleotide sequence surrounding the translation initiation codon (known as Kozak consensus sequence (Kozak, 1987a)). While strong Kozak sequences are normally recognized by the ribosome, weak Kozak sequences are often missed.
Leaky scanning is a common mechanism to translate viral mRNAs and uORF-containing transcripts (Ryabova et al., 2006; Lorenzo-Orts et al., 2019b). uORF translation often causes ribosome stalling during translational elongation or termination. Ribosome stalling can either block the recruitment of additional ribosomes to the mRNA or promote transcript degradation by nonsense-mediated decay (NMD) (Barbosa et al., 2013). mORF expression thus relies on the strength of the uORF Kozak consensus sequence: while a strong uORF Kozak sequence favors uORF rather than mORF translation, a weak uORF Kozak sequence can promote mORF translation
by leaky scanning (Ferreira et al., 2014). For instance, the human mRNA encoding the molybdopterin synthase catalytic subunit (MOCO1-A) contains an overlapping ORF encoding for a second subunit of this enzyme (MOCO1-B). The ribosome can reach the MOCO1-B start codon by leaky scanning of MOCO1-A (Sloan et al., 1999).
Ribosome reinitiation
After terminating translation, ribosomes are ultimately released from the mRNA. In transcripts containing multiple ORFs, as uORF-containing transcripts or viral mRNAs, the ribosome can remain attached to the mRNA after termination and resume translation at a downstream start codon (Kozak, 1999). The ability of ribosomes to reinitiate translation depends on (i) the distance between uORF and mORF coding sequences (being more efficient when the intergenic distance is long and both coding sequences are in frame (Kozak, 1987b)), (ii) uORF length (reinitiating preferentially after translating short rather than long uORFs) (Kochetov et al., 2008), and (iii) the sequence of the 5’ and 3’ UTRs (Kochetov et al., 2008).
The most famous example of ribosome reinitiation is found in the yeast GCN4 transcript (Thireos et al., 1984). General control protein 4 (GCN4) is a transcription factor which activates the expression of genes involved in purine and amino acid synthesis in response to nutrient deprivation.
The GCN4 mRNA contains four short uORFs; while translation of the first two uORFs allows ribosome reinitiation at the GCN4 start codon, translation of the last two uORFs promotes termination. Under normal nutrient conditions, the ribosome reinitiates translation at the start codon of the last uORF, which promotes termination before reaching the GCN4 start codon. Under specific stress conditions or starvation, the factors required for translation initiation (complexes composed of Met-tRNAiMet and eIF2·GTP) are limited. Recruitment of these factors to initiate translation takes longer, and the ribosome will bypass the last uORF and will reinitiate translation at the downstream GCN4 start codon.
The eukaryotic initiation factor 3 (eIF3) is a large protein complex required for translation initiation. One of the conserved subunits, eIF3h, was shown to facilitate ribosome reinitiation (Roy et al., 2010). In plants, TOR and the protein kinase S6K1 can directly bind eIF3, and TOR can promote eiF3h phosphorylation. When active, TOR is targeted to polysomes, leading to the phosphorylation and subsequent dissociation of S6K1. eIF3h phosphorylation and S6K1 dissociation facilitates ribosome reinitiation. TOR thus mediates active translation of uORF- containing transcripts in polysomes, and facilitates translation of multiple ORFs by ribosome reinitiation (Schepetilnikov et al., 2013; Schepetilnikov and Ryabova, 2018).
5’ UTR m6A
It has been shown that N6-methylation of adenosine residues located in the 5’UTR can promote cap-independent translation of the respective mRNA (Meyer et al., 2015). Interestingly, adenosine N6-methylation is more frequent upon certain stresses, when eiF4F formation (required for cap-dependent translation) is compromised. For instance, the mammalian mRNA encoding the heat shock protein 70 (HSP70) harbors m6A methylation sites in its 5’ UTR (Schwartz et al., 2014) which can be methylated upon heat stress (Dominissini et al., 2012), promoting the cap-independent translation of this protein (Meyer et al., 2015).
3’ CITEs
3’ Cap-independent translation elements (3’-CITEs) are sequences commonly present in the 3’ end of mRNAs found in some plant (Truniger et al., 2017) and animal (Garcia-Moreno et al., 2016) viruses. 3’-CITES can bind either translation initiation factors or ribosomal subunits to the 3’
end of mRNAs and bring them to the 5’ end. There are seven different types of 3’-CITES based on their RNA structure. For most of them, the interaction of the 3’ end with the 5’ UTR or the beginning of the ORF occurs via complementary sequences. Binding of these complementary
sequences forms a RNA loop, known as kissing stem-loop, and can occur within very distant sequences.
Only viral mRNAs have been reported to contain 3’-CITEs thus far. However, some DNA sequences from virus have been found in eukaryotic nuclear genomes (Bejarano et al., 1996; Liu et al., 2010). Hence, it is possible that 3’CITES would also exist in non-viral mRNAs, providing an alternative route for cap-independent translation in eukaryotes.
Circular RNA (circRNA)
mRNAs normally form circular structures due to the interaction of the polyA-binding protein (PABP) with translation initiation factors bound to the 5’ cap (Wells et al., 1998) (Fig. 2B).
It has been proposed that his interaction stimulates protein translation by (i) promoting ribosome recycling, and (ii) facilitating ribosome binding (Kahvejian et al., 2005). Although most mRNAs can form circular structures, only a particular subset can be truly circularized. mRNA circularization occurs via backsplicing, a mechanism in which the 3’ end of an exon can be covalently linked to the 5’ end of an upstream exon. Circular RNAs (circRNAs) were long thought to be non-coding RNA molecules. Recently, it has been shown that a subset of Drosophila and mammalian circRNAs can be indeed translated into proteins in a cap-independent manner (Legnini et al., 2017; Pamudurti et al., 2017). Translation of circRNA seems to occur via AU-rich motifs acting as IRES-like elements (Fan et al., 2018). Through mRNA circularization, alternative coding sequences can arise from a single mRNA precursor. It is tempting to speculate that circularization of a polycistronic transcript could therefore promote the cap-independent translation of at least one of the genes.
Some circRNAs have further roles in the regulation of gene expression. They can promote RNA-polymerase II-dependent transcription in the nucleus (Zhang et al., 2013; Li et al., 2015), regulate splicing of its cognate mRNA (Conn et al., 2017), bind to microRNAs, or interact with proteins regulating translation.
1.4. Aims of this work
PolyP are energy and Pi-source polymers present in the three domains of life. Although Pi is an essential macronutrient in plants, it is unclear whether plant cells can accumulate polyP.
Interestingly, some protein domains present in polyP-metabolizing enzymes are also present in plants. Based on structural homology to the already characterized polyP-metabolizing enzymes from bacteria and low eukaryotes, we identified putative plant polyP-binding proteins and polyP- metabolizing enzymes. In my thesis, I used genetics, molecular biology, quantitative biochemistry and structural biology to determine the function of two plant proteins with structural homology to the yeast polyP polymerase and to a putative polyP-binding domain.
Aim 1. Understanding the biological function of TTM3 in Arabidopsis.
TTM3 contains a CYTH/TTM domain structurally similar to the yeast polyP polymerase Vtc4. Previous work done by our group (Martinez et al., 2015) and others (Moeder et al., 2013) revealed that TTM3 is cleaving tripolyphosphate molecules rather than producing polyP in vitro.
Knockout mutants of TTM3 showed strong growth phenotypes in vivo (Moeder et al., 2013), suggesting an important role for this protein in plant metabolism. Using genetics and molecular biology, I aimed to understand the biological function of TTM3 in Arabidopsis.
Aim 2. Biochemical and structural analysis of CHAD-containing proteins.
CHAD-containing proteins are often encoded in operons together with polyP metabolizing enzymes (Iyer and Aravind, 2002). Interestingly, ~ 50 % of all CHAD-containing proteins contain an additional N-terminal CYTH/TTM domain (Iyer and Aravind, 2002), reported previously to cleave short polyphosphate molecules (Kohn et al., 2012; Martinez et al., 2015). Since CHAD- proteins co-localize with bacterial polyP granules in vivo (Tumlirsch and Jendrossek, 2017), I wanted to determine whether CHAD can directly bind polyP in vitro using quantitative
biochemistry as well as structural biology. To further investigate polyP metabolism in plants, I used CHAD as a tool to localize polyP in plant cells.
1.5. References
Abramov AY, Fraley C, Diao CT, Winkfein R, Colicos MA, Duchen MR, French RJ, Pavlov E (2007) Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death. PNAS 104: 18091–18096
Ahn K, Kornberg A (1990) Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate. J Biol Chem 265: 11734–11739 Akiyama M, Crooke E, Kornberg A (1993) An exopolyphosphatase of Escherichia coli. The
enzyme and its ppx gene in a polyphosphate operon. J Biol Chem 268: 633–639
Aksoy M, Pootakham W, Grossman AR (2014) Critical function of a Chlamydomonas reinhardtii putative polyphosphate polymerase subunit during nutrient deprivation. Plant Cell 26: 4214–
4229
von Arnim AG, Jia Q, Vaughn JN (2014) Regulation of plant translation by upstream open reading frames. Plant Sci 214: 1–12
Ault-Riché D, Fraley CD, Tzeng C-M, Kornberg A (1998) Novel Assay Reveals Multiple Pathways Regulating Stress-Induced Accumulations of Inorganic Polyphosphate in Escherichia coli. Journal of Bacteriology 180: 1841–1847
Azevedo C, Livermore T, Saiardi A (2015) Protein polyphosphorylation of lysine residues by inorganic polyphosphate. Mol Cell 58: 71–82
Babes V (1895) Beobachtungen über die metachromatischen Körperchen, Sporenbildung, Verzweigung, Kolben-und Kapselbildung pathogener Bakterien. Zeitschr f Hygiene 20:
412–437
Barbosa C, Peixeiro I, Romão L (2013) Gene Expression Regulation by Upstream Open Reading Frames and Human Disease. PLOS Genetics 9: e1003529
Bejarano ER, Khashoggi A, Witty M, Lichtenstein C (1996) Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution. Proc Natl Acad Sci U S A 93: 759–764
Bonfante P, Genre A (2010) Mechanisms underlying beneficial plant–fungus interactions in mycorrhizal symbiosis. Nature Communications 1: 48
Boniecka J, Prusińska J, Dąbrowska GB, Goc A (2017) Within and beyond the stringent response-RSH and (p)ppGpp in plants. Planta 246: 817–842
Browning KS, Bailey-Serres J (2015) Mechanism of Cytoplasmic mRNA Translation. Arabidopsis Book. doi: 10.1199/tab.0176
Bru S, Martínez-Laínez JM, Hernández-Ortega S, Quandt E, Torres-Torronteras J, Martí R, Canadell D, Ariño J, Sharma S, Jiménez J, et al (2016) Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae. Mol Microbiol 101:
367–380
Canadell D, González A, Casado C, Ariño J (2015) Functional interactions between potassium and phosphate homeostasis in Saccharomyces cerevisiae. Molecular Microbiology 95: 555–
572
Castuma CE, Huang R, Kornberg A, Reusch RN (1995) Inorganic polyphosphates in the acquisition of competence in Escherichia coli. J Biol Chem 270: 12980–12983 Christ JJ, Blank LM (2019) Saccharomyces cerevisiae containing 28% polyphosphate and
production of a polyphosphate-rich yeast extract thereof. FEMS Yeast Res. doi:
10.1093/femsyr/foz011
Conn VM, Hugouvieux V, Nayak A, Conos SA, Capovilla G, Cildir G, Jourdain A, Tergaonkar V, Schmid M, Zubieta C, et al (2017) A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation. Nat Plants 3: 17053
Cornelis S, Bruynooghe Y, Denecker G, Van Huffel S, Tinton S, Beyaert R (2000) Identification and Characterization of a Novel Cell Cycle–Regulated Internal Ribosome Entry Site.
Molecular Cell 5: 597–605
Cui Y, Rao S, Chang B, Wang X, Zhang K, Hou X, Zhu X, Wu H, Tian Z, Zhao Z, et al (2015) AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards. Plant Cell Environ 38: 2098–2114
Dinkova TD, Zepeda H, Martínez-Salas E, Martínez LM, Nieto-Sotelo J, de Jiménez ES (2005) Cap-independent translation of maize Hsp101. Plant J 41: 722–731
Docampo R, Ulrich P, Moreno SNJ (2010) Evolution of acidocalcisomes and their role in
polyphosphate storage and osmoregulation in eukaryotic microbes. Philos Trans R Soc Lond B Biol Sci 365: 775–784
Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, et al (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485: 201–206 Dunn T, Gable K, Beeler T (1994) Regulation of cellular Ca2+ by yeast vacuoles. J Biol Chem
269: 7273–7278
Elser JJ (2012) Phosphorus: a limiting nutrient for humanity? Curr Opin Biotechnol 23: 833–838 Elustondo PA, Nichols M, Negoda A, Thirumaran A, Zakharian E, Robertson GS, Pavlov EV
(2016) Mitochondrial permeability transition pore induction is linked to formation of the complex of ATPase C-subunit, polyhydroxybutyrate and inorganic polyphosphate. Cell Death Discovery 2: 16070
Eremina M, Rozhon W, Yang S, Poppenberger B (2015) ENO2 activity is required for the development and reproductive success of plants, and is feedback-repressed by AtMBP-1.
The Plant Journal 81: 895–906
Eskes E, Deprez M-A, Wilms T, Winderickx J (2018) pH homeostasis in yeast; the phosphate perspective. Curr Genet 64: 155–161
Fan X, Yang Y, Wang Z (2018) Pervasive translation of circular RNAs driven by short IRES-like elements. bioRxiv 473207
Ferreira JP, Noderer WL, Diaz de Arce AJ, Wang CL (2014) Engineering ribosomal leaky scanning and upstream open reading frames for precise control of protein translation.
Bioengineered 5: 186–192
Garcia-Moreno M, Sanz MA, Carrasco L (2016) A Viral mRNA Motif at the 3′-Untranslated Region that Confers Translatability in a Cell-Specific Manner. Implications for Virus Evolution. Scientific Reports 6: 19217
García-Ríos M, Fujita T, LaRosa PC, Locy RD, Clithero JM, Bressan RA, Csonka LN (1997) Cloning of a polycistronic cDNA from tomato encoding γ-glutamyl kinase and γ-glutamyl phosphate reductase. Proc Natl Acad Sci U S A 94: 8249–8254
Gerasimaitė R, Mayer A (2017) Ppn2, a novel Zn2+-dependent polyphosphatase in the acidocalcisome-like yeast vacuole. J Cell Sci 130: 1625–1636
Gerasimaitė R, Sharma S, Desfougères Y, Schmidt A, Mayer A (2014) Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity. J Cell Sci 127: 5093–5104
Gibard C, Bhowmik S, Karki M, Kim E-K, Krishnamurthy R (2018) Phosphorylation, oligomerization and self-assembly in water under potential prebiotic conditions. Nature Chemistry 10: 212–217
Gómez-García MR, Kornberg A (2004) Formation of an actin-like filament concurrent with the enzymatic synthesis of inorganic polyphosphate. Proc Natl Acad Sci U S A 101: 15876–
15880
Gray MJ, Jakob U (2015) Oxidative stress protection by polyphosphate--new roles for an old player. Curr Opin Microbiol 24: 1–6
Gray MJ, Wholey W-Y, Wagner NO, Cremers CM, Mueller-Schickert A, Hock NT, Krieger AG, Smith EM, Bender RA, Bardwell JCA, et al (2014) Polyphosphate Is a Primordial Chaperone. Molecular Cell 53: 689–699
Greenwood JS, Bewley JD (1984) Subcellular distribution of phytin in the endosperm of developing castor bean: a possibility for its synthesis in the cytoplasm prior to deposition within protein bodies. Planta 160: 113–120
Hacchou Y, Uematsu T, Ueda O, Usui Y, Uematsu S, Takahashi M, Uchihashi T, Kawazoe Y, Shiba T, Kurihara S, et al (2007) Inorganic polyphosphate: a possible stimulant of bone formation. J Dent Res 86: 893–897
Hijikata N, Murase M, Tani C, Ohtomo R, Osaki M, Ezawa T (2010) Polyphosphate has a central role in the rapid and massive accumulation of phosphorus in extraradical mycelium of an arbuscular mycorrhizal fungus. New Phytologist 186: 285–289
Ho CK, Pei Y, Shuman S (1998) Yeast and Viral RNA 5′ Triphosphatases Comprise a New Nucleoside Triphosphatase Family. J Biol Chem 273: 34151–34156
Holmström KM, Marina N, Baev AY, Wood NW, Gourine AV, Abramov AY (2013) Signalling properties of inorganic polyphosphate in the mammalian brain. Nat Commun 4: 1362 Hothorn M, Neumann H, Lenherr ED, Wehner M, Rybin V, Hassa PO, Uttenweiler A,
Reinhardt M, Schmidt A, Seiler J, et al (2009) Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase. Science 324: 513–516
Iyer LM, Aravind L (2002) The catalytic domains of thiamine triphosphatase and CyaB-like adenylyl cyclase define a novel superfamily of domains that bind organic phosphates. BMC Genomics 3: 33–33
Jimenez-Nuñez MD, Moreno-Sanchez D, Hernandez-Ruiz L, Benítez-Rondán A, Ramos- Amaya A, Rodríguez-Bayona B, Medina F, Brieva JA, Ruiz FA (2012) Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar
transcription. Haematologica 97: 1264–1271
Kahvejian A, Svitkin YV, Sukarieh R, M’Boutchou M-N, Sonenberg N (2005) Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms. Genes Dev 19: 104–113
Kanamori Y, Hayakawa Y, Matsumoto H, Yasukochi Y, Shimura S, Nakahara Y, Kiuchi M, Kamimura M (2010) A eukaryotic (insect) tricistronic mRNA encodes three proteins selected by context-dependent scanning. J Biol Chem 285: 36933–36944
Klemke M, Kehlenbach RH, Huttner WB (2001) Two overlapping reading frames in a single exon encode interacting proteins—a novel way of gene usage. EMBO J 20: 3849–3860 Kochetov AV, Ahmad S, Ivanisenko V, Volkova OA, Kolchanov NA, Sarai A (2008) uORFs,
reinitiation and alternative translation start sites in human mRNAs. FEBS Letters 582:
1293–1297
Kohn G, Delvaux D, Lakaye B, Servais A-C, Scholer G, Fillet M, Elias B, Derochette J-M, Crommen J, Wins P, et al (2012) High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved. PLoS ONE 7: e43879
Kominek J, Doering DT, Opulente DA, Shen X-X, Zhou X, DeVirgilio J, Hulfachor AB,
Kurtzman CP, Rokas A, Hittinger CT (2018) Eukaryotic acquisition of a bacterial operon.
bioRxiv. doi: 10.1101/399394
Kornberg A, Rao NN, Ault-Riché D (1999) Inorganic polyphosphate: a molecule of many functions. Annu Rev Biochem 68: 89–125
Kornberg SR (1957) Adenosine triphosphate synthesis from polyphosphate by an enzyme from Escherichia coli. Biochim Biophys Acta 26: 294–300
Kozak M (1999) Initiation of translation in prokaryotes and eukaryotes. Gene 234: 187–208 Kozak M (1987a) An analysis of 5’-noncoding sequences from 699 vertebrate messenger RNAs.
Nucleic Acids Res 15: 8125–8148
Kozak M (1987b) Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol 7: 3438–3445
Kulakovskaya TV, Lichko LP, Vagabov VM, Kulaev IS (2010) Inorganic polyphosphates in mitochondria. Biochemistry Moscow 75: 825–831
Kumble KD, Kornberg A (1996) Endopolyphosphatases for long chain inorganic polyphosphate in yeast and mammals. J Biol Chem 271: 27146–27151
Kumble KD, Kornberg A (1995) Inorganic Polyphosphate in Mammalian Cells and Tissues. J Biol Chem 270: 5818–5822
Kuroda A, Murphy H, Cashel M, Kornberg A (1997) Guanosine tetra- and pentaphosphate promote accumulation of inorganic polyphosphate in Escherichia coli. J Biol Chem 272:
21240–21243
Kuroda A, Tanaka S, Ikeda T, Kato J, Takiguchi N, Ohtake H (1999) Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid
starvation in Escherichia coli. Proc Natl Acad Sci U S A 96: 14264–14269
Lapis-Gaza HR, Jost R, Finnegan PM (2014) Arabidopsis PHOSPHATE TRANSPORTER1 genes PHT1;8 and PHT1;9 are involved in root-to-shoot translocation of orthophosphate.
BMC Plant Biol 14: 334
Legnini I, Di Timoteo G, Rossi F, Morlando M, Briganti F, Sthandier O, Fatica A, Santini T, Andronache A, Wade M, et al (2017) Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis. Molecular Cell 66: 22-37.e9
Lellis AD, Allen ML, Aertker AW, Tran JK, Hillis DM, Harbin CR, Caldwell C, Gallie DR, Browning KS (2010) Deletion of the eIFiso4G subunit of the Arabidopsis eIFiso4F translation initiation complex impairs health and viability. Plant Mol Biol 74: 249–263 Leyhausen G, Lorenz B, Zhu H, Geurtsen W, Bohnensack R, Müller WE, Schröder HC (1998)
Inorganic polyphosphate in human osteoblast-like cells. J Bone Miner Res 13: 803–812 Li Z, Huang C, Bao C, Chen L, Lin M, Wang X, Zhong G, Yu B, Hu W, Dai L, et al (2015)
Exon-intron circular RNAs regulate transcription in the nucleus. Nat Struct Mol Biol 22:
256–264
